Xenotropic murine leukemia virus (XMuLV), one of the model viruses used in this study. WIKIMEDIA COMMONS (HTTPS://COMMONS.WIKIMEDIA.ORG)

Most recombinant monoclonal antibodies (MAbs) are produced by mammalian cells. Because biopharmaceuticals derived from mammalian tissue culture carry the risk of adventitious virus contamination, regulatory agencies expect risk-mitigation strategies to include validation of purification unit operations for their ability to clear viruses (1). Guidelines from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) describe how to prove viral clearance in downstream purification processes using an orthogonal approach (2). Viral log₁₀ reduction values (LRVs) are determined for orthogonal steps and combined to calculate the cumulative log viral reduction for an entire process (3, 4). Regulatory agencies typically expect drug sponsors to be able to describe their mechanisms of viral clearance in detail (4).

Downstream processing of MAbs commonly starts with protein A capture chromatography. Because most MAbs are eluted at low pH (5), a viral inactivation step follows with incubation between pH 3.0 and 4.0 (4). Low-pH treatment and neutralization leads to precipitation of impurities, so a filtration step is required for particle removal. Depth filters are commonly used to combine removal of precipitates with adsorptive binding of remaining soluble contaminants (6). In addition to removing host cell proteins (HCPs) and DNA (7), viral clearance of >4 LRV has been reported for positively charged depth filters (8–11).

In a previous virus clearance study, we demonstrated the capability of charged 3M Zeta Plus depth filters and 3M Emphaze AEX Hybrid Purifier devices as adsorptive unit operations for virus clearance (8). At pH 5.5, removal of minute virus of mice (MVM) with LRVs of 4.6–7.2 were observed for both at 220 L/m² throughput. Removal of MVM at pH 7.0 decreased significantly to LRVs of 2.4–2.7. Clearance of the larger xenotropic murine leukemia virus-related model virus (X-MuLV) also was determined at common process conditions (pH 5.5 and pH 7.0).

Additionally, we tested pH 7.0 at high conductivity (1 mol/L NaCl) to suppress ionic interactions. Under low-salt conditions at pH 5.5 and 7.0, X-MuLV clearance ranged from 5.0 to 5.8 LRV for all tested filter types. By comparison, adding NaCl substantially lowered clearances to 1.1 LRV (Emphaze) and 2.8 LRV (Zeta Plus). The difference in LRVs between low- and high-salt conditions were attributed to anionic adsorption. By arithmetric subtraction, the Emphaze device showed X-MuLV clearance relying on ionic interactions of 4.0–4.7 LRV, whereas Zeta Plus depth filters delivered 2–3 LRV. Residual X-MuLV clearance obtained under high-salt conditions (1.1–2.8 LRV) may be attributed to hydrophobic interactions or mechanical retention.

Based on those results, we have investigated the retention mechanisms of charged depth filters in more detail, again using the small nonenveloped model virus MVM (20–25 nm) and the larger enveloped virus X-MuLV (80–100 nm). To differentiate the mode of virus removal related to ionic, hydrophobic, and mechanical retention, we performed filtration runs at low conductivity, high conductivity, high propylene glycol (PG) concentration, and high conductivity with PG. Additionally, we compared the results of our virus spiking study with HCP removal levels determined at those four experimental conditions. The results provide a detailed understanding of mechanisms involved in virus retention by charged depth filters.

Table 1: Overview of the used filter types and process parameters

Materials and Methods
Materials and Instruments: For our adsorptive depth filtration studies, we used ÄKTAexplorer 10 systems with Unicorn software from GE Healthcare. All buffers were 0.2-µm filtered before use. Table 1 lists the adsorptive filters with anion-exchange functionality.

Table 2: Virus models used for the virus clearance study

Load Material and Virus Assay: Virus-spiking experiments were performed at Charles River Laboratories (CRL) in Cologne, Germany, using X-MuLV and MVM as model viruses. Table 2 provides more detailed information. The load material for our study was prepared by processing a MAb-containing harvest. Culture harvest was clarified with a Zeta Plus 60SP02A device and 0.2-µm filtered before protein A affinity chromatography eluted with a phosphate–citrate–Tris buffer. The eluate was adjusted to pH 5.5 and diluted to MAb concentrations of 10 g/L and 15 g/L. Samples were 0.2-µm filtered and stored at –70 °C until the spiking study could be performed. Before the adsorptive depth filtration runs, load samples were thawed and adjusted to our desired loading conditions regarding NaCl and PG concentration (Table 3). Those adjustments were made with buffer stock solutions containing 4 mol/L NaCl and 80% PG, respectively, leading to a final MAb concentration of 7.5 g/L for all sample loads.

Table 3: Experiments were performed in duplicate except for run #1 condition already tested in a previous study).

To exclude any influence of test items on cell growth or virus replication, CRL also performed cytotoxicity and viral interference assays. Providing the virus stock solutions, the test facility further analyzed samples with 50% tissue culture infective dose (TCID₅₀) assays. The detection limit of such assays depends on the volume of sample incubated with indicator cells. Those cells were cultivated for a specific incubation period and inspected microscopically for virus-induced changes in cell morphology.

Experimental Methods: Virus-clearance experiments on adsorptive depth filters used four different buffer conditions (Table 3). Virus-reduction values for X-MuLV and MVM were determined using phosphate–citrate–Tris buffer (pH 5.5) but without adding NaCl (“pH 5.5 low salt”), representing an appropriate condition for further processing in a MAb purification process. Second, viral reduction was determined using a phosphate–citrate–Tris buffer at pH 5.5 with 1 mol/L NaCl (“pH 5.5 high salt”) to minimize ionic interactions between viruses and the adsorptive filters. Next, experiments using a phosphate–citrate–Tris buffer at pH 5.5 with 20% PG (“pH 5.5 high PG”) were carried out to minimize hydrophobic interactions. Finally, viral reduction was determined using a phosphate–citrate–Tris buffer at pH 5.5 with 1 mol/L NaCl and 20% PG (“pH 5.5 high salt, high PG”) to minimize ionic and hydrophobic interactions for assessing a possible mechanical virus-retention effect with these filters.

Equation 1: Calculation of virus log₁₀ reduction value (LRV), with C_L = virus concentration of the load, V_L = volume of the load, C_F = virus concentration of the filtrate, and V_F = volume of the filtrate

Filtration runs were performed in parallel with two chromatography systems. CRL spiked 180 mL of protein-A–purified and conditioned MAb solutions with 5% (v/v) of ultracentrifuged and prefiltered virus stock solution (0.45 µm for X-MuLV, 0.1 µm for MVM). After mixing, the laboratory immediately analyzed a sample from each spiked pool for virus titer (“load sample”). For X-MuLV, CRL stored a second load sample until the end of the filtration (“hold sample”). MVM is physicochemically resistant, so no hold sample was analyzed for it (10).

Analysts loaded 220 L/m² (84 mL) of starting material at 158 L/m²/h (1 mL/min) onto equilibrated adsorptive depth filters, then flushed them with 63 L/m² (24 mL) equilibration buffer. Finally, they analyzed virus titers of total filtrates (flow-through and flush, the “filtrate sample”) and hold samples. Virus log₁₀ reduction values (LRV) were calculated as described in Equation 1.

Figure 1: Log₁₀ reduction values (LRVs) for (A) X-MuLV and (B) MVM at low- and high-salt conditions with three filter devices (8). Difference in X-MuLV clearance between low- and high-salt concentration (red arrows) is attributed to electrostatic adsorption. Residual LRV at 1 mol/L NaCl might be related to hydrophobic or mechanical retention. Because MVM is much smaller than the filters’ nominal pore size, mechanical retention is excluded, and runs at high-salt conditions were not performed.

Results and Discussion
Cytotoxicity and viral interference tests performed by CRL determined necessary dilutions that would not influence cell growth or virus replication. A recovery assay showed that all X-MuLV–spiked starting materials could be held at room temperature for up to four hours without significant loss of virus titer (data not shown).

Removal of X-MuLV: Clearance of X-MuLV by a Zeta Plus 90ZB05A depth filter showed comparable log₁₀ reduction values (LRVs) for all four tested conditions (Figure 2, top). Regardless whether NaCl, PG, or both were added at pH 5.5, the determined LRVs ranged from 1.8 to 3.0. Because NaCl and PG did not influence virus clearance, the results indicated that removal of the remaining 2 LRV could be attributed to size exclusion. Previously stated importance of hydrophobic interactions in X-MuLV retention by Zeta Plus depth filters (12) could not be confirmed in our study. Compared with promising results obtained in our first study — which delivered X-MuLV clearance of 6 LRV at pH 5.5 under lowsalt conditions (Figure 1A) — X-MuLV clearance was significantly lower (2.2 LRV) in the present study. Because the results under high-salt conditions are comparable (2.8 log, 1.8 log), our proposed hypothesis of mechanical retention by size is supported.

Figure 2: Log₁₀ reduction values (LRVs) for (top) X-MuLV and (bottom) MVM at low-salt, high-salt, high-PG, and high-salt + high-PG conditions with two filter devices

Several factors could alter the effectiveness of virus clearance by depth filtration. Lower clearance determined under process conditions (pH 5.5) could relate to the three parameters varied from the first to the second study: filter media lot, MAb molecule, and total MAb load (which was 825 g/m² in the first study and 1,650 g/m² in the second). The higher total MAb load could have led to hydrodynamic-radius–based shielding of binding sites on the filter media, decreasing the number of available binding sites for viruses (9). Because filtration runs in both studies were performed in duplicate using the same filter lots, some lot-to-lot variation between the two lots used in these studies is conceivable. Zeta Plus depth filters are made of natural ingredients (diatomaceous earth and cellulose), so some variation between lots is possible. That could cause differences in pore structure inside the filter media and ultimately influence mechanical entrapment (13).

Evaluated in separate experiments, Zeta Plus 90ZB05A depth filters are composed of two different filter layers (Table 1), with the upstream 30ZB layer having pore sizes significantly larger (0.5–2.0 µm) than the X-MuLV model virus (80–100 nm). Because size-related retention thus can be excluded, we considered virus clearance by that single filter layer to be easier to validate than that of the whole filter. So we had the laboratory fractionate the filtrate and flush material from its 30ZB runs into three fractions (virus-removal capacity of a single 30ZB layer could be lower than that of another because of the half thickness of the filter media).

Figure 3: Log₁₀ reduction values (LRVs) for X-MuLV at different filter loadings for low-salt or high-salt–high-PG conditions with 30ZB filter layer (upstream layer of the Zeta Plus 90ZB05A device)

X-MuLV clearance of the first fraction was high after loading of 55 L/m² at pH 5.5, with a calculated LRV of 3.8 (Figure 3). It significantly decreased at a higher loading of 110 L/m², and further at a complete loading of 220 L/m², down to a 1.1 LRV. That decrease in virus removal can be explained by exhausting the electrostatic adsorptive capacity of the depth-filter media. With simultaneously high salt and PG concentrations, consistently low LRVs (0.6–1.1) confirmed that the retention was not based on a size-exclusion effect. Nevertheless, the loading capacity for higher viral clearance is limited to 55 L/m² (corresponding to a MAb load of 412.5 g/m²), but that could be compensated in practice with increased filter area. However, it would be necessary to prove whether viral clearance capacity remains as high as measured when a flush step is carried out after a loading of 55 L/m².

By contrast, results from the synthetic Emphaze device confirmed the X-MuLV results in our first study (Figure 1A). Again, we determined virus removal of 5 LRV under low-salt conditions (pH 5.5), whereas adding NaCl suppressed ionic interactions and reduced LRV to 0.32. That observation again was supported by results from the high-PG (LRV 5.5) and the high-salt–high-PG (LRV 0.2) conditions. Because only the addition of NaCl suppressed virus removal, we believe that the retention mechanism is based predominantly on ionic adsorption, with no hydrophobic effects. We expected that based on the construction of the device, two-thirds of its functionalized nonwoven mass holding the cationic polymer was responsible for an electrostatic adsorption.

Removal of MVM: Because the nominal pore sizes of Zeta Plus 90ZB05A and Emphaze AEX Hybrid Purifier filters are significantly larger (>0.2 µm) than MVM (20–25 nm), we did not expect virus removal by size exclusion. Nevertheless, we tested all four conditions to determine whether the retention is related to ionic and/or hydrophobic interactions. 90ZB05A results showed MVM clearance of 2.3–2.4 LRV for pH 5.5 + low salt and pH 5.5 with 20% PG conditions (Figure 2, bottom). Thus, we can assume that hydrophobic interactions are not relevant for MVM retention. By contrast, virus clearance significantly decreased for both conditions spiked with NaCl to LRV <0.7, a level considered to be insignificant. Thus, the determined LRVs under low-salt conditions (2.3–2.4) are associated with ionic interactions. Moreover, a size-exclusion effect could be excluded for MVM because no substantial removal of viruses could be observed when electrostatic adsorption was suppressed by NaCl.

So the 90ZB05A filter removed MVM in the range of 1.5–2.0 logs based on anionic adsorption. Comparing these results with the high LRV of 6.7 at pH 5.5 determined in our previous study (Figure 1B), we see that the clearance is substantially lower herein. This finding is in accordance with results obtained for X-MuLV above. Again, possible reasons include higher total MAb load and lot-to-lot variability among depth filters.

An Emphaze AEX Hybrid Purifier device delivered MVM clearance of 3.7–3.9 LRV at pH 5.5 under low-salt conditions (Figure 2, bottom). By contrast, with a 1-mol/L NaCl spike, LRVs were insignificant (LRV <0.03). Adding PG to suppress hydrophobic effects made no difference. These results confirm that retention of MVM on this depth filter also is based predominantly on electrostatic adsorption, as already shown for X-MuLV above. Clearances were similar to the result of our first study with a determined LRV of 4.6 at pH 5.5 (Figure 1B). As a result, higher MAb loads and different filter lots appeared to have no significant influence on the device’s virus-removal capacity.

Removal of Host Cell Proteins (HCPs): To broaden our knowledge about retention mechanisms for the conventional and synthetic depth filters, we performed additional tests to determine HCP clearance for all conditions we tested.

Figure 4: Host cell protein (HCP) clearance at low-salt, high-salt, high-PG, and high salt + high PG conditions for MAb 2 (CHO HCP Kit #F550, third generation, from Cygnus Technologies)

We found it interestingly that HCP clearance was influenced by hydrophobic interactions in neither filter. HCP removal at pH 5.5 was comparable with that at pH 5.5 using 20% PG (Figure 4). That result is in accordance with results of the virus study above (Figure 2). Under conditions including 1 mol/L NaCl, HCP clearance by with the 90ZB05A filter decreased by about 50%, whereas removal of HCP with the synthetic device decreased by about 85% (Figure 4). Thus, HCP clearance of the latter fully relies on anionic adsorption, whereas HCP removal capability of the conventional filter involves multiple retention modes.

Even though HCPs represent a mix of proteins of different sizes and electrical charges, these results clearly support our proposed retention mechanisms for both model viruses on the two filters.

Electrostatic Adsorption
To improve our understanding of the mechanisms of virus retention on both Zeta Plus and Emphaze depth filters, we had a contract laboratory perform virus-spiking runs under four different conditions. We chose those feed stream conditions — pH 5.5 with low conductivity, pH 5.5 with high conductivity, pH 5.5 with high PG, and pH 5.5 with high conductivity and high PG concentration — to evaluate which interactions contributed to viral clearance: ionic, hydrophobic, or mechanical. In addition, we investigated mechanical retention of the enveloped X-MuLV virus through experiments using the upstream filter layer of the Zeta Plus 90ZB05A filter with its higher nominal pore size (30ZB) as a reference.

By contrast with previously published suggestions concerning the binding mechanisms on conventional depth filters (9, 12, 14, 15) we found that hydrophobic effects are not important for the retention of MVM and X-MuLV on the Zeta Plus 90ZB05A depth filter because the use of PG did not influence determined LRVs. Current results showed only minor ionic retention, whereas a significant virus clearance based on electrostatic adsorption was reported in our previous study (8). This indicated that the virus clearance capability of the depth filter is subjected to variations. Not even Zeta Plus filters are completely specified, although they are characterized by defined specifications with respect to the amount of accessible charge, the uniformity of the media, and/or the charge distribution at submicron level (7). Therefore, it is possible that lot-to-lot variations or a strong dependency on the MAb loading are dominantly responsible for these observations. The determined LRVs at high conductivity and high PG indicated a residual retention based on the physical entrapment of X-MuLV (2 LRV). As presumed, no size-exclusion effect was determined for the smaller nonenveloped model virus MVM.

We have shown that the synthetic Emphaze device’s excellent viral clearances for both MVM and X-MuLV (3.7–5.5 LRV) are driven by electrostatic adsorption. These results are in accordance with previously reported results obtained for another MAb, suggesting robust viral clearance (8). They also support the mechanistic operation mode of anion-exchange modes, which operate almost exclusively by charge-based separation considered to be robust in terms of viral clearance (16). Our results also could be verified by HCP removal results, which confirmed an electrostatic interaction as the only retention mechanism for the fully synthetic device. Furthermore, results of a previous virus-spiking study of a conventional Q-functionalized membrane adsorber showed no significant MVM removal even with a 4.5× lower MAb load under the same buffer-system conditions (data not published).

We hypothesize that the Emphaze AEX Hybrid Purifier device can withstand the concentration of polyvalent ions in a buffer system through its high ion-exchange capacity, offering a significant advantage over conventional Q-functionalized filter media. The X-MuLV clearance of 5 LRV by the nonwoven component of the Emphaze filter is higher than the published average X-MuLV clearance of 2–4 LRV by AEX resins and adsorbers (16). Key process economic advantages include higher flow rates, reduced process time, disposability, and lower buffer consumption.

References
1 Miesegaes G, et al. Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies. Biotechnol. Bioeng. 106(2) 2010: 238–246; doi:10.1002/bit.22662.

2 ICH Q5A. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin. US Fed. Reg. 63(185) 1998: 51074.

3 Brown M, et al. A Step-Wise Approach to Define Binding Mechanisms of Surrogate Viral Particles to Multi-Modal Anion Exchange Resin in a Single Solute System. Biotechnol. Bioeng. 114(7) 2017: 1487–1494; doi:10.1002/bit.26251.

4 Shukla A, et al. Viral Clearance for Biopharmaceutical Downstream Processes. Pharm. Bioprocess. 3(2) 2015: 127–138.

5 Shukla A, et al. Downstream Processing of Monoclonal Antibodies: Application of Platform Approaches. J. Chromatogr. B 848(1) 2007: 28–39; doi:10.1016/j.jchromb.2006.09.026.

6 Yinges Y, et al. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal During Monoclonal Antibody Purification. Biotechnol. Prog. 22(1) 2006: 288–296; doi:10.1021/bp050274w.

7 Singh N, et al. Development of Adsorptive Hybrid Filters to Enable Two-Step Purification of Biologics. MAbs 9(2) 2017: 350–364; doi:10.1080/19420862.2016.1267091.

8 Metzger M, et al. Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal. BioProcess Int. 13(2) 2015: 36–44.

9 Venkiteshwaran A, et al. Mechanistic Evaluation of Virus Clearance By Depth Filtration. Biotechnol. Prog. 31(2) 2015: 431–437; doi:10.1002/btpr.2061.

10 Zhou JX, et al. Viral Clearance Using Disposable Systems in Monoclonal Antibody Commercial Downstream Processing. Biotechnol. Bioeng. 100(3) 2008: 488–496; doi:10.1002/bit.21781.

11 Wang M. Zeta + VR Filters for Viral Reduction. BioProcess Int. 9(7) 2011: 62.

12 Zhou JX, et al. Orthogonal Virus Clearance Applications in Monoclonal Antibody Production. Process Scale Purification of Antibodies. Gottschalk U, Ed. John Wiley and Sons: Hoboken, NJ, 2009: 169–186.

13 Trilisky E, et al. Flow-Dependent Entrapment of Large Bioparticles in Porous Process Media. Biotechnol. Bioeng. 104(1) 2009: 127–133; doi:10.1002/bit.22370.

14 Singh N, et al. Clarification Technologies for Monoclonal Antibody Manufacturing Processes: Current State and Future Perspectives. Biotechnol. Bioeng. 113(4) 2016: 698–716; doi:10.1002/bit.25810.

15 Michen B, et al. Virus Removal in Ceramic Depth Filters Based on Diatomaceous Earth. Environ. Sci. Technol. 46(2) 2012: 1170–1177; doi:10.1021/es2030565.

16 Miesegaes G, et al. Viral Clearance By Flow-Through Mode Ion-Exchange Columns and Membrane Adsorbers. Biotechnol. Prog. 30(1) 2014: 124–131; doi:10.1002/btpr.1832.

Corresponding author Anja Trapp (anja.trapp@rentschler.de) is a scientist in bioprocessing technology and innovation, Laura Igl is a technical assistant, Sabine Faust is a process engineer, Alexander Faude is group leader, Roland Wagner is senior advisor and head of production, and Stefan Schmidt is chief scientific officer at Rentschler Biopharma SE in Laupheim, Germany. Corresponding author Dr. Sophie Muczenski is a specialist application engineer, and Nicole Mang is a sales account executive in the separation and purification sciences division at 3M Deutschland GmbH, Carl-Schurz-Straße 1, 41453 Neuss, Germany; 49-2131-145133; smuczenski@mmm.com. Zeta Plus and Emphaze are registered trademarks of 3M. AKTAexplorer and Unicorn are registered trademarks of GE Healthcare.

The post Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal appeared first on BioProcess International.

Table 1: Traditional viral clearance (VC) execution (percent virus spiking without large-volume testing) compared with total viral challenge (including large-volume testing) — anticipated VC results

Biologics derived from mammalian organisms have been accepted for therapeutic use for almost a century (1). However, these pharmaceuticals have the potential for contamination with pathogenic adventitious agents such as viruses. With cell-line–derived recombinant proteins, the viral risks commonly include viruses in the Retroviridae and Parvoviridae families (2). As patient safety and manufacturing facility suitability became significant concerns in the 1980s and 1990s, several industry and regulatory bodies reached consensus on how to approach the unique challenges of viral safety in biotherapeutics (3–5).

The resulting “safety tripod” brings together three principles in a systematic approach that provides both patient safety and risk mitigation in the manufacturing of biotherapeutics. First, a risk assessment for viral contaminants is conducted that builds strategies to reduce or eliminate risks through selection and sourcing of raw materials or implementation of testing strategies. Second, testing of raw materials (including cell banks) and in-process samples is implemented to identify virus contamination events before batch release.

Third — and arguably the most important for production of biotherapeutic proteins — viral clearance processes (validated virus reduction or inactivation) are introduced to downstream manufacturing for clearance of a wide-ranging panel of relevant and/or model viruses (5). Sourcing of cell banks and raw materials and quality control (QC) tests have limits and sometimes fail to detect all adventitious agents, which has resulted in a number of virus contamination events over the years (6–8). Viral clearance provides a consistent and robust level of protection for both patients and biomanufacturing facilities when combined with appropriate validation and manufacturing plans.

Typical viral clearance strategies include validation of virus inactivation through low-pH or solvent/detergent holds and virus reduction through chromatography steps as well as virus-retentive filtration. Multiple approaches can satisfy the requirement to use orthogonal clearance steps in a given process (4, 5). Low-pH holds, solvent/detergent treatments, and chromatography steps depend on specific chemical properties of viruses and can prove difficult to implement for good clearance of small nonenveloped viruses (9). Nanofiltration provides a well-characterized size-exclusion mechanism for retention of all but the smallest viruses, regardless of their chemical attributes (10). So it is a highly robust viral clearance step that provides a logarithmic reduction value (LRV) >4 for even difficult-to-clear small (18–24 nm) nonenveloped parvoviruses under a broad range of process and operating conditions (11).

Traditionally, virus-retentive filtration unit operations have been validated by conducting laboratory-scale spiking studies. Testing of small-scale filtration processes uses a virus-spiked feed solution prepared as a function of spike percentage of total volume (5). That methodology historically has provided sufficient results for virus stocks produced with typical purification strategies (nonspecific or nonoptimized methods). However, percentage spiking with more highly purified, higher titer virus stocks could yield improved viral clearance results but also may produce inconsistent and unacceptable viral clearance results that can affect critical study filing dates (12).

Here, we explore the advantages of implementing a total viral challenge approach in conjunction with large-volume testing over the traditional percentage-spiking method without large-volume testing (Table 1). In our virus-removal filtration studies, we used protein solutions spiked with ultrapurified minute virus of mice (MVM, 18–24 nm) or xenotropic murine leukemia virus (X-MuLV, 80–130 nm). It is important to note that extra-volume sample testing can have a significant impact on claimable LRVs in cases of complete or near-complete clearance. The resulting data in Table 1 show that with modern approaches to spiking methodologies, expected LRVs have increased significantly across separate and distinct unit operations. That potentially allows for fewer process steps to be evaluated in viral clearance studies.

Materials and Methods
Selection of Virus-Removal Filters: The two virus-retentive filters we selected for this study (both from Asahi Kasei Medical Co., Inc.) clear parvoviruses and are made of two different membrane materials. Whereas the Planova 20N filter is made of a regenerated cellulose hollow fiber, the Planova BioEX filter is made of a modified (hydrophilized) polyvinylidene fluoride (PVDF) membrane.

Advanced Database: To gain valuable insights into the specific unit operations of individual viral clearance studies, we consulted a comprehensive database with entries from more than 3,500 studies spanning over 25 years (WuXi AppTec). For this study, we reviewed records with the following criteria:

• parvovirus-grade virus-retentive filters
• virus spikes of ultrapurified MVM or X-MuLV
• operating parameters (e.g., load concentration, volume, throughput, and
  pressure) made available to account for atypical products and processes.

We included study results covering a broad range of virus LRVs. From this extensive array of data, we could make recommendations for spiking and testing requirements to produce optimal filtration performance and the potential for high viral clearance (14).

Selection of Virus Stocks: In this study, we used both X-MuLV and MVM because they are widely accepted model viruses in viral clearance studies for biotherapeutics. MVM is a relevant small (18–24 nm) nonenveloped virus that has caused a number of documented bioreactor contaminations; X-MuLV is considered to be a model virus for many processes because certain cell lines have been shown to have endogenous retroviral-like particles (15). For this study, we used ultrapurified virus stocks of both types.

To generate such virus preparations, chromatographic techniques are used as the main purification method, with an additional proprietary purification step included in preparation of ultrapurified MVM virus stocks. QC analysis of the ultrapurified viruses reveals that both stock preparations contain fewer contaminants than other grades of purified virus and consist of mostly monodispersed forms of viruses of known size for each virus type (16, 17).

Using ultrapurified virus stocks ultimately enables testing with lower spiking volumes and minimally affects virus-removal filter performance while yielding high viral-clearance values of 5–6 log₁₀ or more (16, 17). Modern parvovirus preparations are roughly 0.5–1.0 log₁₀ (plaque forming units, PFU) different from their historical counterparts, a difference that can represent a three- to 10-fold increase in infectious particles and thus should be taken into account when spiking parvoviruses into sample load materials.

Study Design and Execution: In this study we conducted 16 filtration runs: eight using Planova 20N filters and eight using Planova BioEX filters. For each filter type, we tested high and low operating pressures with MVM (duplicate runs), X-MuLV (single runs), and without virus (a single mock run). The high and low filtration operating pressures were

• 14 psi and 10 psi, respectively, for Planova 20N filters
• 45 psi and 30 psi, respectively for Planova BioEX filters.

First, we thawed the feed material (human IgG from Equitech-Bio), diluted it to 0.1 g/L in 10-mM sodium phosphate and 40-mM sodium chloride buffer (pH 7, 6.4 mS/cm), and stored it at 2–8 °C. Before filtration, MVM or X-MuLV stocks were spiked into room-temperature protein solution at a target total challenge of 7.5 log₁₀ PFU/filter based on our review of previous study results from the viral clearance database. We processed spiked load material through a 0.2-µm prefilter (MVM-spiked solution) and 0.45-µm prefilter (X-MuLV–spiked solution). Load and processing controls were removed from the spiked material for each run.

Equation 1

For all run conditions, we applied the feed material in the same manner to each filter and collected filtrate in two 100-L/m² fractions followed by a 10-minute complete system depressurization, then collected a single 15-L/m² buffer flush at the initial operating pressure in a separate fraction. Each fraction was assayed separately. Additionally, we created a representative pool with proportional amounts of each of the three fractions for large-volume analysis. To determine volumetric throughput and flux of each filtration run by mass, we used Asahi Kasei Bioprocess data-acquisition software.

Virus Titer Quantification: We used standard plaque-assay methodologies for determining virus titer of both MVM and X-MuLV. Rapid large-volume testing was conducted on simulated pool samples for reducing the assay limit of detection (LoD) and increase reported virus LRV for samples in which no virus was detected.

Figure 1: Flux curves for Planova 20N filtrations at high pressure (14 psi)

Briefly, the plaque assays involved producing a monolayer growth of either 324K cells (for MVM) or PG4 cells (for X-MuLV) in six-well plates or large-volume dishes. We incubated the cell monolayers with run sample dilutions at 37 °C — one hour for MVM samples and two hours for X-MuLV samples. After removing the samples from the plates and dishes, we overlaid the cell monolayers with an agarose/culture media mixture and incubated them at 37 °C for either six days (X-MuLV) or 10 days (MVM). Following that final sample incubation, we fixed each cell monolayer with a formalin solution and stained it with crystal violet. Plaques (voids in the cell monolayer) were counted and converted into a plaque-forming unit per milliliter (PFU/mL) measurement for each sample. We calculated virus LRV using Equation 1.

Table 2: Viral clearance data for minute virus of mice (MVM) sorted by total viral challenge

Results and Discussion
Database Findings: Analysis of data obtained from the viral clearance database revealed artifacts of variable data or poorly optimized clearance (several presented in Table 2). Viral clearance was still effective for runs in studies A and B (internal data), but inconsistent breakthrough was observed and produced variation in virus LRV of more than 1 log₁₀ between duplicate runs. In studies C and D (internal data), nonrobust viral clearance was obtained with MVM LRV <4. Cases E and F (internal data) showed the potential to achieve consistent and higher MVM LRV for studies conducted within the normal bounds of traditional process parameters.

Figure 2: Flux curves for Planova 20N filtrations at low pressure (10 psi)

Even though viral clearance artifacts typically are observed only in studies using the smallest parvoviruses, significant risk remains that less satisfactory results could compromise the development and regulatory approval of biopharmaceuticals substantially. Thus, recommendations for using optimized virus preparations in virus-filtration studies have been made (14). Note that a correlation was observed between lower spiking challenges and more consistent viral clearance results, suggesting that virus load may be a critical factor in ensuring predictable outcomes (Table 2). Limiting the total viral challenge to 7.5 log₁₀ PFU/run could mitigate the risk of such artifacts in viral clearance studies, as observed in studies E1 and F1.

Figure 3: Flux curves for Planova BioEX filtrations at high pressure (45 psi)

Process Flux: All filtrations were executed successfully and demonstrated minimal impact of virus spikes on process performance (Figure 1–4). For high-pressure runs with both filter types, spiked runs and mock runs had equivalent starting flux and experienced little to no flux decay, indicating that the virus spike did not affect filter performance. For low-pressure runs with both filter types, the spiked runs had lower starting flux than the mock runs but did maintain similarly near-zero flux decay by comparison. Although we did observe differences in initial flux, the filters performed adequately throughout all runs, with all target filter throughputs achieved.

Figure 4: Flux curves for Planova BioEX filtrations at low pressure (30 psi)

Viral Clearance: Table 3 reports viral clearance data for MVM runs, and Table 4 shows X-MuLV results. No virus was detected in any filtrate sample during this study, and there was no measurable impact of low pressure or process pause on the filters’ viral clearance capability. For all runs conducted with the total viral challenge approach, the simulated pool showed complete clearance with a virus LRV ≥5.9, demonstrating the benefits of this approach. Our dataset strongly supports the use of the total viral challenge approach in conjunction with large-volume testing for viral clearance studies: No viral breakthrough was observed, and consistent robust viral clearance was achieved for all tests.

Table 3: Viral clearance data resulting from filtrations spiked with minute virus of mice (MVM); “≥” indicates complete clearance

To provide context for setting virus-spiking levels, considering virus titers that could arise during a contamination event is important to ensuring that the virus challenge presented during validation studies provides a relevant or worst-case scenario. In recombinant bioprocesses, contaminants usually are identified first in a bioreactor because of their deleterious impacts on cell culture performance. Even when they are not detected at such an early stage, broad in vitro testing or contaminant-specific molecular testing of unprocessed bulk materials usually have LoDs ≤1 log10 PFU/mL (18).

Table 4: Viral clearance data from filtrations spiked with xenotropic murine leukemia virus (X-MuLV); “≥” indicates complete clearance

Therefore, a gross contamination event is likely to be detected. However, in the unlikely case that virus contamination had no observable impact in a bioreactor and was not detected with bulk testing procedures, other virus-removal steps (column chromatography and/or chemical inactivation) before virus filtration certainly would reduce the contaminating virus load.

A Virus-Filtration Example: The only published report of parvovirus titer from a bioreactor contamination indicated a MVM titer of 6 log₁₀ copies/mL by quantitative polymerase chain reaction (qPCR) (8). In this case, the contaminant was discovered through MVM-specific testing. Regardless, the manufacturing process probably would include chromatography steps that could be expected to reduce that level by 2–6 log₁₀, resulting in a worst-case MVM titer of 4 log₁₀ copies/mL at the virus-filtration step. Spiking virus at around 5 log₁₀ PFU/mL thus still provides a greater challenge than the worst-case level for that step.

Understanding relevant viral challenge situations during plasma-product manufacturing is complicated by variations in potential viral clearance steps used for different products and different virus classes. However, it is helpful to note that robust molecular testing regimes are used to limit potential virus loads in plasma pools. For instance, the US Food and Drug Administration (FDA) has provided guidance that B19 parvovirus levels should be <4 log₁₀ copies/mL (19). Without any additional virus-removal steps, a 5 log₁₀ PFU/mL parvovirus spike still represents a worst-case scenario for removal of that contaminant.

In our study, we attempted to reproduce that viral load titer. In so doing, we believe we have reduced the likelihood of observing aberrant viral clearance study artifacts.

A Modern Approach
Our study demonstrates the benefits of using the total viral challenge approach in designing viral clearance studies. By limiting total viral challenge to 7.5 log₁₀ PFU per virus filter, you can achieve highly robust virus LRV while minimizing the risk of study artifacts. The effect is further amplified when this technique is used in conjunction with contemporary virus preparations and large-volume sample testing. Although we have discussed the application of implementing the use of total virus challenge for virus-filtration runs, note that the same methodology has been implemented in other unit operations such as chromatography or low-pH inactivation. In future studies, investigators should consider the tools described here for guidance on achieving appropriate viral clearance as needed for their own downstream purification processes.

References
1 Lalonde R, Honig P. Clinical Pharmacology in the Era of Biotherapeutics. Clin. Pharmacol. Ther. 84, 2008: 533–536.

2 Stuckey J, et al. A Novel Approach to Achieving Modular Retrovirus Clearance for a Parvovirus Filter. Biotechnol. Prog. 30(1) 2014: 79–85; doi:10.1002/btpr.1820.

3 Sofer G, et al. PDA Technical Report No. 41: Virus Filtration. PDA J. Pharm. Sci. Technol. 59(S-2) 2005: 1–42.

4 CPMP BWP 268/95. Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses. European Medicines Agency: London, UK, 14 February 1996.

5 ICH Q5A. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin. US Fed. Reg. 63(185) 1998: 51074.

6 Victoria JG, et al. Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus. J. Virology 84(12) 2010: 6033–6040; doi:10.1128/JVI.02690-09.

7 Skrine J. A Biotech Production Facility Contamination Case Study — Minute Mouse Virus. PDA J. Pharm. Sci. Technol. 65(6) 2011: 599–611; doi:10.5731/pdajpst.2011.00823.

8 Moody M, et al. Mouse Minute Virus (MMV) Contamination — A Case Study: Detection, Root Cause Determination, and Corrective Actions. PDA J. Pharm. Sci. Technol. 65(6) 2011: 580–588; doi:10.5731/pdajpst.2011.00824.

9 Aranha H, Forbes S. Viral Clearance Strategies for Biopharmaceutical Safety, Part 2: A Multifaceted Approach to Process Validation. BioPharm 14(5) 2001: 43–54, 90.

10 Yamamoto A, et al. Effect of Hydrodynamic Forces on Virus Removal Capability of Planova Filters. AIChE J. 60(6) 2014: 2286–2297 ; doi:10.1002/aic.14392.

11 Hongo-Hirasaki T, Komuro M, Ide S. Effect of Antibody Solution Conditions on Filter Performance for Virus Removal Filter Planova 20N. Biotechnol. Prog. 26(4) 2010: 1080–1087; doi:10.1002/btpr.415.

12 Asher D, et al. PDA Technical Report No. 47: Preparation of Virus Spikes Used for Virus Clearance Studies. Parenteral Drug Association: Bethesda, MD, 2010.

13 Chen D, Chen Q. Virus Retentive Filtration in Biopharmaceutical Manufacturing. PDA Letters 15 April 2016: www.pda.org/pda-letter-portal/archives/full-article/virus-retentive-filtration-in-biopharmaceutical-manufacturing Accessed on 21FEB2016.

14 Hongo-Hirasaki T, et al. Effects of Varying Virus-Spiking Conditions on a VirusRemoval Filter Planova 20N in a Virus Validation Study of Antibody Solutions. Biotechnol. Prog. 27(1) 2011: 162–169; doi:10.1002/btpr.533.

15 Stauss DM, et al. Removal of Endogenous Retrovirus-Like Particles from CHO-Cell Derived Products Using Q Sepharose Fast Flow Chromatography. Biotechnol. Prog. 25(4) 2009: 1194–1197; doi:10.1002/btpr.249.

16 Slocum A, et al. Impact of Virus Preparation Quality on Parvovirus Filter Performance. Biotechnol. Bioeng. 110(1) 2013: 229–239; doi:10.1002/bit.24600.

17 Roush D, et al. Limits in Virus Filtration Capability? Impact of Virus Quality and Spike Level on Virus Removal with Xenotropic Murine Leukemia Virus. Biotechnol. Prog. 31(1) 2015:135–144; doi:10.1002/btpr.2020.

18 Gombold J, et al. Systematic Evaluation of In Vitro and In Vivo Adventitious Virus Assays for the Detection of Viral Contamination of Cell Banks and Biological Products. Vaccine 32(24) 2014: 2916–2926; doi:10.1016/j.vaccine.2014.02.021.

19 US Food and Drug Administration. Guidance for Industry: Nucleic Acid Testing (NAT) to Reduce the Possible Risk of Human Parvovirus B19 Transmission by Plasma-Derived Products. US Fed. Reg. 74(143) 2009: 37231–37232.

20 Lute S, et al. Phage Passage After Extended Processing in Small Virus Retentive Filters. Biotechnol. Appl. Biochem. 47(Part 3) 2007: 141–151; doi:10.1042/BA20060254.

Corresponding author Michael Burnham is a senior principal scientist in process development and commercialization, Alexander Schwartz is a viral clearance scientist, and Joseph Hughes is vice president of biologics testing at WuXi AppTec, Inc., 4751 League Island Boulevard, Philadelphia, PA 19112; 1-215-218-7100 x5542; mike. burnham@wuxiapptec.com. Esha Vyas is field applications manager, Nanna Takahashi is an account manager, Pauline Nemitz was field applications manager (now with Sartorius Stedim Biotech), Daniel Strauss is a principal scientist, and Naokatsu Hirotomi is executive vice president and general manager of Asahi Kasei Bioprocess America, Inc., 1855 Elmdale Avenue, Glenview, IL 60026.

The post Advanced Viral Clearance Study Design: A Total Viral Challenge Approach to Virus Filtration appeared first on BioProcess International.

Figure 1: Fouling of chromatography particle surfaces by compound contaminant associations

One of the early disappointments in development of immunoglobulin G (IgG) purification technology was ultrafiltration on membranes with 50–100 kDa cutoffs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that most host cell proteins were smaller than that. IgG was retained. Parallel concentration and buffer exchange could be performed going into a follow-on polishing step. These features made it an obvious candidate for initial capture, but it did not perform as hoped. Membrane fouling sabotaged its concentration–diafiltration potential, and prohibitive levels of host contaminants remained in the IgG fraction. Protein A affinity chromatography became established as the preferred capture method. Ultrafiltration was consigned to the supporting role of concentration and diafiltration after purification was essentially complete.

Protein A has since proven to be consistent and highly competent, but like ultrafiltration, it should be capable of much better performance than it generally delivers. In its prechromatography days, it was hailed for its exquisite specificity, binding only IgG. The expectation was that the affinity chromatography version would achieve essentially complete purification in a single step. In practice, host cell protein (HCP) contamination often is observed in the range of 1,000–10,000 ppm. DNA also persists. It does not seem reasonable that protein A should have affinity for either, and it doesn’t. Their persistence points instead to a nonspecific contamination pathway.

Depressed protein A performance is now understood to result mainly from chemical fouling of chromatography media during sample application. Chemical fouling is distinct from physical fouling. Physical fouling involves cells and debris of a size sufficient to clog filters and columns. Chemical fouling involves nonspecific chemical interactions of contaminants with the surfaces of purification media; 20–40% of host contaminants in cell culture harvests are associated in compound assemblages that include hundreds of species (1–3). Some are weakly associated, some strongly. Some of their components bind more strongly to protein A than IgG does and act as anchors for associated species that individually lack affinity for the chromatography surface.

Figure 2: Dissociation of contaminant subsets coincident with IgG elution

Accumulation of such assemblages on chromatography surfaces interferes with performance in two major ways. Aggregates of 50–400 nm block IgG access to the diffusive pores in chromatography particles. That reduces dynamic capacity on protein A as much as 20% compared with loading purified IgG (Figure 1). IgG elution conditions subsequently dissociate contaminant subsets from still-anchored components, and the dissociated subsets cooccupy the IgG fraction (Figure 2). 99% of host contaminants in IgG eluted from protein A derive from this pathway (1).

The effects of chemical fouling are not limited to protein A. They compromise all chromatography methods. They reduce capacity on traditional and multimodal cation exchangers by 50–65% (4, 5). Chemical fouling burdens even size-exclusion chromatography (SEC), in which the chemical surfaces of chromatography particles are generally assumed to be inert. In fact, SEC interacts so strongly with some contaminant heteroassociations that they smear all the way across the elution profile instead of being restricted to the aggregate fractions corresponding to their actual size class (6).

Removing chemical foulants before column loading enables dramatic improvement: HCP removal by protein A improves more than 100-fold and DNA removal more than 1,000-fold. IgG binding capacity is elevated to the same levels obtained when protein A is loaded with purified IgG (3). Improvements with ion-exchange and multimodal capture are even greater (4, 5).

Given that chemical fouling depresses performance of all chromatography methods and that advance removal of chemical foulants enables them to fulfill their fractionation potential, could it also enable ultrafiltration to deliver the potential envisioned in the 1980s? This article addresses that question and goes a step further. It coordinates capture by ultrafiltration with an in-line polishing chromatography step that takes advantage of ultrafiltration’s abilities to perform parallel concentration and diafiltration.

Removing Chemical Foulants from Cell Culture Harvests
Advance removal of chemical foulants from cell culture harvests targets the most reactive species: the most negatively charged, the most positively charged, the most hydrophobic, largest and least soluble. This is accomplished by adding a combination of flocculating agents to cell-free or cell-containing harvests. Allantoin crystals bind aggregates, viruses, and endotoxins by hydrogen bonding (7–9). Octanoic (caprylic) acid precipitates a variety of HCP and viruses (3–5). Electropositive polymers, particles, or depth filters target DNA, virus, and acidic HCP. Removal of solids eliminates 40–70% of HCP, 99% of DNA, 2–3 logs of endotoxin, 5–9 logs of virus, and 75–95% of aggregates (1–5). Typical IgG losses of about 10% mostly represent misfolded product associated with aggregates. Other approaches are discussed in the literature (10–12).

Figure 3: Size distribution of host cell protein (HCP) before and after foulant removal; note the different scales on the left and right frames.

Figure 3 illustrates SEC profiles before and after flocculation of a Chinese hamster ovary (CHO) cell culture harvest. The aggregate population — which has nearly the same cumulative mass as the IgG peak — contains misfolded IgG, but the dominant species are HCP associated with chromatin nucleation centers (1–5). This illustrates why ultrafiltration initially failed to meet expectations as an IgG capture method. The aggregates would have been coretained with the IgG where they interfered with pore flux and remained in the retentate with the IgG after processing. The right-hand frame in Figure 3 shows why advance foulant removal should enable ultrafiltration to deliver outstanding performance.

Figure 4: Flow diagrams through an apparatus configured to support ultrafiltration with a single adsorbent channel

Ultrafiltration-Adsorption for Integrated IgG Capture–Polishing
Figure 4 illustrates a basic apparatus for coordinating ultrafiltration with in-line adsorption (13–15). The first frame shows the system with only an ultrafiltration unit in line. The second frame illustrates the flow path with an adsorptive chromatography unit also in line. Additional adsorbent channels can be added, and any given adsorbent can be operated in either bind–elute or flow-through mode. Figure 5 shows an early prototype with two adsorbent channels.

Figure 5: An early laboratory prototype ultrafiltration-adsorption system has two adsorbent channels, each equipped with a hollow-fiber membrane adsorber. Changes in flow path were controlled on this unit by manual three-way valves.

Equilibration: Processing begins by equilibrating the system. The equilibration buffer is formulated to the loading conditions for the chromatography adsorbent. The adsorbent is put off line. Ultrafiltration remains on line during the entire process. Concentration and diafiltration begin coincident with sample introduction. Most small contaminants are eliminated through the permeate during this phase. The chromatography adsorbent goes on line before the retentate is fully equilibrated, and the retentate is recycled through the adsorbent while buffer exchange and concentration continue. The process is complete when buffer conditions in the system match the equilibration buffer.

Monoliths and Membrane Adsorbers: The chromatography adsorbent can be a monolith, a membrane adsorber, or a packed column. Monoliths and membranes have lower protein-binding capacity per unit volume than columns with similar selectivity, but the ultrafiltration step reduces that capacity requirement. Monoliths and membranes, meanwhile, maintain their binding efficiency at flow rates over 10× higher than columns. That has value when integrating ultrafiltration with solid-phase adsorption because the filter area is relatively large and requires high volumetric flow rates to support reasonable process times.

Figure 6: Efficiency of media use by different chromatography formats; arrow shows the direction of flow. Monoliths and membrane adsorbers both achieve virtually complete saturation with only a single pass because mass transport is convective. Columns packed with porous particles seldom achieve much better than 50% saturation in a single pass because of the inefficiency of diffusive mass transport.

Monoliths and membrane adsorbers are also preferred because they support much better media use. Saturation can be achieved with only a single pass because both types of adsorbers rely on convective mass transport. Convective mass transport efficiency is independent of both flow rate and solute size. Columns packed with porous particles rely on diffusion for product to enter the pores, and diffusive efficiency is dependent on both flow rate and solute size. This explains why single-pass loading fails to saturate packed columns in a single pass (Figure 6) and why they benefit from multiple-pass loading.

Porous-particle columns remain an important option despite their slowness because they come with a higher diversity of ligands than monoliths or membrane adsorbers, especially including multimodal adsorbents. Their linear flow-rate restrictions can be compensated for with short beds in either axial or radial-flow formats. Another benefit of a porous-particle column is that its bed volume can be customized to match the capacity requirements of a separation.

Figure 7: Experimental results from foulant removal, followed by ultrafiltration-adsorption with a single adsorbent channel in flow-through mode; the tangential-flow filtration (TFF) cartridge was a regenerated cellulose membrane with a 30-kDa cutoff. The adsorbent was a strong anion-exchange monolith.

Figure 7 illustrates results from an experiment with a prospective Herceptin (trastuzumab) biosimilar. Chemical foulants were extracted in advance with allantoin, octanoic acid, and electropositive particles or depth filters as described elsewhere (1–5, 14). The buffer was 50 mM Tris, pH 8.0. Antibody was concentrated to 20 g/L over the course of the experiment. The adsorbent was put in line after 2.5 diavolume (DV) of concentration/ buffer exchange, and the experiment was complete at 5 DV. Process time was 4–6 h. HCP were reduced to <37 ppm, DNA to <1 ppb, and aggregates to about 0.1%. Process recovery was 86%.

Table 1: Ultrafiltration with a single adsorbent channel in flow-through mode

Table 1 shows results from different harvests of the same cell line using different foulant removal methods and different ultrafiltration media and adsorbents. In brief, 30-kDa cellulose ultrafiltration membranes gave the same results as 50-kDa PES membranes. Membranes with 100-kDa ratings caused antibody losses with this antibody but might be suitable for other antibodies. Monoliths, membrane adsorbers, and porous-particle columns were interchangeable. Strong and weak anion-exchange adsorbents were interchangeable. A multimodal cation exchanger (Capto MMC) and a phenyl membrane adsorber gave results comparable to those of anion-exchangers.

This is not to suggest that the technique will work with any adsorbent for any antibody. Conditions were optimized for each adsorbent. With other antibodies, performance among adsorbents varied more substantially. In all cases, however, performance was affected most dramatically by the efficiency of foulant removal. Omitting foulant removal resulted in universal failure, with HCP contamination commonly persisting at 10,000–30,000 ppm.

Figure 8: Experimental results from foulant removal followed by capture with protein A, then polishing ultrafiltration-adsorption with a single adsorbent channel in flow-through mode; the TFF cartridge was a PES membrane with a 50-kDa cutoff. The adsorbent was a weak anion exchange monolith (CIM EDA).

Ultrafiltration–Adsorption for Polishing After Stand-Alone Capture
Figure 8 summarizes a purification in which a protein A eluate still at pH 3.5 was introduced to the system. It was concentrated to 25 g/L and buffer-exchanged to 50 mM MES, pH 5.5, while it recycled through a weak anion-exchange monolith. A 0.22-µm membrane filter was placed in line ahead of the monolith to remove turbidity that developed during pH adjustment. This particular antibody was a rare outlier that still contained >2,000 ppm HCP after the protein A step despite having been treated in advance to remove foulants. Coordinated ultrafiltration–adsorption still reduced HCP to <10 ppm, DNA to <1 ppb, and aggregates to <0.1% (13–15).

The ability of the method to reduce HCP from 2,000 ppm to less than 10 ppm highlights two important points: First, by suspending the mechanisms that compromise purification performance, advance foulant removal enables purification methods to achieve the results they should be able to deliver. Second, keeping the ultrafiltration channel in line eliminates small contaminants throughout the entire process cycle. This is an especially important enhancement for flow-through applications because it removes nonadsorbed small contaminants from the system. By contrast, nonadsorbed contaminants in conventional flow-through chromatography applications stay with the antibody.

Coordination of Multistep Chromatography Processes
Flow-through steps with a single adsorbent channel are most compelling: They involve fewer buffers and less process time, but additional adsorbent channels can be added to enable complete multistep purification processes on a single instrument.

For two flow-through channels, the process begins with sample equilibration/concentration. The first adsorbent is put in line and kept there until the sample is equilibrated. Then that adsorbent is put off-line, and the second is brought in-line. The system is equilibrated with a buffer designed to optimize contaminant removal with the second adsorbent. Then rinsing the system with clean buffer displaces product from the internal flow path, after which the system is sanitized with 1 M NaOH.

Using adsorbents in bind–elute mode involves more buffer inputs and takes more time but still conserves the benefits of keeping sample concentrations high throughout the process. It also suspends the difficulty of buffer exchange between process steps. Once your protein is in the system, you can buffer-exchange it in conjunction with adsorption. Large non-IgG proteins such IgM, factor VIII, and von Willebrand factor particularly suggest themselves as natural subjects for ultrafiltration-adsorption because they could accommodate membranes with larger pore-size distributions, potentially removing an even broader spectrum of contaminants.

Figure 9: Experimental size-exclusion chromatography (SEC) results showing purification of bacteriophage M13 with a two-channel ultrafiltrationadsorption (UA) system; baseline triangles mark the point at which sample salts elute from the column; ELISA = enzyme-linked immunosorbent assay, qPCR = quantitative polymerase chain reaction, QA = quaternary amine

Virus particles for vaccines, gene therapy vectors, or antibiotic replacement also are obvious candidates. Figure 9 summarizes purification of a bacteriophage with a two-channel apparatus (13–15). The steps were concentration and elimination of small contaminants through the membrane in parallel with equilibration to 50 mM MES, pH 6, then binding to a cation-exchange monolith. The cation exchanger was eluted and put off-line, and an anion-exchange monolith put in-line. The buffer was exchanged to 50 mM HEPES, pH 7.0, causing the virus to bind; then it was eluted.

Future Directions
The biopharmaceutical industry’s evolution toward continuous processing raises the obvious question of whether coordinated ultrafiltration-adsorption can fulfill this ideal. It can, but in a fundamentally different way than approaches such as simulated moving-bed (SMB) chromatography. SMB represents genuine continuous processing: It processes feed continuously at the rate produced and continuously delivers processed product. SMB, however, supports only a single chromatography method per instrument. Multiple instruments are required to conduct multistep processes. Each instrument contains dozens of moving parts and coordinates thousands of mechanical events per day. Method development requires sophisticated simulation software to model the
nonintuitive retrograde order of chromatography events across multiple columns.

Processing with ultrafiltration-adsorption occurs in batch mode, but it can support a complete multistep purification process on a single instrument. All the system’s components can be sanitized ahead of use in a single step and at the end of a process in a single step. A simple surge tank to accumulate harvest between cycles enables round-the-clock production. Even systems configured for multiple adsorption steps involve only a fraction of the moving parts and monitoring devices required by SMB and thousands of fewer mechanical events per process cycle. Development involves established intuitive concepts and familiar guidelines.

Equipment access could be considered a limitation for ultrafiltration-adsorption systems to the extent that they are not presently available commercially. But the components are. Data discussed in this article were obtained with commercially available integrated tangential-flow filtration units, modified to include UV, pH, and conductivity sensors for in-line process monitoring. Those components have been available at a number of process scales for decades, as have the chromatography media, so scalability seems unlikely to be an issue. Experienced chromatographers can assemble a functioning unit within a few hours.

It is impossible to predict whether or when the industry will be ready to embrace another new instrument technology, but several key points are already clear: Ultrafiltration is absolutely capable of providing the processing benefits envisioned by the industry’s early founders, and we can certainly use it more effectively than tradition has taught us. The evolution of chemistry and instrumentation since those days has put us in better-than-ever position to do so. The increasing productivity and economic demands being placed on the industry meanwhile demand that we not overlook opportunities that lie easily within our reach.

Acknowledgments
Thanks to the Bioprocessing Technology Institute in Singapore, where the foulant removal and ultrafiltration-adsorption technology discussed in this article were developed under a grant from the Singaporean Agency for Science, Technology and Research (A*STAR).

References
1 Gagnon P, et al. Nonspecific Interactions of Chromatin with Immunoglobulin G and Protein A, and Their Impact on Purification Performance. J. Chromatogr. A 1340, 2014: 68–78; doi:10.1016/j.chroma.2014.03.010.

2 Gagnon P, et al. Non-Immunospecific Association of Immunoglobulin G with Chromatin During Elution from Protein A Inflates Host Contamination, Aggregate Content, and Antibody Loss. J. Chromatogr. A 1408, 2015: 151–160; https://doi.org/10.1016/j.chroma.2015.07.017.

3 Nian R, et al. Advance Chromatin Extraction Improves Capture Performance of Protein A Affinity Chromatography. J. Chromatogr. A 1431, 2016: 1–7; doi:10.1016/j.chroma.2015.12.044.

4 Nian R, Gagnon P. Advance Chromatin Extraction Enhances Performance and Productivity of Cation Exchange Chromatography-Based Capture of Immunoglobulin G Monoclonal Antibodies. J. Chromatogr. A 1453, 2016: 54–61; doi:10.1016/j.chroma.2016.05.029.

5 Gagnon P, et al. Chromatin-Mediated Depression of Fractionation Performance on Electronegative Multimodal Chromatography Media, Its Prevention, and Ramification for Purification of Immunoglobulin G. J. Chromatogr. A 1374, 2016: 145–155; doi:10.1016/j.chroma.2014.11.052.

6 Tan L, et al. Characterization of DNA in Cell Culture Supernatant By Fluorescence-Detection Size-Exclusion Chromatography. Anal. Bioanal. Chem. 407, 2015: 4173–4181; doi:10.1007/s00216-015-8639-9.

7 Vagenende V, et al. Amide-Mediated Hydrogen Bonding at Organic Crystal Interfaces Enables Selective Endotoxin Binding with Picomolar Affinity. ACS Appl. Mat. Interfaces 5(10) 2013: 4472–4478; doi:10.1021/am401018q.

8 Vagenende V. et al. Self-Assembly of Lipopolysaccharide Layers on Allantoin Crystals. Colloids Surf. B Biointerfaces 120, 2014: 8–14; doi: 0.1016/j.colsurfb.2014.04.008.

9 Vagenende V. et al. Allantoin As a Solid Phase Adsorbent for Removing Endotoxins. J. Chromatogr. A 1310, 2013: 15–20; doi:10.1016/j.chroma.2013.08.043.

10 Singh N, et al. Clarification Technologies for Antibody Manufacturing Processes: Current State and Future Perspectives. Biotechnol. Bioeng. 113(4) 2016: 698–716; doi:10.1002/bit.25810.

11 McNerney T, et al. PDADMAC Flocculation of Chinese Hamster Ovary Cells: Enabling a Centrifuge-Less Harvest Process for Monoclonal Antibodies. mAbs 7(2) 2015: 413–427; doi:10.1080/19420862.2015.1007824.

12 Kang Y, et al. Development of a Novel and Efficient Cell Culture Flocculation Process Using a Stimulus Responsive Polymer to Streamline Antibody Purification. Biotechnol. Bioeng. 110(11) 2013: 2928–2937; doi:10.1002/bit.24969.

13 Gagnon P, et al. Flow-Through. Turbocharged. Oral presentation, Prep XVIII, Philadelphia, July 26–29, 2015.

14 Gagnon P. Apparatus and Methods for Fractionation of Biological Products. United States Patent Application 20170173537, 2017.

15 Gagnon P, et al. The Nokia Syndrome. Can Protein A Survive? Oral presentation, IBC Bioprocess Development and Production Week, Huntington Beach, March 30–April 2, 2015.

Pete Gagnon is CSO at BIA Separations and a member of BPI’s Editorial Advisory Board; pete.gagnon@biaseparations.com.

The post IgG Purification By Ultrafiltration: Time for Another Look appeared first on BioProcess International.

Figure 1: Usual clarification options for viral vaccines and vectors

Viral vaccines rely on the antigen properties of a virus or virus-like entity to trigger an immune response and induce immune protection against a forthcoming viral infection. Through development of recombinant viral vaccines, developers can reduce risks associated with the presence of live and inactivated viruses. Instead, recombinant vaccines induce immunity against a pathogen by relying on the capacity of one or more antigens delivered by means of viral vectors or the baculovirus/plasmid system (1). Viral vaccines are formulated with or without adjuvants and categorized as shown in Table 1.

Table 1: Classification of viral vaccines

Viral vaccine manufacturing processes can be templated. They follow a general scheme, starting with production in either an embryonated egg or mammalian/insect cell culture. After production, the bulk harvest material is processed to purify a vaccine of interest. Following upstream production and lysis (optional), a clarification step typically is introduced to start the purification process by either centrifugation or filtration. This is a critical unit operation because it strongly affects product recovery and subsequent downstream purification.

Selection of the clarification methodology depends on the type of cells involved, the nature of the virus, and properties of the process fluids. Filtration-based technologies have gained prominence in vaccine clarification following the increasing implementation of single-use technologies upstream. Filtration methods include membrane technology (microfiltration operated in normal-flow filtration (NFF) mode, tangential-flow filtration (TFF), or depth filters operated as NFF.

Here we provide a comprehensive overview of different filtration technologies and their application in viral vaccine clarification. We also outline challenges and present current best practices.

Considerations for Clarification of Viral Vaccines: Type of Substrate
Composition, type, and level of contaminants to be removed during clarification mainly depend on the upstream process and expression system used for production. In most cases, the virus particles must be kept integral during the clarification step.

For decades, embryonated chicken eggs have been used to produce both human and animal vaccines. However, the resulting allantoic fluid harvest (rich in virus particles and cellular debris) is a challenging feed for clarification. This fluid has a high solids content (>25%, which increases with embryo age) and high mineral and protein content, hence its highly viscous consistency. It also contains rudimentary tissue compounds from chicken embryos such as feathers, beaks, blood vessels, and/or blood cells.

Several viral vaccines have moved away from an egg-based process toward the use of live cells (e.g., plant, microbial, avian, or mammalian). The composition of these feed streams can vary significantly. Based on cell viability  — and lysis method selected (e.g., chemical or mechanical), if applicable — the impurity profile in the fluid to be clarified can differ considerably. For example, low cell viability can indicate high levels of contaminants released into a feed stream from cell lysis. Typical contaminants such as host cell DNA, host cell proteins (HCPs), lipids, and bigger particles such as cell debris can be identified. The proportion of solids in the feed usually is an indicator of purifying challenges ahead (~6–8% mammalian cells or up to 40% in yeast). Compared with allantoic fluid, however, cell culture harvests are considerably cleaner in terms of solids load and soluble content.

Another expression method is the baculovirus expression vector system (BEVS) used with insect cells. This is gaining interest particularly for producing viral vectors and virus-like particles (VLPs). Other expression systems such as bacteria, yeast, and plant cells also can be used to produce viral particles.

Considerations on Key Product Quality Criteria and Control Strategies
Yield: In most cases, yield is an off-line measurement conducted at the end of a number of process steps. Depending on the success criteria for each step, yield can be the main parameter to consider when selecting one option over another for a given step. Based on the size and properties of viral particles, yield could be affected by the clarification method used. For example, whereas positive charges increase nucleic acids and HCP capture, diatomaceous earth can retain viruses by adsorption.

Some viruses are shear sensitive and can be damaged by high shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages through TFF. Due to their large size, viruses larger than 100 nm also can be retained simply by tight filters. Thus, companies should take such factors into consideration when selecting depth filters because some depth filter devices include a 0.1-μm membrane that could cause retention-driven product loss. Other process elements such as aggregation and an excess of impurities can compromise viral particle recovery.

Final Product Purity Levels: Regulatory agencies provide recommendations and requirements regarding acceptable residual amounts of contaminants in final drug products. For reasons of patient safety and tolerance, host cell DNA in a final product must be reduced to appropriate levels. In 1998, the World Health Organization (WHO) specified the maximum residual DNA content in a vaccine to be <10 ng/dose. Since then, the European Medicines Agency (EMA) proposed more stringent conditions based on the type of cell line (tumorigenic origin) used in vaccine manufacturing. The US Food and Drug Administration (FDA) follows a case-by-case evaluation approach and recommends that manufacturers reduce both the size (~200 bp) and amount of DNA per dose.

To date, a final DNA content of <10 ng/ dose commonly is accepted for most biologics. Similarly, a recombinant monoclonal antibody (MAb) product must reach clearance of impurities to <100 ppm of HCP, ≤10 ng/dose of DNA, and <5% of immunogenic aggregates (Table 2).

Table 2: Acceptable remaining impurities in vaccine products

Feed Quality Evaluation Criteria and Processing Parameters: Several parameters can be used to assess the clarity or quality of a product, either during process development or after each manufacturing process step. Turbidity is an easy parameter to monitor and provides an immediate assessment of feed quality. For example, it enables the detection of depth filter breakthrough.

The two primary methods of ascertaining the effectiveness of the clarification step are centrifugation and filtration. Turbidity can be monitored simply by absorbance/scatter in the visible range, providing an immediate assessment of particle load within the filtrate or centrate. Turbidity also relates to filter capacity, which is the volume of feed a depth filter can process before the pressure drop breaches specifications. Capacity relating to both pressure drop and turbidity breakthrough are linked, and specifications for both should be set during process development. For some processes (particularly those with a smaller average particle size in the feed), turbidity breakthrough is the limiting factor in sizing a filtration train. Often, capacity limit is attributed to pressure drop. But because high pressures or flow rates can cause premature turbidity breakthrough, both mechanisms can be related.

Technology Options for Clarification of Virus Vaccines: Because of the extreme diversity of viral vaccines in terms of size, structure, shape, and expression system, no unified template exists for their production and purification. Those processes can be divided into four different phases: upstream/production, clarification, purification, and formulation.

To reduce burden on downstream purification steps, the main objective of a clarification process is to remove undesirable materials, including whole cells, cell debris, colloids, and large aggregates. As the first downstream process, clarification should be optimized to maximize product yield and purity. Several serial operational steps often are required to achieve a desired level of clarification. The first operation (often referred to as primary clarification) removes larger particles, and the second (often referred to as secondary clarification) removes colloids and other submicron particles (Figure 1).

In theory, all available technologies (low-speed centrifugation, microfiltration TFF, NFF) can be selected and potentially combined to clarify viruses. As with other manufacturing process steps, a clarification process should have predictable scalability, be manufacturing-friendly (e.g., be easy to use, reduce holdup volume, provide operator safety), and have a low cost of goods (CoG). However, the clarification process of viral vaccines has two unique characteristics that can require more tailored solutions:

• low solids content with a high nucleic acid and colloid content, requiring higher retention capacity
• high feed variability and cell culture enhancements, requiring more robustness (2).

Although centrifugation can handle a high solids load and traditionally has been used in batch and continuous modes, it requires large capital investment and high maintenance costs. More important, centrifugation scale-up can be problematic because of unreliable scale-down models with nonlinear scalability and high-shear operation for shear-sensitive vaccines. But NFF and TFF have gained interest for vaccine clarification because they are significantly easier to scale-up and implement.

NFF: Primary clarification using NFF typically involves depth filters that often contain positively charged material and filter aids (e.g., diatomaceous earth) that improve retention of cell debris, colloids, and negatively charged contaminants. NFF relies on two main mechanisms for particle retention: size exclusion and adsorption.

NFF membrane filters can be used in secondary filtration because they retain particles by size exclusion. Certain grades of depth filters have a tighter pore-size distribution (which offers greater colloidal particle retention) but do not have high holding capacity. Noncharged depth filters also can be used for clarification while offering higher cost-effectiveness for small batches (≤1,000 L). They typically use three media types:

• melt-blown media fabricated in a pleated format to achieve higher flow rates and holding capacities
• graded density of concentrically wrapped media to allow the filter to remove finer contaminants progressively
• membranes to provide higher retention efficiency.

TFF membranes with retention ratings in the range of 0.1–0.65 μm have been used to retain cells, cell debris, and other large contaminants. Most TFF devices are linearly scalable and reusable after cleaning, and hence greatly reduce consumable costs. However, certain viruses (e.g., extracellularly produced enveloped virus-like particles) can be damaged by high-shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages in TFF processes. Open-channel TFF devices (cassette format without screen) are preferred to minimize shear.

Case Studies
Egg-Based Vaccines: In influenza vaccine processes, a typical allantoic fluid harvest is rich in proteins (e.g., ovalbumin, lysozyme, ovomucin) and contains 45 μg hemagglutinin antigen per egg (~3–4 μg HA and 10⁸–10⁹ infectious units/mL of allantoic fluid). The typical gravity-settled turbidity of a virus-containing allantoic fluid (VCAF) generally is 46–132 NTU. Low-speed zonal continuous centrifugation around 4,000–5,000g often is the preferred option to remove large particles and thus gets used for primary clarification, typically providing a recovery yield of 70% (3, 4). Many vaccine manufacturers use sucrose gradient zonal centrifugation to purify and concentrate viruses. However, polypropylene and cellulose-based depth filters also can be implemented for filtration. Fair capacities of 150–210 L/m² and up to 3× reduction of feed stream turbidity can be achieved with those allantoic fluid harvests. That option is appropriate for influenza vaccines, which are prone to adsorption loss during clarification on charged filters.

NFF also can be used for secondary clarification. Combinations of polypropylene, cellulose, and glass-fiber materials generally demonstrate good efficiency (5). Using a salt solution can reduce association between a virus vaccine and solid debris, resulting in a yield increase of about twofold without compromising viral particle integrity.

One study demonstrated that higher ionic strength on allantoic debris increased influenza virus yields (6). In that case, 1.5 M NaCl was applied to pooled allantoic fluids of various influenza strains, and the sample was centrifuged for different durations to understand the amount of virus partitioned in the supernatant and pellet. Control samples were kept at 0.15M NaCl. This test scope was expanded to include other influenza strains — A/New Caledonia (H1N1), A/Texas (H3N2), B/Jiangsu and B/Hong Kong — to demonstrate an average twofold yield increase. Further work verified the integrity of the purified virus in control and higher ionic strengths. Data showed that higher ionic strength did not adversely affect the live titer of the tested influenza virus strains (2). Specifically, studies have reported use of a 1.2-μm cellulose nitrate (CN) filter polypropylene media followed by 0.45-μm filter polyvinylidene fluoride (PVDF) membrane for clarification of cell-based influenza with a loading of 111 L/m² and 105 L/m², respectively (7).

Another option is use of TFF with a 0.65-μm or 0.45-μm microfiltration membrane device operated with permeate flux control (8). Using a “two-pump process” with a permeate pump in addition to the standard TFF feed pump allows for permeate flux control to manage/reduce polarization and fouling. That provides better characterization of the “critical” flux — the limiting flux above which a process becomes unstable (9).

Moreover, researchers conducted primary clarification of allantoic fluid using a 40-μm bag filter followed by an open-channel microfiltration device (Prostak 0.65 μm; data not shown). Using a crossflow of 3 LPM/channel, with transmembrane pressure (TMP) at 0.2 bar and dP at 0.4 bar, a capacity of 33 L/m² was achieved. The MF process was designed for 10× concentration and 5× diafiltration, and the Prostak allowed for 60× reuse.

Viruses in Adherent Cells on Microcarriers: Microcarriers can be used as a support matrix for the growth of adherent cells such as Vero cells. Primary clarification of these cells grown on microcarriers can be performed using a 75-μm stainless steel sieve to remove microcarriers from harvest. A Millistak+ C0HC depth filter medium then can be used for secondary clarification and directly followed by a sterilizing-grade filter (10). This study was reported for a cell density of 0.78 x 10⁶ cells/mL. Results showed varying filter capacity based on cell density or cell viability of harvest.

Viral Vectors: Trial results on lentiviruses from the supernatant of HEK293T cells show positive performance (data not shown). The negative charge of lentiviruses is known to be responsible for poor recoveries with positively charged depth filters. Therefore, despite the fact that no sign of plugging was observed using a Millistak+ C0HC depth filter, very low viral vector recovery was recorded. However, use of a 1.0/0.5 μm Polysep II filter led to both a high capacity and high 84% recovery. The Polygard CN filter, on the other hand, behaved quite well in terms of recovery (75%) but showed more signs of plugging. An extra 10–20% lower virus recovery should be considered for the following 0.45 μm or 0.22μm (sterile) filtration step.

Two other studies on lentivirus feed streams confirm the positive recovery performance using the Polygard CN filters (CN25, CN12, CN10, and CN06 with more than 80% recovery). Other studies report an interesting result with recoveries reaching up to 90% for the Millistak+ CE50 filter that does not contain inorganic filter aid (e.g., diatomaceous earth; data not shown).

Adenoviruses also can be prone to adsorption, but divergent results have been reported. In some cases, good adenovirus recovery is observed even when a positively charged depth filter medium containing diatomaceous earth such as the Millistak+ HC medium is used (11). If adenovirus is lost, Polygard and Clarigard filters can be used instead, but a secondary clarification might be needed to reduce turbidity (12).

Clarification Options
Different approaches are used to produce viral vaccines, making designing a typical template for their clarification difficult. Indeed, these products can be produced by different expression systems and possess a range of physicochemical properties. The clarification method selected should take those factors into account to ensure that yield and contaminants removal are sufficient.

NFF and microfiltration TFF technologies are increasingly preferred to centrifugation because of their more predictable scalability and robustness. Low CoG also can be achieved by tailoring the choice of media chemistry and porosity to gain high recovery and productivity with satisfactory impurity removal. Continuing innovation in this field by suppliers of purification and filtration systems will help vaccine manufacturers meet their current and future challenges on the road to developing novel and efficient therapies.

References
1 Nascimento IP, Leite LCC. Recombinant Vaccines and the Development of New Vaccine Strategies. Braz. J. Med. Biol. Res. 45(12) 2012: 1102–1111.

2 Besnard L, et al. Clarification of Vaccines: An Overview of Filter-Based Technology Trends and Best Practices. Biotechnol. Advances 34(1) 2016: 1–13.

3 Hendriks J, et al. An International Technology Platform for Influenza Vaccines. Vaccine 29 Suppl 1, 2011: A8–11.

4 Eichhorn U. Influenza Vaccine Composition. US Patent US7316813 B2, 2008.

5 Lampson GP, Machlowitz RA. Process for Producing Purified Concentrated Influenza Virus. US Patents US3547779 A, 1970.

6 Hughes K, et al. Yield Increases in Intact Influenza Vaccine Virus from Chicken Allantoic Fluid through isolation from Insoluble Allantoic Debris. Vaccine 25(22) 2007: 4456–4463.

7 Thompson M, Wee J, Nagpal A. Methods for Purification of Viruses. European Patent EP2334328 A4, 2012.

8 Lau SY, et al. Impact of Process Loading on Optimization and Scale-Up of TFF Microfiltration. BioProcess J. 13(2) 2014: 46–55; doi:10.12665/J132.Pattna.

9 Raghunath B, et al. Best Practices for Optimization and Scale-Up of Microfiltration TFF Processes. Bioprocess. J. 11(1) 2012: 30–40.

10 Thomassen YE, et al. Scale-Down of the Inactivated Polio Vaccine Production Process. Biotechnol. Bioeng. 110(5) 2013: 1354–1365.

11 Namatovu HH, et al. Evaluation of Filtration Products in the Production of Adenovirus Candidates Used in Vaccine Production: Overview and Case Study. BioProcess J. 5(3) 2006: 67–74.

12 Weggeman M, van Corven EJJM. Virus Purification Methods. US Patent: US8124106 B2, 2012.

Corresponding author Anissa Boumlic (anissa.boumlic@merckgroup.com) is associate director of the vaccine segment at Millipore S.A.S.

The post Filter-Based Clarification of Viral Vaccines and Vectors appeared first on BioProcess International.

Single-step harvesting of a high-density cell culture from a Biostat STR bioreactor with Sartoclear Dynamics body-feed filtration from Sartorius Stedim Biotech (WWW.SARTORIUS.COM)

Downstream processing has been considered a “bottleneck” in the manufacture of protein biotherapeutics ever since cell culture engineers began dramatically improving production efficiencies around the turn of the century. And as single-use technologies have grown in importance and acceptance, offering more solutions every year, their biggest challenges too have been in the separation, purification, and processing that follows product expression in cell culture. Many of the technologies familiar to process engineers — e.g., centrifugation and chromatography — present technical and economic problems.

In a recent white paper (1), BioPlan Associates reported that respondents to its 13th annual survey of biomanufacturers reported downstream processing as requiring the most technological improvement of any part of bioprocessing. But productivity is improving. For the coming year, less than half (45%) of respondents expect capacity problems — compared with 88% of the industry in 2005.

Where would user companies like to see suppliers direct their development efforts? Nearly three quarters of respondents (especially contract manufacturers) cited downstream continuous bioprocessing and disposables for purification. Our featured report in May will address the former more specifically — but in general, although interest is high, not many relevant products are available yet. In 2016, bioprocessing facility budgets were up 5% for downstream technologies. “Essentially, nearly all respondents want improvements in downstream technologies,” the report claims (1).

Separation and purification technologies are slowly catching up to upstream processing, however, and vendors are filling the gaps in their offerings. Filtration continues to advance, of course, with some options even encroaching on the adsorption mechanics of chromatography. And now even drug-product filling operations can choose single-use options.

Harvest and Clarification
Modern bioproduction technologies have given us expression titers measured in grams per liter of culture — whereas before the turn of the century, milligrams per liter were common. High-density culturing is one reason, but other advancements include more complicated media and feeds, culture strategies, and optimization efforts. Some of those improvements upstream can cause trouble for harvest, clarification, and beyond.

The first phase of downstream processing typically includes centrifugation or primary filtration steps followed by secondary filtration before purification involving chromatography (2).

Harvesting here is just the same as in agriculture: collecting the material produced by your hard-working life forms (in this case, animal cells or microbes). Along with the expressed protein of interest come host-cell proteins that are interesting only as contaminants; nucleic acids; leftover nutrients, supplements, and byproducts; secondary metabolites; and water. Clarification follows to prepare this messy product stream for downstream chromatography and purification. With solids making up 3–5% of the culture volume, the clarification process alone typically takes the yield of active protein down by 10–15% (2). The most demanding processes involve high turbidities and increased particle/contaminant loads as well as high densities and titers (3).

Filtration: Complications related to downstream processing are ironic in one sense: Disposability in bioprocessing pretty much began with filter cartridges, although upstream/production single-use applications surpassed downstream technologies along the way. Filters remain the most popular single-use technologies, with 91% of companies currently using disposables citing filter cartridges in the latest BioPlan Associates survey, with robust growth continuing (1). Use of depth filters is are widespread (82%), although only 5% of respondents report using disposable tangential flow filtration (TFF) devices.

Another up-and-coming filtration technology is “body-feed filtration,” which incorporates filter-aids such as diatomaceous earth (DE) to increase filter capacity — of particular use with the highly concentrated feed streams that can come from high–cell-density cultures. Adsorptive depth filtration (ADF) incorporates DE into the filter itself (3). Depth filters have particular utility in clarification, which increases with the help of filter aids, flocculating agents that settle impurities out of a harvest solution, or protective prefilters. Sartorius Stedim and MilliporeSigma are well-known proponents of such approaches (2–4). See the next article in this featured report for more discussion.

Filters play many roles in downstream processing — beyond harvest and clarification to virus reduction, buffer exchange, volume reduction, and final sterile filtration just to name a few. Filtration systems are highly automatable, as well (see the box, below), which is increasingly needed in modern biomanufacturing.

Centrifugation has been problematic for conversion to single use, but some suppliers do offer solutions. Most notable are kSep systems (now part of Sartorius Stedim Biotech) and Carr Centritech UniFuge systems from PneumaticScaleAngelus. The former are based on fluidized-bed centrifuge technology originally developed by KBI Biopharma; the latter comprise more traditional technology with irradiated and disposable product-contact surfaces.

The Sartorius technology can be used both for harvesting cells as product or discarding them as by-products. Balanced centrifugal and fluid-flow forces retains particles as a concentrated fluidized bed under a continuous flow of media or buffer. Some companies are applying it toward continuous processing. The Carr system offers continuous operation as well. Both are highly automatable, although they face limitations in scalability and process monitoring (5).

Other Options: Harvest clarification methods such as feed pretreatment involve single-use technology only in that they require tubing to move harvested material and treatments to and from a mixing system (which may or may not be disposable). Acids and salts can cause solutes to precipitate out, but they also can denature proteins; cationic polymers bind contaminants together into cloudy flocs that can be filtered out, but the polymers themselves become contaminants that must be removed later. If such methods are used, they are likely to be combined with the above technologies, whether single-use or multiuse forms thereof.

Chromatography
When you ask about single-use chromatography, the answer usually comes in the form of prepacked columns (e.g., ReadyToProcess brand from GE Healthcare, OPUS columns from Repligen, and Chromabolt and Mobius FlexReady brands from MilliporeSigma). They aren’t strictly single-use in nature, though: Most resins, gels, and other chemistry-based separation media are too expensive to use only once. Instead, they are washed and equilibrated for repeated use with the same product stream, then discarded along with their polymer columns once a batch is complete. In addition to the usual benefits related to cleaning and cleaning validation, however, this approach saves users the time, cost, and fussiness of column packing — giving them consistent results from expert suppliers instead.

Column volumes currently available (e.g., ≤20 L) require several cycles to purify a 1,000-L batch. And that takes time, thus adding cost. The alternative of adding more columns also costs more — lots more — unless you’re talking about continuous multicolumn processes such as that described in this special section by authors from CMC Biologics and Pall Life Sciences. The key with disposable chromatography is to balance cost of goods (CoG) of materials and time/labor. The more expensive the medium (e.g., protein A affinity resins), the more sense it makes to use traditional multiuse technology. So too with frequent harvesting and media with long lifetimes. However, mixed-mode sorbents and sequential chromatography are improving performance while reducing costs. Meanwhile, smaller columns are becoming more popular thanks to high-capacity, high-flow resins and smaller production batches (e.g., from high-density cultures).

A new column-free chromatography technology is drawing publicly stated interest from companies such as Medimmune (Astrazeneca) and Regeneron: Continuous Countercurrent Tangential Chromatography (CCTC) from ChromaTan Corporation. A slurry of resin sequentially binds product as it flows through a series of mixers and hollow-fiber membranes, where it is also washed, eluted, and stripped in a continuous process. The “countercurrent” refers to buffers flowing through in the opposite direction, both lessening the amount of buffer used and improving resin use efficiency. Like the Pall process highlighted elsewhere in this report, CCTC has potential for continuous processing — and more on this small company’s progress is coming in our May featured report.

Alternatives to chromatography resins — in columns or otherwise — are available from membrane suppliers. Functional filtration and membrane adsorbers typically are limited in dynamic binding capacity (DBC) compared with column/resin technology, but they handle significantly higher flow rates.

Nearly one in five respondents to BioPlan Associates’ 13th annual survey cited chromatography columns as currently causing them significant or severe capacity constraints (1). Membrane adsorbers, however, have yet to take over a significant portion of the market. But their adoption is growing, with first use in respondents’ facilities reportedly up 13% for 2016 and 10-year market growth of 31%. The concept is not entirely new technology, but recent introductions — e.g., salt-tolerant devices and new ligand technologies — offer improvements in robustness and efficiency. The article from Renaud Jacquemart and James G. Stout in this special insert provides more discussion.

Formulation, Fill and Finish 
Mixing and storage systems are another single-use technology making inroads with biomanufacturers, at least in part because of their many uses. Their annual adoption rate was up 16% for 2016, and their 10-year growth has been about 50% (1). Downstream applications include viral safety (e.g., detergent treatment), storage of process intermediates, and product formulation, to name a few.

Fill and Finish: BioPlan Associates identified single-use technology as the key trend in biopharmaceutical fill and finish operations, with nearly two-thirds of their respondents ranking it number one (1). Over a third of respondents plan to implement new fill–finish technologies at their facilities in the next two years.

Major suppliers such as MilliporeSigma and Pall have introduced solutions to meet this need, and companies such as Biotest, Disposable-Lab, and Merck have implemented them (6–8). The basic idea has been to adapt fluid-path technology with metal filling needles, combining them with bag containment and pumping systems. As you’ll see in the interview at the end of this featured report, facility design and engineering firm NNE Pharmaplan is a major proponent of these ideas.

Pumps are important throughout downstream bioprocessing, of course. And although polymer tubing and connectors are established single-use components for fluid handling, pumps thus far have proven to be more of a challenge (9). Solutions are in the works to address these needs, and companies such as PSG Dover already have put forth some options. It offers a line of positive displacement diaphragm pumps (Quattroflow) with product-wetted plastic chambers that can be replaced. Other options include a rotary pump from Quantex Arc and a disposable pump-head system for Masterflex peristaltic pumps from Cole Parmer.

Challenges Yet to Be Overcome
Finally, another gap that remains to be filled completely for downstream processing relates to sensing and sampling. Most such solutions that have been made available so far are meant for upstream production applications. PendoTech offers pressure sensors, however, as well as those for ultraviolet absorbance and conductivity (10). Sartorius Stedim Biotech has incorporated such options into its own product offerings (11).

But what might be the biggest challenge facing downstream processors who want to use disposable systems and components isn’t so much technical as it is business related. What users really want from their single-use technology providers is standardization of designs that would allow them to mix and match components to put together systems that work best for their own processes. Bioprocessors say that this would improve adoption and implementation of disposables; suppliers are reluctant to share with their competitors. Not long ago, in fact, if you brought up the question of single-use standardization at an industry conference, you might get a lot of laughs but not much real discussion. However, companies on both sides are taking the idea more seriously now. And the “alphabet soup” of organizations concerned with single-use technology are helping to make it happen (12). Standards could reduce the risk of process failures and allow suppliers to stick with proven features while focusing their attention on needed innovations.

References
1 Top 15 Trends in Biopharmaceutical Manufacturing, 2016. BioPlan Associates, Inc.: Rockville, MD, 2016.

2 LeMerdy S. Evolving Clarification Strategies to Meet New Challenges. BioProcess Int. October 2014: insert.

3 Minow B, et al. High–Cell-Density Clarification By Single-Use Diatomaceous Earth Filtration. BioProcess Int. 12(4) 2014: S36–S46.

4 Schreffler J, et al. Characterization of Postcapture Impurity Removal Across an Adsorptive Depth Filter. BioProcess Int. 13(3) 2015: 36–45.

5 Pattasseril J, et al. Downstream Technology Landscape for Large-Scale Therapeutic Cell Processing. BioProcess Int. 11(3) 2013: S38–S47.

6 Camposano D, Mills A, Piton C A Single-Use, Clinical-Scale Filling System: From Design to Delivery. BioProcess Int. 14(6) 2016: 50–59.

7 Gross R, et al. Establishing Single-Use Assemblies on Filling Equipment. BioProcess Int. 12(4) 2014: S48–S54.

8 Zambaux J-P, Barry J. Development of a Single-Use Filling Needle. BioProcess Int. 12(5) 2014: 46–53.

9 Wittkoff W, Prasad R. Single-Use Pumps Take Center Stage. BioProcess Int. 11(4) 2013: S18–S23.

10 Annarelli D. Novel Single-Use Sensors for Biopharmaceutical Applications. BioProcess Int. 12(7) 2014: 58–59.

11 Weichert H, et al. Integrated Optical Single-Use Sensors: Moving Toward a True Single-Use Factory for Biologics and Vaccine Production. BioProcess Int. 12(8) 2014: S20–S24. 1

2 Vogel JD, Eustis M. The Single-Use Watering Hole: Where Innovation Needs Collaboration, Harmonization, and Standardization. BioProcess Int. 13(1) 2015: insert.

Further Reading
Bird P, Hutchinson N. Automation of a Single-Use Final Bulk Filtration Step: Enhancing Operational Flexibility and Facilitating Compliant, Right-First-Time Manufacturing. BioProcess Int. 13(3) 2015: S40–S43, S52.

Blomberg M. The New Hybrid: Single-Use Systems Enabled by Process Automation. BioProcess Int. 13(3) 2015: S34–S39.

Grier S, Yakabu S. Prepacked Chromatography Columns: Evaluation for Use in Pilot and Large-Scale Bioprocessing. BioProcess Int. 14(4) 2016: 48–53.

McGlaughlin MS. An Emerging Answer to the Downstream Bottleneck. BioProcess Int. 10(5) 2012: S58–S61.

Metzger M, et al. Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal. BioProcess Int. 13(2) 2015: 36–44.

Mok Y, et al. Best Practices for Critical Sterile Filter Operation: A Case Study. BioProcess Int. 14(5) 2016: 28–33.

O’Brien TP, et al. Large-Scale, Single-Use Depth Filtration Systems. BioProcess Int. 10(5) 2012: S50–S57.

Quinlan A. Advances in Chromatography Automation. BioProcess Int. 13(1) 2015: 16–17.

Cheryl Scott is cofounder and senior technical editor of BioProcess International, PO Box 70, Dexter, OR 97431; 1-646-957-8879; cscott@bioprocessintl.com.

The post Downstream Disposables: The Latest Single-Use Solutions for Downstream Processing appeared first on BioProcess International.

Figure 1: Harvest clarification options

Steadily increasing demand for biopharmaceutical drugs has led the industry to examine its manufacturing scales while pressuring research and development groups to produce high-yielding clones and processes. Improved media, feed supplements, bioreactor designs, and control of process parameters have helped biomanufacturers achieve multifold increases in volumetric productivity from production bioreactors.

However, cell culture processes are significantly affected by their bioreactor’s ability to support cells at higher densities and sustain cultures at lower viabilities. With the implementation of a number of new approaches, cell densities have been increased from 5–7 × 10⁶ cells/mL to >25 × 10⁶ cells/mL. Such increased densities — and improved specific productivity of the modern clones — has increased productivity as well, with expression titers rising from 1.0 g/L to 5–8 g/L in fed-batch cell culture processes. Moreover, concentrated fed-batch and perfusion cell culture processes have further increased cell density up to >50 × 10⁶ cells/mL, which has further complicated clarification as well as overall downstream processing of recombinant proteins. A high-density cell culture process always poses a challenge to clarification techniques for separating product from cells without compromising its yield or quality.

In addition to cell culture processes, clarification and purification techniques are under tremendous pressure to deliver high-quality biological products while controlling cost of goods (CoG). Choice of clarification technique directly affects final-product quality because it paves the way to downstream purification processes that build product quality and ensure product safety.

The basic intention of a cell-separation unit operation is to remove whole cells and their components for further processing of a culture’s supernatant. No single technology fits all needs of such applications for all products and processes. The choice of clarification technique varies for different cell densities and viabilities, cell type, culture styles and modes, and harvest viscosities. As cell density and product titer have improved over the past decade, clarification techniques also have improved to meet resulting process and product requirements. We recommend screening all commercially available technologies to understand the best available option for manufacturing of a given biopharmaceutical. We also prefer single-use devices, performing screening studies of high-capacity depth filters as reported herein.

The objective of the study described below was to evaluate different single-use technologies and understand the impact of high cell density and lowered viability on existing devices. We challenged different available depth filters with similar feed streams to evaluate and compare their performance.

Impact of High Cell Density and Low Viability
The harvested supernatants of today’s fed-batch processes, with very high cell densities and low viabilities, show increased solid materials such as whole cells, cell debris of different sizes, colloids (lipid components of media and cell walls), nucleic acids, and other particulate matter. Those increased levels can cause early plugging of traditional depth filters. In many cases, multifold increases in filter area are required, leading to poor scalability and expense at manufacturing scale.

Traditional primary clarification techniques such as centrifugation, tangential-flow filtration (TFF), and early single-use depth filtration have been used in biomanufacturing to achieve desired quality outputs. They are still integral to many commercial manufacturing processes. However, downstream process engineers are looking for alternatives that can provide more efficient clarification of high-density cultures. Combinations of the familiar techniques have been applied in some cases, depending on the need of the cell culture process and its scale of operations (1). All these techniques — or combinations thereof — have served us at some point. Figure 1 illustrates some combinations of clarification options.

Most downstream processes in biopharmaceutical manufacturing include two major categories of technology: chromatography and filtration. The latter includes clarification, virus filtration, and tangential-flow filtration. Clarification process can be separated further into two broad categories: primary recovery and secondary recovery, also referred as primary and secondary clarification steps. The primary step removes the bulk of large particles, whole cells, and cell debris; the secondary step removes smaller particles present in the resulting filtrate. Centrifugation, TFF, and depth filtrations are common choices for primary clarification; depth filtration and bioburden-reduction filters often provide secondary clarification.

Primary Clarification Options
Tangential-flow microfiltration (TFF-MF) separates particles based on size exclusion using microfiltration membranes with a pore size of ≤0.65 μm. This process is highly efficient for removing whole cell mass and fragments. It provides the most consistent separation by retaining particle sizes larger than the membrane cutoff size.

TFF-MF devices also offer advantages for scaling up downstream processing based on modularity. With higher cell densities, however, ruptured and fragmented cells often are observed in recirculation loops, making a secondary clarification necessary to reduce those smaller particles before further downstream processes (e.g., chromatography). High cell density can increase polarization on cell membrane surfaces, leading to frequent filter clogging and complicating manufacturing-scale operations.

High-yielding cell culture processes with higher cell densities and specific productivities produce large amounts of solid material (>6%). TFF is best suited for such cultures with solid content <3%. Beyond that, however, TFF becomes inefficient.

Centrifugation can be applied to feed streams with high solids levels, concentrating cell mass ≥40% v/v. Product recovery can be low, however, because of increased pellet volumes and high desludging, which are common for cell harvests with very high solids content. Additionally, low cell viability generates particles of different sizes, drastically decreasing their removal efficiency. Although this technique can handle high concentrations of insoluble materials, its ability to produce a clear product is limited. Thus, it often requires the support of secondary filters.

In most cases, loading a cleaner material on a column requires depth filtration after centrifugation and before chromatography. The area requirement for such a depth filter would depend on the clarity of the centrifugation output and the amount of suspended material present after primary clarification.

Depth filters typically are made of cellulose, with a porous filter aid such as diatomaceous earth (DE) and an ionic charged resin binder. These filters function by retaining particles within their porous matrices. Depth filtration has been a single-use choice for clarification in manufacturing many biopharmaceutical products.

Depth-filter pore sizes vary for primary and secondary applications. Different types of depth filters are now available with single or multiple layers and gradient pore distributions. These filters can be applied either directly to whole cell broth (for cleaner output) or used in two stages: primary depth filters to remove larger particles, followed by secondary depth filters to remove finer suspended particles. Depth filters fit well with the emerging trend of single-use technology, making them our primary choice as a single-use clarification solution in biopharmaceutical development.

With the advancement of single-use technology, primary and secondary filters are merged into one single step that reduces cycle times and required filter area for efficient clarification. Furthermore, lower buffer volumes are required for flushing in modern processes, enabling improved economics for clarification of high-density cell cultures. Finally, hardware requirements for depth filtration at higher scale are simple and highly flexible, which makes depth filters an attractive choice as a single-use clarification technique.

Key features needed for a filter to qualify its use from laboratory scale through pilot to commercial scale are high capacity with minimal area, low cost, consistent performance, high product quality and yield, scale-up ease and flexibility, and a small overall footprint for efficient use of space in a biomanufacturing facility.

Depth Filter Study
For evaluation of existing depth filters and other improved technologies, we divided our experiments into two phases. In phase 1, we obtained and evaluated available depth filters and additives capable of handling higher cell densities from different vendors. Filters were challenged with ~28 × 10⁶ cells/mL of cell culture harvest having ~75% cell viability. We analyzed the performance of all filters to select a few best candidates for our phase 2 study. Then, we challenged those shortlisted filters with a bit lower cell density (~24 × 10⁶ cells/mL) and even lower viability ~65%. Phase 2 was meant to study reproducibility and robustness of filter performance with some variation in an upstream process.

In most bioprocess applications using depth filters, flow rate in relation to membrane cross-section area has been found to affect process capacity significantly (1). It’s now known that the increased flux reduces the capacity of a membrane (L/m²) (1). Therefore, to achieve acceptable depth-filter performance, we applied a constant flow method. This keeps flux throughout a process, during which pressure and turbidity are monitored until they reach their maximum allowable operating limits. We used different endpoints for each filter, as specified and recommended by its supplier. Finally, we compared data from all experiments and interpreted them to determine the best-suited depth filtration technique for our process.

Materials and Methods
In our phase 1 study, we evaluated depth filters from four suppliers — Pall Life Sciences, Sartorius Stedim Biotech, MilliporeSigma, and 3M — with different pore sizes and multiple layers or both. Each filter was tested for its ability to reduce turbidity to a desired level: <15 NTU, preferably <10 NTU. We used a LaMotte turbidity meter throughout this experiment to measure sample turbidity.

We also compared an alternative clarification technology: flocculation, which relies on aggregation of particles for easier separation. In this method, a cationic polymer is added to harvested supernatant to trigger floccule formation. The polymer binds to whole cells, cell debris, and other components, then attracts other nearby components by the van der Waals force to form aggregates of varied size and density. We tested this method along with depth filters of different pore sizes as recommended by their vendors. The polymer dose varies from harvest to harvest, and it’s important to determine the optimum concentration of flocculant for each cell culture process. To determine an appropriate concentration, we added an increasing volume of flocculant solution to a constant volume of harvest, checking all samples to determine which reached the lowest turbidity. We thus considered the concentration at which the sample showed lowest turbidity to be the optimum flocculant concentration for filtration. Then we challenged a harvest with this optimum concentration further using depth filters of different pore size to discover the best-performing depth filter.

Finally, we evaluated another technology: addition of diatomaceous earth (DE) powder to harvested supernatant before filtration. This technique increases the surface area of a depth filter by making a porous cake of DE over it. In normal depth filtration, a filter cake forms on the surface of a filter and clogs its available surface area. But with DE added, it forms a permeable layer over the filter, which increases its depth and surface area for filtration. The porous layer traps whole cells and debris through both adsorption and sieving to yield a clear filtrate. Again, the amount of DE required varies with harvested cell mass. It can be determined by measuring pelleted cell weight and using the result to calculate a recommended percentage of DE. We also assessed this method’s performance using recommended filters.

So the different types of single-use clarification technologies we evaluated are as follows: a single depth filter (single-stage clarification), primary and secondary depth filters (two-stage clarification), flocculation followed by depth filtration, and DE additive (to enhance filtration efficiency) followed by filtration.

Results and Discussion

Figure 2: Comparative profile of all the depth filters at constant flow (phase 1 study)

Figure 2 shows a comparative profile of all filters tested in phase 1, plotting the correlation of filter performance (capacity) with differential pressure. Table 1 summarizes the values of reduced turbidity and capacity for those filters. And Figures 3A and 3B provide a comprehensive summary of the phase 1 study.

We performed two different experiments on filters provided by Pall Life Sciences. In the first experiment, the HPPDH4 depth filter showed a capacity of 47.3 L/m² and a pooled turbidity of <12 NTU. The second experiment used primary and secondary depth filters SC050PDK5 and SC050PDE2.

Table 1: Performance comparison of depth filters (phase 1)

The main aim of the first-stage filter SC050PDK5, with its wider pore size, is to remove larger particles with a turbidity cutoff of <25 NTU. Its output then goes to the secondary depth filter SC050PDE2 (narrow pore size) to get a final pooled turbidity value of <10. Capacity for the primary filter was 63.6 L/m²; a correct capacity of secondary filter SC050PDE2 could not be determined because of limited output from primary filter.

Figure 3: Comparative performance of depth filters from different vendors (phase 1)

Among the MilliporeSigma filters, those having wider pores (60 HX) showed a capacity of 143 L/m² with no increase in pressure, whereas the 40 MS filter with intermediate pore size showed a very high capacity of 383 L/m², and the 20 MS filter with the smallest pores showed a filtration capacity of 315 L/m², within the allowed pressure limit. Turbidity reduction was acceptable for both the 20 MS and 40 MS units, but the 20 MS filter showed lower pool turbidity than the 40 MS filter.

In our DE-based filtration study, we mixed 40% and 50% DE material from Sartorius Stedim to the harvest cell weight. Results suggest that the harvest with 40% DE showed a better capacity and turbidity reduction than the harvest with 50% DE addition. Final turbidity with both harvests was comparable and >10 NTU.

Within a set pressure limit, we used a primary depth filter (10SP02A) provided by 3M to generate material for a secondary depth filter (90ZB08A). The first showed a low capacity value of 80 L/m² and a high turbidity value of 264 NTU; the second, with its tighter pores, showed a better capacity of 130 L/m² and pooled turbidity of <5 NTU.

Figure 4: Comparative performance of selected depth filters phase 2

To select the best-performing filters for our phase 2 study, we considered the capacity, turbidity reduction, and process recovery data from all filters used in the phase 1 study. Two new technologies — from Sartorius Stedim and MilliporeSigma — showed promising results for handling high-density cell culture harvests. Therefore, we used Sartoclear Dynamics with 40% DE added and Clarisolve 20 MS and 40 MS filters for our phase 2 study (Figures 4A and 4B).

At the end of the clarification study, we recovered the filter hold-up by allowing air to push product out of the filter to prevent further dilution. Alternately, hold-up volume can be recovered by flushing a filter by two or three hold-up volumes of buffer. Product recoveries for the selected filters were nearly comparable at ≥95%. Actual recovery of the Seitz HPPDH4 primary filter could not be determined, however, due to clogging of primary filters that occurred during material generation.

Overall performance of selected filters in this case was comparable at small scale. But we recommend calculating and optimizing recoveries at pilot scale during scale-up. Pilot-scale batches requires an additional cassette holder, tubings, valves, and connectors — all of which further increases the hold-up volume of filter cassettes.

We found the performance of both Clarisolve 20 MS and 40 MS filters to be comparable and better than that of Sartoclear 40% DE. We observed an increased capacity for all three options than what was seen in our phase 1 results, which could be attributed to a decrease in cell density. But turbidity reduction for all filters in phase 2 was comparable and consistent with our phase 1 data.

An Upcoming Technology
Acoustic wave separation promises to handle very high cell densities from both concentrated fed-batch and perfusion cultures, in which cell density can reach >50 × 10⁶ cells/mL of harvest. We look forward to testing the new Cadence Acoustic Separation (CAS) technology from Pall Corporation as a potential solution to the associated with a very high-density cell cultures.

Figure 5: (A) Direction of cell movement and increase in size of cells at acoustic zone; (B) how accumulated cells at the base are collected and removed as sludge

In a CAS system, continuous standing waves retard the flow of cells. As cells increase in number, they aggregate into bigger and bigger clumps. Those experience higher gravitation and cannot remain in the flow, so they settle to the conical end of the system’s flow cells (Figure 5). Multiple acoustic flow-cell chambers in series can further reduce turbidity as required for a given process. This seems to be a good option for cell removal in continuous processing as well as harvest of very high-density cell cultures. However, a secondary depth filter may be required to decrease turbidity further downstream of the CAS system.

An Iterative Approach
Several new technologies are available now for clarification and handling high-density cell culture harvests. But no single technique fits all cell culture processes and products. Therefore, it is important to evaluate all available technologies to determine the best clarification solution for an efficient and economical biomanufacturing process. For example, turbidity reduction and filter capacity are important criteria in selection of single-use depth filters for clarification purposes.

In our study, Clarisolve filters (20 MS and 40 MS) showed better performance than others tested. However, the choice of clarification technology for any commercial application must be critically evaluated before its implementation in a final process. Pros and cons of each technology and their implications in a given manufacturing process must be understood at laboratory scale — and preferably at pilot scale — before implementing a final choice at commercial manufacturing scale.

Furthermore, other factors also should be taken into consideration: e.g., cost of each technology, facility requirements, hardware needs, process scalability and flexibility, vendor support, and regulatory requirements. In our study, we found that the higher-density cell culture could be handled with existing depth filtration techniques to get a desired end product. But an extensive study of available options is the most appropriate way to determine your own needs.

Acknowledgments
The authors are thankful to Ipca Laboratories Ltd. (Mumbai, India) for necessary support. They also give special thanks to Dr. Ashok Kumar (president of Ipca laboratories Ltd.) for his immense motivation and necessary support.

References
1 Yavorsky D, et al. The Clarification of Bioreactor Cell Culture for Biopharmaceuticals. Pharm. Technol. March 2003: 62–76.

Further Reading
Collins M, Levison P. Development of High Performance Integrated and Disposable Clarification Solution for Continuous Bioprocessing. BioProcess Int. 14(6) 2016: S30– S33.

Dhanasekharan K, et al. Emerging Technology Trends in Biologics Development: A Contract Development and Manufacturing Perspective. BioProcess Int. 14(9) 2016: 32–37.

Gronemeyer P, Ditz R, Strube J. Trends in Upstream and Downstream Process Development for Antibody Manufacturing. Bioengineering 1(4) 2014: 188–212.

Le Merdy S. Evolving Clarification Strategies to Meet New Challenges. BioProcess Int. 12(9) 2014: I10–I12.

Pegel A, et al. Evaluating Disposable Depth Filtration Platforms for MAb Harvest Clarification. BioProcess Int. 9(9) 2011: 52–54.

Tomic S, et al. Complete Clarification Solution for Processing High Cell Culture Harvests. Sep. Purif. Technol. 141, 2015: 269– 275.

Manish K. Sharma is senior manager, Snehal Raikar is a research associate, Smriti Srivastava is a junior research associate, and corresponding author Sanjeev K. Gupta is general manager of the advanced biotech laboratory at Ipca Laboratories, Ltd. in Mumbai, India; +91-22-6210-5820; sanjeev.gupta@ipca.com; www.ipca.com.

The post Examining Single-Use Harvest Clarification Options: A Case Study Comparing Depth-Filter Turbidities and Recoveries appeared first on BioProcess International.

Figure 1: Conventional and M-pleat pattern

Maximizing filtration-area density is a design strategy to minimize filter footprint and improve filtration process economics. Pleated membrane formats commonly are used to achieve that goal for sterilizing-grade filters operating in dead-end mode (also known as normal-flow filtration). Although high-density pleat geometries increase productivity for a device, such formats can present unique challenges. One of the most common concerns is that pleat formats can introduce flow resistance that impedes a device’s filtration efficiency, particularly for high–pleat-density geometries (1, 2). Filtration efficiency can be affected by pleat length, membrane permeability, and thickness and flow characteristics of upstream and downstream supports as well as other aspects of a pleat configuration.

One approach for overcoming those challenges has been the development of an M-pleat pattern that allows for a nearly 100% increase in membrane area over conventional designs (Figure 1). The M-pleat design has been optimized for maximum area density, device manufacturability, and a high level of device robustness. Other improvements include the ratio of long to short pleats, pleat compression, and upstream and downstream support materials (see “Design Improvements” box below). M-pleat designs help address the challenges of filtration efficiency while matching existing cartridge sleeve and length dimensions.

Ultimately, filter-design efficiency depends on how well a membrane performs when it is in a filtration device. One method of measuring a device’s filtration efficiency is to compare the performance of a membrane in a filtration device with that of one in a flat-sheet format (where the flow resistance is dominated by the membrane itself). The ratio of commercial-scale filter performance to small-scale, flat-sheet performance is often referred to as the scaling factor. Ideally, that should be at or close to one.

The scalability factor of a device must be defined with respect to a set of specified conditions. In particular, there can be a compromise among other filter requirements (including high-area density) and good scalability. Scalability also can be sensitive to filter operating conditions, challenge stream characteristics, and the particulars of a selected filtration endpoint. A filter can exhibit a range of scaling factors depending on the combination of those other conditions.

Here we discuss variations in scalability in newly developed high-area versions of sterilizing-grade filters. We describe a model that was developed to predict filter device efficiency as a function of the filtration properties of a filtered stream. The model can be used to assess which applications can benefit most from high-area pleated devices and which should use conventional pleat configurations.

Our study evaluated filters for scalability against a nonplugging stream (water) and three different plugging streams that represented a wide range of particle-size distributions. We assessed scalability as a function of particle size and degree of plugging. Filters were tested primarily for constant pressure operation and measured for constant-flux operation.

Theoretical Background
Several factors must be considered when scaling from discs to pleated devices. Applicable to all pleated filter devices, these factors include flow resistance from upstream and downstream supports, pressure losses from housings and plumbing, and variability in filter properties, stream characteristics, and operating conditions (3). Although some theoretical treatments of flow restrictions in conventional pleat patterns have been studied, no known similar treatments have been published for the M-pleat pattern evaluated in this work. Here we focus on the effect of the M-pleat pattern on filter performance, because factors such as membrane and process variability are not specific to high-area devices.

Rather than develop a model that predicts the impact of the M-pleat pattern on filter efficiency from first principles, we used a semiempirical approach. Flow resistance associated with the pleat pattern is inferred from clean-water scaling data and then applied to a model that predicts filtration efficiency as a function of membrane plugging.

The flux through a filter can be described using Darcy’s law (Equation 1), in which J is the flux, ΔP is the pressure differential across a filter, and R_t is the total resistance to flow. R_t includes flow resistance from the membrane (R_m), upstream and downstream supports (R_s), and filter housing (R_h) (Equation 2). Substituting Equation 2 into Equation 1 gives Equation 3.

Figure 2: Components of total filtration resistance as a function of membrane plugging

For a plugging stream, R_m will increase with throughput while R_s will be unchanged (assuming that upstream and downstream supports do not plug). R_h will change with flow rate (predictably).

As a membrane plugs, resistance from the membrane becomes an increasingly larger fraction of the total resistance (Figure 2). R_s and R_h decrease relative to R_m as membrane plugging increases and the scaling factor increases with increasing filtrate volume (and degree of membrane plugging).

Table 1: Ten-inch cartridges evaluated in this study

Materials and Methods
Membranes and Devices: Four high-area cartridges were evaluated in this study (Table 1). They included 10-inch high-area sterile high-capacity (SHC) (referred to as SHC-HA) and SHC standard-area (SHC-SA) cartridge filters (from MilliporeSigma). Those products contain the same types of membranes: one layer each of 0.5-µm and 0.2-µm polyethersulfone (PES) asymmetric membrane. Standard-area versions of those devices contain about 0.5 m² of effective filtration area. Two additional 10-inch cartridge filters from different manufacturers also were evaluated. For small-scale tests, 25-mm membrane discs were installed into OptiScale 25 devices (from MilliporeSigma), which contain 3.5 cm² of effective filtration area.

Table 2:  List of challenge streams for throughput tests (PBS = polybutylene succinate)

Challenge Streams: Three challenge streams were used in this study (Table 2) to represent small, medium, and large particle sizes and particle-size distributions (Figure 3). The challenge streams were concentrated to achieve a high degree of plugging (>90% flux decay at <1,000 L/m² of filtrate) within about 30 minutes at the process conditions.

Figure 3: Particle-size distributions of the challenge streams used in this study; particle sizing data were gathered using a Malvern MasterSizer particle size analyzer.

Test Method: Both OptiScale 25 devices and 10-inch cartridges were tested for clean-water permeability at 10 psi (690 mbar) and 21–25 °C. Following the water permeability test, throughput tests using one of the challenge streams were conducted at 10 psi (690 mbar). That throughput testing ran until the membrane permeability was reduced by at least 95% compared with the clean water permeability.

Table 3: Cartridge-to-disc scaling factors for water permeability (LMH/psi)

Results and Discussion
Water permeability data were collected for each membrane and device type (Table 3). For water, scalability factors for three of the four filters tested were about 0.5. The flow resistance from the supports was about the same as that of the membrane, resulting in about a 50% loss in productivity compared with that of the OptiScale 25 device. In the fourth device (SHRp-HA, containing the tighter 0.1-μm membrane) the membrane was responsible for a higher fraction of the total filter resistance, resulting in a higher scaling factor.

Figure 4: Throughput curves for SHC-HA challenged with soy peptone

Throughput was evaluated using multiple feed streams representing a wide range of particle-size distributions. The soy peptone (papainic digest of soymeal from MilliporeSigma) stream had a relatively small median particle size (about 0.2 µm) and relatively narrow particle-size distribution. This stream would be expected to foul membranes through an internal pore-plugging mechanism. The study found that for this stream, the initial flux-scaling factor was similar to that of water. But as the membrane fouled, the flux-scaling factor converged toward one (Figure 4). That is a result of the membrane becoming an increasingly larger fraction of the total filter resistance as the membrane fouls.

Figure 5: Comparing data and model predictions for filtration of soy peptone with SHC-HA; (A) scaling factor over time; (B) 10-inch cartridge throughput over time.

We plotted scaling factor as a function of filtration time for the SHC-HA cartridge and the model prediction (Figure 5). Figure 6 shows throughput scaling factors for the cartridges tested in this study at 30 minutes filtration time (about 95–99% flux decay). For that stream (using 2 g/L soy peptone), scaling factors are all within about 15% of one (within the approximate measurement uncertainty).

The Hy-Soy T (Kerry) stream had a large particle size and wide distribution. Figure 7 shows throughput and flux decay curves for this stream, and Figure 8 shows scaling factors at 30 minutes. This stream showed initial flux scaling factors as expected (similar to water). But as the filters plugged, the scaling factors diverged away from one rather than converging. That result is explained by the larger particle size in the Hy-Soy T stream. These particles cannot enter the membrane pore structure and therefore accumulate at the membrane surface, forming a cake.

Figure 6: Scaling factors at 30 minutes filtration time using 2 g/L soy peptone

The OptiScale 25 format includes an open space above the membrane surface, resulting in unhindered cake buildup with the Hy-Soy T stream. In a densely pleated format, a nonwoven support is in contact with the membrane surface, bounded by an adjoining pleat, which limits the available space for a cake to form. Furthermore, in a densely pleated format, particles must travel laterally through the nonwoven support before reaching the membrane. Any particle buildup at the entrance to the pleat pack prevents subsequent particles from accessing the membrane surface.

Figure 7: Throughput curves for SHC-HA challenged with Hy-Soy-T

Previous studies have shown that standard-area devices suffer less than high-area devices from large-particle accessibility to the membrane surface (3). As a result, high-area devices may not be preferred to standard devices if challenged directly with a Hy-Soy T (or large particle size and wide distribution) stream. An appropriately sized prefilter removes large particles that would otherwise form a cake on the final-filter membrane surface. With the large particles removed, the throughput advantage of high-area devices can be restored.

Figure 8: Scaling factors at 30 minutes filtration time using 0.1 g/L Hy-Soy T product

We also tested a whey stream for throughput with intermediate particle size (Figure 9). As with the smaller particle-size stream, the flux scaling factors converged toward one as predicted (Figure 10). Those data demonstrate that for plugging streams in which caking is not a predominant fouling mechanism, high-area devices scale close to one if a high level of plugging is achieved. Alternatively, if the level of plugging is low or intermediate, then the scaling factor also is intermediate between that of what it is with water and one. In such a case, the model described in this study can be used to estimate scaling factor.

Figure 9: Throughput curves for SHC-HA challenged with whey

Normal-flow filtration often is operated at constant pressure, but constant flow is another common mode of operation. To evaluate scalability for constant-flow operation, we tested an SHC-HA cartridge at a flux of 500 LMH using a whey stream. In that test, the initial ΔP for the cartridge was about double that of the OptiScale 25 devices. But as the membrane plugged and reached the 20 psi (1,400 mbar) filtration endpoint, the pressure profiles of the cartridge and OptiScale 25 devices converged (Figure 11). This trend is similar to that in constant-pressure operation. Results demonstrate that the scalability principles demonstrated for constant-pressure operation also apply to constant-flow operation.

Figure 10: Comparing data and model predictions for filtration of whey with SHC-HA; (A) scaling factor over time; (B) 10-inch cartridge throughput over time

Advantages of Using High-Area Devices
Nonconventional pleat configurations can provide increased filtration areas and greater process efficiencies in cartridge devices. A larger area has the advantage of lower cost per unit of filtration area, and potentially higher productivity per device. In some cases however, a high pleat density within a device can lead to decreased efficiency in use of a contained area.

This study examined a model developed to predict scalability as a function of membrane plugging when caking is not predominant. That model can be used to quantify the advantage of high-area devices for a given set of operating conditions, membrane-fouling properties, and filtration endpoint. It also provides an understanding of mechanisms underlying measured scaling factors.

Figure 11: Scalability at constant flow operation for filtration of whey with SHC-HA

We also found that for plugging streams for which caking is not a predominant fouling mechanism, high-area devices exhibited near-linear scalability. This was because, as the membrane fouls, it becomes the dominant resistance to flow. In that circumstance, using high-area devices offers a clear advantage over standard-area devices. An exception to that advantage can come with plugging streams in which caking is the primary fouling mechanism. When particles are larger than membrane pores, the volume available in a dense pleat pattern to form a cake is limited. In such cases, prefiltration is recommended to remove large particles.

References
1 Gollan A, Parekh BS. Hydrodynamic Aspects of Semidense Pleat Designs in Pleated Cartridges. Filtr. Separat. 22(5) 1985: 326–329.

2 Giglia S, Yavorsky D. Scaling from Discs to Pleated Devices. PDA J. Pharm. Sci. Technol. 61(4) 2007: 314–323.

3 Giglia S, et al. Improving the Accuracy of Scaling from Discs to Cartridges for Dead-End Microfiltration of Biological Fluids. J. Membrane Sci. 365(1) 2010: 347–355.

Corresponding author Sal Giglia is R&D manager, filtration applications, at MilliporeSigma, 80 Ashby Road, Bedford, MA 01730; 1-781-533-2564; sal.giglia@emdmillipore.com. Songhua Liu and Ryan Sylvia are both development engineers at MilliporeSigma.

The post Scaling Considerations to Maximize the High-Area Advantage appeared first on BioProcess International.

The ready-to-use MaxiCaps MR system is entirely disposable and enables the integration of three, six, or nine 30-inch filter capsules.

Whether viral vectors are clarified or the bioburden after cell harvest needs to be reduced to recover antibodies, such applications in biopharmaceutical production require large filtration areas. Single-use technologies are indispensable in many such bioprocesses. Although some single-use filter assemblies have reached their limits, Sartorius Stedim Biotech has made developments to revolutionize these production steps.

Scale-Up Limitations in Single-Use technology
Conventional stainless steel process systems have been established for decades in the pharmaceutical industry. They are the basis of safe and reliable manufacturing processes for both classical pharmaceuticals and advanced biologics. Complexity and cost efficiency in biopharmaceutical production require great flexibility. Thus, most leading biopharmaceutical manufacturers are increasingly relying on modular single-use systems designed for flexible use.

But the rapid introduction of technologies that enable such flexibility has led to a number of different components and solutions over the past years. As batch volumes produced using single-use technologies increase, the processes for such batches become more complex and involve higher risks. The challenges of a complete single-use process are inherent in the diversity and large number of parts to be connected. Particularly when scaling up batch sizes to 1,000–2,000 L, users discover limitations in some process steps and thus face significant problems.

Minimizing Risk Even in Large-Scale Filtration
In cooperation with a multinational pharmaceutical company, Sartorius Stedim Biotech faced the task of developing a single-use solution for large-volume filtration steps that would minimize risk, time, and effort. The challenge was to incorporate up to 27 m² of filter area into a single, closed, and ready-to-use device to minimize the risk of faulty connections between filter capsules and thus lessen the risk of leakage for users.

Figure 1: Stainless steel multiround housings are the established solution for conventional large-scale filtration processes. Complex filter assemblies with multiple cable-tie connections, T-pieces and complex tubing manifolds are the single-use equivalent. MaxiCaps MR combines all advantages of a single-use solution with those of a conventional system.

Standardization, Installation, Automation
The result of this collaboration was the development of MaxiCaps MR filtration system, which replaces complex filter and tubing assemblies consisting of several 30-inch capsules and sterile connections used so far. This preassembled and presterilized device enables three, six, or nine 30-inch capsules to be integrated into a single system with only one connection in a process. That saves not only time and expense, but also 90% of sterile connections, 90% of tubing, and up to 90% of integrity testing time. The system saves process time because it is ready to use, unlike conventional filtration systems. Its efficient design minimizes footprint in cleanrooms.

Moreover, simultaneous flow through the entire filter area of this closed system ensures efficient filter use, thus optimizing the filtration process. The MaxiCaps MR system is the logical next step in standardization, easy installation, and risk mitigation in single-use systems. It is completely made of plastic, with a design that systematically implements the single-use concept for large volumes. Single-use pressure and flow-rate sensors
can be connected easily to the MaxiCaps MR system. This is the first step in the direction of a fully automated system that is already being assessed by users in a production environment.

Large-scale filter assemblies today: multiple cable-tie connections, T-pieces, complex tubing manifolds

Applications
The MaxiCaps MR system provides a filter area of up to 27 m² for prefilters and sterilizing-grade filters and has a number of applications. It is tailored to accommodate large-scale filtration of media and buffers in volumes of 5,000 L and more. Contract manufacturing organizations (CMOs) that use modular single-use processes to adapt to changing products tend to prefer generously sized filtration areas for initial bioburden reduction and sterile filtration as post–cell-harvest steps for monoclonal antibodies (MAbs), for example. In this case, the system already in use has proved its efficiency downstream of 2,000-L single-use bioreactors.

For new (not perfectly defined) processes in which centrifugation is not yet qualified, higher filtration areas are needed to ensure safety and reliability. A polypropylene or glass-fiber prefilter with a nominal pore size of 1.2 µm often is used in the first post–cell-harvest clarification step for viral vaccines, such as for human cytomegalovirus (CMV) in the range of 150–200 nm. In such processes, the closed, completely single-use, and ready-to-go MaxiCaps MR system is a significant improvement over traditional filters and already being used for batch sizes of 250 L and higher.

For most applications, the major focus is on total throughput and less so on flow
rate. The MaxiCaps MR system minimizes risk, time, and effort for applications in which filters tend to become readily blocked, requiring a large filtration area.

System Details
The large-scale single-use MaxiCaps MR filtration system is based on 30-inch MaxiCaps filter capsules. Filters and materials have been established for decades and are already in use in today’s single-use filter-and-tubing assemblies. The abbreviation “MR” stands for “multiround” and is intended to be associated with conventional multiround housing configurations used in classical stainless steel processes. Filter solutions for large filtration areas evolved from large stainless steel MR systems to complex single-use constructions to the MaxiCaps MR system, which offers all the advantages but none of the disadvantages of a single-use solution. The gamma-irradiated system is presterilized, preassembled, and doublepackaged, so it minimizes time and effort for installation.

Figure 2: Simultaneous flow through a multicapsule assembly is decisive when it comes to efficient use of filtration area. The central distribution pipe ensures optimal flow through all filter capsules. This flow also has been validated.

The new system is configurable based on standard options. Users can choose three, six, or nine filter capsules. Either prefilters or sterile filters of different types can be used in one system. Therefore, the filtration area can be between 4 m² and 27 m². Three capsules each are held adjacent to one another by a plastic holder, ensuring simultaneous flow through all filter elements (Figure 2). Halogen-free materials are used exclusively, eliminating the drawbacks of fluorinated or chlorinated compounds that occasionally result in high extractables and require more complicated disposal. Regardless of the number of filters in the MaxiCaps MR system, there is only one inlet and one outlet and only one air filter for central sterile venting. Single-use valves on those three ports ensure optimal control of various steps such as flushing, venting, filtration, and integrity testing. Likewise, tubing is used only at those three points for the inlet, outlet, and sterile venting.

Central sterile venting using the Sartopore Air filter allows a completely closed single-use system design.

Only two tube connections need to be made to integrate the system into a user’s process, thereby minimizing the risk of a handling error or leakage. The system has undergone comprehensive validation testing, and its packaging for transportation meets ASTM D4169-09 requirements. Waste disposal also has been taken into account. Although the system appears to be complex at first glance, a minimum amount of polypropylene material has been used, which facilitates disassembly and disposal.

The central sterilizing-grade air filter (Sartopore Air) ensures simple, sterile venting of the completely contained system. If an integrity testing system (such as Sartocheck) is connected to the MaxiCaps MR system, integrity can be verified by running only one test (as opposed to the need for individual testing required for individual capsules). So the system reduces the time needed for integrity testing by up to four hours.

A New Dimension
The MaxiCaps MR system is the first single-use equivalent to large-scale, multiround filter configurations provided by stainless steel systems. It combines all the advantages of a single-use solution with those of a conventional system, without increasing the time and effort of installation or the risk of user error during scale-up. Its innovative design meets the requirements of today’s challenging pharmaceutical environment and the need for simple and safe single-use solutions for large-volume production.

Corresponding author Nikolai von Knauer is product manager of Single-Use Filtration Solutions; nikolai.vonknauer@sartorius-stedim.com; 49-551-308-2473. Dr. Thomas Loewe is director of R&D Filtration Devices, and Dr. Jens Meyer is marketing manager of Filtration Technologies at Sartorius Stedim Biotech GmbH, Goettingen, Germany.

Sartopore and MaxiCaps are trademarks of Sartorius Stedim Biotech.

The post New Dimensions in Single-Use Filtration appeared first on BioProcess International.

Scale-down models (SDM) are physical, small-scale models of commercial-scale unit operations or processes that are used throughout the biopharmaceutical industry for validation studies, commercial deviation investigations, and postapproval process improvements. To support these studies, regulatory guidelines state that SDMs should be representative of the commercial process. For some downstream unit operations such as column chromatography, developing a representative SDM is straightforward because a linear scale-down approach can be used. However, developing a representative SDM for other downstream unit operations such as ultrafiltration/diafiltration (UF/DF) is more difficult because of scale-down equipment limitations and associated process challenges. The authors present a systematic (stepwise), science-based approach used to overcome these limitations during the development of a UF/DF SDM. Just fill out the form to view and download the complete eBook.

Get the eBook Now

The post eBook: Development of a Representative Scale-Down UF/DF Model: Overcoming Equipment Limitations and Associated Process Challenges appeared first on BioProcess International.

Xenotropic murine leukemia virus (XMuLV), one of the model viruses used in this study. WIKIMEDIA COMMONS (HTTPS://COMMONS.WIKIMEDIA.ORG)

Most recombinant monoclonal antibodies (MAbs) are produced by mammalian cells. Because biopharmaceuticals derived from mammalian tissue culture carry the risk of adventitious virus contamination, regulatory agencies expect risk-mitigation strategies to include validation of purification unit operations for their ability to clear viruses (1). Guidelines from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) describe how to prove viral clearance in downstream purification processes using an orthogonal approach (2). Viral log₁₀ reduction values (LRVs) are determined for orthogonal steps and combined to calculate the cumulative log viral reduction for an entire process (3, 4). Regulatory agencies typically expect drug sponsors to be able to describe their mechanisms of viral clearance in detail (4).

Downstream processing of MAbs commonly starts with protein A capture chromatography. Because most MAbs are eluted at low pH (5), a viral inactivation step follows with incubation between pH 3.0 and 4.0 (4). Low-pH treatment and neutralization leads to precipitation of impurities, so a filtration step is required for particle removal. Depth filters are commonly used to combine removal of precipitates with adsorptive binding of remaining soluble contaminants (6). In addition to removing host cell proteins (HCPs) and DNA (7), viral clearance of >4 LRV has been reported for positively charged depth filters (8–11).

In a previous virus clearance study, we demonstrated the capability of charged 3M Zeta Plus depth filters and 3M Emphaze AEX Hybrid Purifier devices as adsorptive unit operations for virus clearance (8). At pH 5.5, removal of minute virus of mice (MVM) with LRVs of 4.6–7.2 were observed for both at 220 L/m² throughput. Removal of MVM at pH 7.0 decreased significantly to LRVs of 2.4–2.7. Clearance of the larger xenotropic murine leukemia virus-related model virus (X-MuLV) also was determined at common process conditions (pH 5.5 and pH 7.0).

Additionally, we tested pH 7.0 at high conductivity (1 mol/L NaCl) to suppress ionic interactions. Under low-salt conditions at pH 5.5 and 7.0, X-MuLV clearance ranged from 5.0 to 5.8 LRV for all tested filter types. By comparison, adding NaCl substantially lowered clearances to 1.1 LRV (Emphaze) and 2.8 LRV (Zeta Plus). The difference in LRVs between low- and high-salt conditions were attributed to anionic adsorption. By arithmetric subtraction, the Emphaze device showed X-MuLV clearance relying on ionic interactions of 4.0–4.7 LRV, whereas Zeta Plus depth filters delivered 2–3 LRV. Residual X-MuLV clearance obtained under high-salt conditions (1.1–2.8 LRV) may be attributed to hydrophobic interactions or mechanical retention.

Based on those results, we have investigated the retention mechanisms of charged depth filters in more detail, again using the small nonenveloped model virus MVM (20–25 nm) and the larger enveloped virus X-MuLV (80–100 nm). To differentiate the mode of virus removal related to ionic, hydrophobic, and mechanical retention, we performed filtration runs at low conductivity, high conductivity, high propylene glycol (PG) concentration, and high conductivity with PG. Additionally, we compared the results of our virus spiking study with HCP removal levels determined at those four experimental conditions. The results provide a detailed understanding of mechanisms involved in virus retention by charged depth filters.

Table 1: Overview of the used filter types and process parameters

Materials and Methods
Materials and Instruments: For our adsorptive depth filtration studies, we used ÄKTAexplorer 10 systems with Unicorn software from GE Healthcare. All buffers were 0.2-µm filtered before use. Table 1 lists the adsorptive filters with anion-exchange functionality.

Table 2: Virus models used for the virus clearance study

Load Material and Virus Assay: Virus-spiking experiments were performed at Charles River Laboratories (CRL) in Cologne, Germany, using X-MuLV and MVM as model viruses. Table 2 provides more detailed information. The load material for our study was prepared by processing a MAb-containing harvest. Culture harvest was clarified with a Zeta Plus 60SP02A device and 0.2-µm filtered before protein A affinity chromatography eluted with a phosphate–citrate–Tris buffer. The eluate was adjusted to pH 5.5 and diluted to MAb concentrations of 10 g/L and 15 g/L. Samples were 0.2-µm filtered and stored at –70 °C until the spiking study could be performed. Before the adsorptive depth filtration runs, load samples were thawed and adjusted to our desired loading conditions regarding NaCl and PG concentration (Table 3). Those adjustments were made with buffer stock solutions containing 4 mol/L NaCl and 80% PG, respectively, leading to a final MAb concentration of 7.5 g/L for all sample loads.

Table 3: Experiments were performed in duplicate except for run #1 condition already tested in a previous study).

To exclude any influence of test items on cell growth or virus replication, CRL also performed cytotoxicity and viral interference assays. Providing the virus stock solutions, the test facility further analyzed samples with 50% tissue culture infective dose (TCID₅₀) assays. The detection limit of such assays depends on the volume of sample incubated with indicator cells. Those cells were cultivated for a specific incubation period and inspected microscopically for virus-induced changes in cell morphology.

Experimental Methods: Virus-clearance experiments on adsorptive depth filters used four different buffer conditions (Table 3). Virus-reduction values for X-MuLV and MVM were determined using phosphate–citrate–Tris buffer (pH 5.5) but without adding NaCl (“pH 5.5 low salt”), representing an appropriate condition for further processing in a MAb purification process. Second, viral reduction was determined using a phosphate–citrate–Tris buffer at pH 5.5 with 1 mol/L NaCl (“pH 5.5 high salt”) to minimize ionic interactions between viruses and the adsorptive filters. Next, experiments using a phosphate–citrate–Tris buffer at pH 5.5 with 20% PG (“pH 5.5 high PG”) were carried out to minimize hydrophobic interactions. Finally, viral reduction was determined using a phosphate–citrate–Tris buffer at pH 5.5 with 1 mol/L NaCl and 20% PG (“pH 5.5 high salt, high PG”) to minimize ionic and hydrophobic interactions for assessing a possible mechanical virus-retention effect with these filters.

Equation 1: Calculation of virus log₁₀ reduction value (LRV), with C_L = virus concentration of the load, V_L = volume of the load, C_F = virus concentration of the filtrate, and V_F = volume of the filtrate

Filtration runs were performed in parallel with two chromatography systems. CRL spiked 180 mL of protein-A–purified and conditioned MAb solutions with 5% (v/v) of ultracentrifuged and prefiltered virus stock solution (0.45 µm for X-MuLV, 0.1 µm for MVM). After mixing, the laboratory immediately analyzed a sample from each spiked pool for virus titer (“load sample”). For X-MuLV, CRL stored a second load sample until the end of the filtration (“hold sample”). MVM is physicochemically resistant, so no hold sample was analyzed for it (10).

Analysts loaded 220 L/m² (84 mL) of starting material at 158 L/m²/h (1 mL/min) onto equilibrated adsorptive depth filters, then flushed them with 63 L/m² (24 mL) equilibration buffer. Finally, they analyzed virus titers of total filtrates (flow-through and flush, the “filtrate sample”) and hold samples. Virus log₁₀ reduction values (LRV) were calculated as described in Equation 1.

Figure 1: Log₁₀ reduction values (LRVs) for (A) X-MuLV and (B) MVM at low- and high-salt conditions with three filter devices (8). Difference in X-MuLV clearance between low- and high-salt concentration (red arrows) is attributed to electrostatic adsorption. Residual LRV at 1 mol/L NaCl might be related to hydrophobic or mechanical retention. Because MVM is much smaller than the filters’ nominal pore size, mechanical retention is excluded, and runs at high-salt conditions were not performed.

Results and Discussion
Cytotoxicity and viral interference tests performed by CRL determined necessary dilutions that would not influence cell growth or virus replication. A recovery assay showed that all X-MuLV–spiked starting materials could be held at room temperature for up to four hours without significant loss of virus titer (data not shown).

Removal of X-MuLV: Clearance of X-MuLV by a Zeta Plus 90ZB05A depth filter showed comparable log₁₀ reduction values (LRVs) for all four tested conditions (Figure 2, top). Regardless whether NaCl, PG, or both were added at pH 5.5, the determined LRVs ranged from 1.8 to 3.0. Because NaCl and PG did not influence virus clearance, the results indicated that removal of the remaining 2 LRV could be attributed to size exclusion. Previously stated importance of hydrophobic interactions in X-MuLV retention by Zeta Plus depth filters (12) could not be confirmed in our study. Compared with promising results obtained in our first study — which delivered X-MuLV clearance of 6 LRV at pH 5.5 under lowsalt conditions (Figure 1A) — X-MuLV clearance was significantly lower (2.2 LRV) in the present study. Because the results under high-salt conditions are comparable (2.8 log, 1.8 log), our proposed hypothesis of mechanical retention by size is supported.

Figure 2: Log₁₀ reduction values (LRVs) for (top) X-MuLV and (bottom) MVM at low-salt, high-salt, high-PG, and high-salt + high-PG conditions with two filter devices

Several factors could alter the effectiveness of virus clearance by depth filtration. Lower clearance determined under process conditions (pH 5.5) could relate to the three parameters varied from the first to the second study: filter media lot, MAb molecule, and total MAb load (which was 825 g/m² in the first study and 1,650 g/m² in the second). The higher total MAb load could have led to hydrodynamic-radius–based shielding of binding sites on the filter media, decreasing the number of available binding sites for viruses (9). Because filtration runs in both studies were performed in duplicate using the same filter lots, some lot-to-lot variation between the two lots used in these studies is conceivable. Zeta Plus depth filters are made of natural ingredients (diatomaceous earth and cellulose), so some variation between lots is possible. That could cause differences in pore structure inside the filter media and ultimately influence mechanical entrapment (13).

Evaluated in separate experiments, Zeta Plus 90ZB05A depth filters are composed of two different filter layers (Table 1), with the upstream 30ZB layer having pore sizes significantly larger (0.5–2.0 µm) than the X-MuLV model virus (80–100 nm). Because size-related retention thus can be excluded, we considered virus clearance by that single filter layer to be easier to validate than that of the whole filter. So we had the laboratory fractionate the filtrate and flush material from its 30ZB runs into three fractions (virus-removal capacity of a single 30ZB layer could be lower than that of another because of the half thickness of the filter media).

Figure 3: Log₁₀ reduction values (LRVs) for X-MuLV at different filter loadings for low-salt or high-salt–high-PG conditions with 30ZB filter layer (upstream layer of the Zeta Plus 90ZB05A device)

X-MuLV clearance of the first fraction was high after loading of 55 L/m² at pH 5.5, with a calculated LRV of 3.8 (Figure 3). It significantly decreased at a higher loading of 110 L/m², and further at a complete loading of 220 L/m², down to a 1.1 LRV. That decrease in virus removal can be explained by exhausting the electrostatic adsorptive capacity of the depth-filter media. With simultaneously high salt and PG concentrations, consistently low LRVs (0.6–1.1) confirmed that the retention was not based on a size-exclusion effect. Nevertheless, the loading capacity for higher viral clearance is limited to 55 L/m² (corresponding to a MAb load of 412.5 g/m²), but that could be compensated in practice with increased filter area. However, it would be necessary to prove whether viral clearance capacity remains as high as measured when a flush step is carried out after a loading of 55 L/m².

By contrast, results from the synthetic Emphaze device confirmed the X-MuLV results in our first study (Figure 1A). Again, we determined virus removal of 5 LRV under low-salt conditions (pH 5.5), whereas adding NaCl suppressed ionic interactions and reduced LRV to 0.32. That observation again was supported by results from the high-PG (LRV 5.5) and the high-salt–high-PG (LRV 0.2) conditions. Because only the addition of NaCl suppressed virus removal, we believe that the retention mechanism is based predominantly on ionic adsorption, with no hydrophobic effects. We expected that based on the construction of the device, two-thirds of its functionalized nonwoven mass holding the cationic polymer was responsible for an electrostatic adsorption.

Removal of MVM: Because the nominal pore sizes of Zeta Plus 90ZB05A and Emphaze AEX Hybrid Purifier filters are significantly larger (>0.2 µm) than MVM (20–25 nm), we did not expect virus removal by size exclusion. Nevertheless, we tested all four conditions to determine whether the retention is related to ionic and/or hydrophobic interactions. 90ZB05A results showed MVM clearance of 2.3–2.4 LRV for pH 5.5 + low salt and pH 5.5 with 20% PG conditions (Figure 2, bottom). Thus, we can assume that hydrophobic interactions are not relevant for MVM retention. By contrast, virus clearance significantly decreased for both conditions spiked with NaCl to LRV <0.7, a level considered to be insignificant. Thus, the determined LRVs under low-salt conditions (2.3–2.4) are associated with ionic interactions. Moreover, a size-exclusion effect could be excluded for MVM because no substantial removal of viruses could be observed when electrostatic adsorption was suppressed by NaCl.

So the 90ZB05A filter removed MVM in the range of 1.5–2.0 logs based on anionic adsorption. Comparing these results with the high LRV of 6.7 at pH 5.5 determined in our previous study (Figure 1B), we see that the clearance is substantially lower herein. This finding is in accordance with results obtained for X-MuLV above. Again, possible reasons include higher total MAb load and lot-to-lot variability among depth filters.

An Emphaze AEX Hybrid Purifier device delivered MVM clearance of 3.7–3.9 LRV at pH 5.5 under low-salt conditions (Figure 2, bottom). By contrast, with a 1-mol/L NaCl spike, LRVs were insignificant (LRV <0.03). Adding PG to suppress hydrophobic effects made no difference. These results confirm that retention of MVM on this depth filter also is based predominantly on electrostatic adsorption, as already shown for X-MuLV above. Clearances were similar to the result of our first study with a determined LRV of 4.6 at pH 5.5 (Figure 1B). As a result, higher MAb loads and different filter lots appeared to have no significant influence on the device’s virus-removal capacity.

Removal of Host Cell Proteins (HCPs): To broaden our knowledge about retention mechanisms for the conventional and synthetic depth filters, we performed additional tests to determine HCP clearance for all conditions we tested.

Figure 4: Host cell protein (HCP) clearance at low-salt, high-salt, high-PG, and high salt + high PG conditions for MAb 2 (CHO HCP Kit #F550, third generation, from Cygnus Technologies)

We found it interestingly that HCP clearance was influenced by hydrophobic interactions in neither filter. HCP removal at pH 5.5 was comparable with that at pH 5.5 using 20% PG (Figure 4). That result is in accordance with results of the virus study above (Figure 2). Under conditions including 1 mol/L NaCl, HCP clearance by with the 90ZB05A filter decreased by about 50%, whereas removal of HCP with the synthetic device decreased by about 85% (Figure 4). Thus, HCP clearance of the latter fully relies on anionic adsorption, whereas HCP removal capability of the conventional filter involves multiple retention modes.

Even though HCPs represent a mix of proteins of different sizes and electrical charges, these results clearly support our proposed retention mechanisms for both model viruses on the two filters.

Electrostatic Adsorption
To improve our understanding of the mechanisms of virus retention on both Zeta Plus and Emphaze depth filters, we had a contract laboratory perform virus-spiking runs under four different conditions. We chose those feed stream conditions — pH 5.5 with low conductivity, pH 5.5 with high conductivity, pH 5.5 with high PG, and pH 5.5 with high conductivity and high PG concentration — to evaluate which interactions contributed to viral clearance: ionic, hydrophobic, or mechanical. In addition, we investigated mechanical retention of the enveloped X-MuLV virus through experiments using the upstream filter layer of the Zeta Plus 90ZB05A filter with its higher nominal pore size (30ZB) as a reference.

By contrast with previously published suggestions concerning the binding mechanisms on conventional depth filters (9, 12, 14, 15) we found that hydrophobic effects are not important for the retention of MVM and X-MuLV on the Zeta Plus 90ZB05A depth filter because the use of PG did not influence determined LRVs. Current results showed only minor ionic retention, whereas a significant virus clearance based on electrostatic adsorption was reported in our previous study (8). This indicated that the virus clearance capability of the depth filter is subjected to variations. Not even Zeta Plus filters are completely specified, although they are characterized by defined specifications with respect to the amount of accessible charge, the uniformity of the media, and/or the charge distribution at submicron level (7). Therefore, it is possible that lot-to-lot variations or a strong dependency on the MAb loading are dominantly responsible for these observations. The determined LRVs at high conductivity and high PG indicated a residual retention based on the physical entrapment of X-MuLV (2 LRV). As presumed, no size-exclusion effect was determined for the smaller nonenveloped model virus MVM.

We have shown that the synthetic Emphaze device’s excellent viral clearances for both MVM and X-MuLV (3.7–5.5 LRV) are driven by electrostatic adsorption. These results are in accordance with previously reported results obtained for another MAb, suggesting robust viral clearance (8). They also support the mechanistic operation mode of anion-exchange modes, which operate almost exclusively by charge-based separation considered to be robust in terms of viral clearance (16). Our results also could be verified by HCP removal results, which confirmed an electrostatic interaction as the only retention mechanism for the fully synthetic device. Furthermore, results of a previous virus-spiking study of a conventional Q-functionalized membrane adsorber showed no significant MVM removal even with a 4.5× lower MAb load under the same buffer-system conditions (data not published).

We hypothesize that the Emphaze AEX Hybrid Purifier device can withstand the concentration of polyvalent ions in a buffer system through its high ion-exchange capacity, offering a significant advantage over conventional Q-functionalized filter media. The X-MuLV clearance of 5 LRV by the nonwoven component of the Emphaze filter is higher than the published average X-MuLV clearance of 2–4 LRV by AEX resins and adsorbers (16). Key process economic advantages include higher flow rates, reduced process time, disposability, and lower buffer consumption.

References
1 Miesegaes G, et al. Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies. Biotechnol. Bioeng. 106(2) 2010: 238–246; doi:10.1002/bit.22662.

2 ICH Q5A. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin. US Fed. Reg. 63(185) 1998: 51074.

3 Brown M, et al. A Step-Wise Approach to Define Binding Mechanisms of Surrogate Viral Particles to Multi-Modal Anion Exchange Resin in a Single Solute System. Biotechnol. Bioeng. 114(7) 2017: 1487–1494; doi:10.1002/bit.26251.

4 Shukla A, et al. Viral Clearance for Biopharmaceutical Downstream Processes. Pharm. Bioprocess. 3(2) 2015: 127–138.

5 Shukla A, et al. Downstream Processing of Monoclonal Antibodies: Application of Platform Approaches. J. Chromatogr. B 848(1) 2007: 28–39; doi:10.1016/j.jchromb.2006.09.026.

6 Yinges Y, et al. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal During Monoclonal Antibody Purification. Biotechnol. Prog. 22(1) 2006: 288–296; doi:10.1021/bp050274w.

7 Singh N, et al. Development of Adsorptive Hybrid Filters to Enable Two-Step Purification of Biologics. MAbs 9(2) 2017: 350–364; doi:10.1080/19420862.2016.1267091.

8 Metzger M, et al. Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal. BioProcess Int. 13(2) 2015: 36–44.

9 Venkiteshwaran A, et al. Mechanistic Evaluation of Virus Clearance By Depth Filtration. Biotechnol. Prog. 31(2) 2015: 431–437; doi:10.1002/btpr.2061.

10 Zhou JX, et al. Viral Clearance Using Disposable Systems in Monoclonal Antibody Commercial Downstream Processing. Biotechnol. Bioeng. 100(3) 2008: 488–496; doi:10.1002/bit.21781.

11 Wang M. Zeta + VR Filters for Viral Reduction. BioProcess Int. 9(7) 2011: 62.

12 Zhou JX, et al. Orthogonal Virus Clearance Applications in Monoclonal Antibody Production. Process Scale Purification of Antibodies. Gottschalk U, Ed. John Wiley and Sons: Hoboken, NJ, 2009: 169–186.

13 Trilisky E, et al. Flow-Dependent Entrapment of Large Bioparticles in Porous Process Media. Biotechnol. Bioeng. 104(1) 2009: 127–133; doi:10.1002/bit.22370.

14 Singh N, et al. Clarification Technologies for Monoclonal Antibody Manufacturing Processes: Current State and Future Perspectives. Biotechnol. Bioeng. 113(4) 2016: 698–716; doi:10.1002/bit.25810.

15 Michen B, et al. Virus Removal in Ceramic Depth Filters Based on Diatomaceous Earth. Environ. Sci. Technol. 46(2) 2012: 1170–1177; doi:10.1021/es2030565.

16 Miesegaes G, et al. Viral Clearance By Flow-Through Mode Ion-Exchange Columns and Membrane Adsorbers. Biotechnol. Prog. 30(1) 2014: 124–131; doi:10.1002/btpr.1832.

Corresponding author Anja Trapp (anja.trapp@rentschler.de) is a scientist in bioprocessing technology and innovation, Laura Igl is a technical assistant, Sabine Faust is a process engineer, Alexander Faude is group leader, Roland Wagner is senior advisor and head of production, and Stefan Schmidt is chief scientific officer at Rentschler Biopharma SE in Laupheim, Germany. Corresponding author Dr. Sophie Muczenski is a specialist application engineer, and Nicole Mang is a sales account executive in the separation and purification sciences division at 3M Deutschland GmbH, Carl-Schurz-Straße 1, 41453 Neuss, Germany; 49-2131-145133; smuczenski@mmm.com. Zeta Plus and Emphaze are registered trademarks of 3M. AKTAexplorer and Unicorn are registered trademarks of GE Healthcare.

The post Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal appeared first on BioProcess International.

Table 1: Traditional viral clearance (VC) execution (percent virus spiking without large-volume testing) compared with total viral challenge (including large-volume testing) — anticipated VC results

Biologics derived from mammalian organisms have been accepted for therapeutic use for almost a century (1). However, these pharmaceuticals have the potential for contamination with pathogenic adventitious agents such as viruses. With cell-line–derived recombinant proteins, the viral risks commonly include viruses in the Retroviridae and Parvoviridae families (2). As patient safety and manufacturing facility suitability became significant concerns in the 1980s and 1990s, several industry and regulatory bodies reached consensus on how to approach the unique challenges of viral safety in biotherapeutics (3–5).

The resulting “safety tripod” brings together three principles in a systematic approach that provides both patient safety and risk mitigation in the manufacturing of biotherapeutics. First, a risk assessment for viral contaminants is conducted that builds strategies to reduce or eliminate risks through selection and sourcing of raw materials or implementation of testing strategies. Second, testing of raw materials (including cell banks) and in-process samples is implemented to identify virus contamination events before batch release.

Third — and arguably the most important for production of biotherapeutic proteins — viral clearance processes (validated virus reduction or inactivation) are introduced to downstream manufacturing for clearance of a wide-ranging panel of relevant and/or model viruses (5). Sourcing of cell banks and raw materials and quality control (QC) tests have limits and sometimes fail to detect all adventitious agents, which has resulted in a number of virus contamination events over the years (6–8). Viral clearance provides a consistent and robust level of protection for both patients and biomanufacturing facilities when combined with appropriate validation and manufacturing plans.

Typical viral clearance strategies include validation of virus inactivation through low-pH or solvent/detergent holds and virus reduction through chromatography steps as well as virus-retentive filtration. Multiple approaches can satisfy the requirement to use orthogonal clearance steps in a given process (4, 5). Low-pH holds, solvent/detergent treatments, and chromatography steps depend on specific chemical properties of viruses and can prove difficult to implement for good clearance of small nonenveloped viruses (9). Nanofiltration provides a well-characterized size-exclusion mechanism for retention of all but the smallest viruses, regardless of their chemical attributes (10). So it is a highly robust viral clearance step that provides a logarithmic reduction value (LRV) >4 for even difficult-to-clear small (18–24 nm) nonenveloped parvoviruses under a broad range of process and operating conditions (11).

Traditionally, virus-retentive filtration unit operations have been validated by conducting laboratory-scale spiking studies. Testing of small-scale filtration processes uses a virus-spiked feed solution prepared as a function of spike percentage of total volume (5). That methodology historically has provided sufficient results for virus stocks produced with typical purification strategies (nonspecific or nonoptimized methods). However, percentage spiking with more highly purified, higher titer virus stocks could yield improved viral clearance results but also may produce inconsistent and unacceptable viral clearance results that can affect critical study filing dates (12).

Here, we explore the advantages of implementing a total viral challenge approach in conjunction with large-volume testing over the traditional percentage-spiking method without large-volume testing (Table 1). In our virus-removal filtration studies, we used protein solutions spiked with ultrapurified minute virus of mice (MVM, 18–24 nm) or xenotropic murine leukemia virus (X-MuLV, 80–130 nm). It is important to note that extra-volume sample testing can have a significant impact on claimable LRVs in cases of complete or near-complete clearance. The resulting data in Table 1 show that with modern approaches to spiking methodologies, expected LRVs have increased significantly across separate and distinct unit operations. That potentially allows for fewer process steps to be evaluated in viral clearance studies.

Materials and Methods
Selection of Virus-Removal Filters: The two virus-retentive filters we selected for this study (both from Asahi Kasei Medical Co., Inc.) clear parvoviruses and are made of two different membrane materials. Whereas the Planova 20N filter is made of a regenerated cellulose hollow fiber, the Planova BioEX filter is made of a modified (hydrophilized) polyvinylidene fluoride (PVDF) membrane.

Advanced Database: To gain valuable insights into the specific unit operations of individual viral clearance studies, we consulted a comprehensive database with entries from more than 3,500 studies spanning over 25 years (WuXi AppTec). For this study, we reviewed records with the following criteria:

• parvovirus-grade virus-retentive filters
• virus spikes of ultrapurified MVM or X-MuLV
• operating parameters (e.g., load concentration, volume, throughput, and
  pressure) made available to account for atypical products and processes.

We included study results covering a broad range of virus LRVs. From this extensive array of data, we could make recommendations for spiking and testing requirements to produce optimal filtration performance and the potential for high viral clearance (14).

Selection of Virus Stocks: In this study, we used both X-MuLV and MVM because they are widely accepted model viruses in viral clearance studies for biotherapeutics. MVM is a relevant small (18–24 nm) nonenveloped virus that has caused a number of documented bioreactor contaminations; X-MuLV is considered to be a model virus for many processes because certain cell lines have been shown to have endogenous retroviral-like particles (15). For this study, we used ultrapurified virus stocks of both types.

To generate such virus preparations, chromatographic techniques are used as the main purification method, with an additional proprietary purification step included in preparation of ultrapurified MVM virus stocks. QC analysis of the ultrapurified viruses reveals that both stock preparations contain fewer contaminants than other grades of purified virus and consist of mostly monodispersed forms of viruses of known size for each virus type (16, 17).

Using ultrapurified virus stocks ultimately enables testing with lower spiking volumes and minimally affects virus-removal filter performance while yielding high viral-clearance values of 5–6 log₁₀ or more (16, 17). Modern parvovirus preparations are roughly 0.5–1.0 log₁₀ (plaque forming units, PFU) different from their historical counterparts, a difference that can represent a three- to 10-fold increase in infectious particles and thus should be taken into account when spiking parvoviruses into sample load materials.

Study Design and Execution: In this study we conducted 16 filtration runs: eight using Planova 20N filters and eight using Planova BioEX filters. For each filter type, we tested high and low operating pressures with MVM (duplicate runs), X-MuLV (single runs), and without virus (a single mock run). The high and low filtration operating pressures were

• 14 psi and 10 psi, respectively, for Planova 20N filters
• 45 psi and 30 psi, respectively for Planova BioEX filters.

First, we thawed the feed material (human IgG from Equitech-Bio), diluted it to 0.1 g/L in 10-mM sodium phosphate and 40-mM sodium chloride buffer (pH 7, 6.4 mS/cm), and stored it at 2–8 °C. Before filtration, MVM or X-MuLV stocks were spiked into room-temperature protein solution at a target total challenge of 7.5 log₁₀ PFU/filter based on our review of previous study results from the viral clearance database. We processed spiked load material through a 0.2-µm prefilter (MVM-spiked solution) and 0.45-µm prefilter (X-MuLV–spiked solution). Load and processing controls were removed from the spiked material for each run.

Equation 1

For all run conditions, we applied the feed material in the same manner to each filter and collected filtrate in two 100-L/m² fractions followed by a 10-minute complete system depressurization, then collected a single 15-L/m² buffer flush at the initial operating pressure in a separate fraction. Each fraction was assayed separately. Additionally, we created a representative pool with proportional amounts of each of the three fractions for large-volume analysis. To determine volumetric throughput and flux of each filtration run by mass, we used Asahi Kasei Bioprocess data-acquisition software.

Virus Titer Quantification: We used standard plaque-assay methodologies for determining virus titer of both MVM and X-MuLV. Rapid large-volume testing was conducted on simulated pool samples for reducing the assay limit of detection (LoD) and increase reported virus LRV for samples in which no virus was detected.

Figure 1: Flux curves for Planova 20N filtrations at high pressure (14 psi)

Briefly, the plaque assays involved producing a monolayer growth of either 324K cells (for MVM) or PG4 cells (for X-MuLV) in six-well plates or large-volume dishes. We incubated the cell monolayers with run sample dilutions at 37 °C — one hour for MVM samples and two hours for X-MuLV samples. After removing the samples from the plates and dishes, we overlaid the cell monolayers with an agarose/culture media mixture and incubated them at 37 °C for either six days (X-MuLV) or 10 days (MVM). Following that final sample incubation, we fixed each cell monolayer with a formalin solution and stained it with crystal violet. Plaques (voids in the cell monolayer) were counted and converted into a plaque-forming unit per milliliter (PFU/mL) measurement for each sample. We calculated virus LRV using Equation 1.

Table 2: Viral clearance data for minute virus of mice (MVM) sorted by total viral challenge

Results and Discussion
Database Findings: Analysis of data obtained from the viral clearance database revealed artifacts of variable data or poorly optimized clearance (several presented in Table 2). Viral clearance was still effective for runs in studies A and B (internal data), but inconsistent breakthrough was observed and produced variation in virus LRV of more than 1 log₁₀ between duplicate runs. In studies C and D (internal data), nonrobust viral clearance was obtained with MVM LRV <4. Cases E and F (internal data) showed the potential to achieve consistent and higher MVM LRV for studies conducted within the normal bounds of traditional process parameters.

Figure 2: Flux curves for Planova 20N filtrations at low pressure (10 psi)

Even though viral clearance artifacts typically are observed only in studies using the smallest parvoviruses, significant risk remains that less satisfactory results could compromise the development and regulatory approval of biopharmaceuticals substantially. Thus, recommendations for using optimized virus preparations in virus-filtration studies have been made (14). Note that a correlation was observed between lower spiking challenges and more consistent viral clearance results, suggesting that virus load may be a critical factor in ensuring predictable outcomes (Table 2). Limiting the total viral challenge to 7.5 log₁₀ PFU/run could mitigate the risk of such artifacts in viral clearance studies, as observed in studies E1 and F1.

Figure 3: Flux curves for Planova BioEX filtrations at high pressure (45 psi)

Process Flux: All filtrations were executed successfully and demonstrated minimal impact of virus spikes on process performance (Figure 1–4). For high-pressure runs with both filter types, spiked runs and mock runs had equivalent starting flux and experienced little to no flux decay, indicating that the virus spike did not affect filter performance. For low-pressure runs with both filter types, the spiked runs had lower starting flux than the mock runs but did maintain similarly near-zero flux decay by comparison. Although we did observe differences in initial flux, the filters performed adequately throughout all runs, with all target filter throughputs achieved.

Figure 4: Flux curves for Planova BioEX filtrations at low pressure (30 psi)

Viral Clearance: Table 3 reports viral clearance data for MVM runs, and Table 4 shows X-MuLV results. No virus was detected in any filtrate sample during this study, and there was no measurable impact of low pressure or process pause on the filters’ viral clearance capability. For all runs conducted with the total viral challenge approach, the simulated pool showed complete clearance with a virus LRV ≥5.9, demonstrating the benefits of this approach. Our dataset strongly supports the use of the total viral challenge approach in conjunction with large-volume testing for viral clearance studies: No viral breakthrough was observed, and consistent robust viral clearance was achieved for all tests.

Table 3: Viral clearance data resulting from filtrations spiked with minute virus of mice (MVM); “≥” indicates complete clearance

To provide context for setting virus-spiking levels, considering virus titers that could arise during a contamination event is important to ensuring that the virus challenge presented during validation studies provides a relevant or worst-case scenario. In recombinant bioprocesses, contaminants usually are identified first in a bioreactor because of their deleterious impacts on cell culture performance. Even when they are not detected at such an early stage, broad in vitro testing or contaminant-specific molecular testing of unprocessed bulk materials usually have LoDs ≤1 log10 PFU/mL (18).

Table 4: Viral clearance data from filtrations spiked with xenotropic murine leukemia virus (X-MuLV); “≥” indicates complete clearance

Therefore, a gross contamination event is likely to be detected. However, in the unlikely case that virus contamination had no observable impact in a bioreactor and was not detected with bulk testing procedures, other virus-removal steps (column chromatography and/or chemical inactivation) before virus filtration certainly would reduce the contaminating virus load.

A Virus-Filtration Example: The only published report of parvovirus titer from a bioreactor contamination indicated a MVM titer of 6 log₁₀ copies/mL by quantitative polymerase chain reaction (qPCR) (8). In this case, the contaminant was discovered through MVM-specific testing. Regardless, the manufacturing process probably would include chromatography steps that could be expected to reduce that level by 2–6 log₁₀, resulting in a worst-case MVM titer of 4 log₁₀ copies/mL at the virus-filtration step. Spiking virus at around 5 log₁₀ PFU/mL thus still provides a greater challenge than the worst-case level for that step.

Understanding relevant viral challenge situations during plasma-product manufacturing is complicated by variations in potential viral clearance steps used for different products and different virus classes. However, it is helpful to note that robust molecular testing regimes are used to limit potential virus loads in plasma pools. For instance, the US Food and Drug Administration (FDA) has provided guidance that B19 parvovirus levels should be <4 log₁₀ copies/mL (19). Without any additional virus-removal steps, a 5 log₁₀ PFU/mL parvovirus spike still represents a worst-case scenario for removal of that contaminant.

In our study, we attempted to reproduce that viral load titer. In so doing, we believe we have reduced the likelihood of observing aberrant viral clearance study artifacts.

A Modern Approach
Our study demonstrates the benefits of using the total viral challenge approach in designing viral clearance studies. By limiting total viral challenge to 7.5 log₁₀ PFU per virus filter, you can achieve highly robust virus LRV while minimizing the risk of study artifacts. The effect is further amplified when this technique is used in conjunction with contemporary virus preparations and large-volume sample testing. Although we have discussed the application of implementing the use of total virus challenge for virus-filtration runs, note that the same methodology has been implemented in other unit operations such as chromatography or low-pH inactivation. In future studies, investigators should consider the tools described here for guidance on achieving appropriate viral clearance as needed for their own downstream purification processes.

References
1 Lalonde R, Honig P. Clinical Pharmacology in the Era of Biotherapeutics. Clin. Pharmacol. Ther. 84, 2008: 533–536.

2 Stuckey J, et al. A Novel Approach to Achieving Modular Retrovirus Clearance for a Parvovirus Filter. Biotechnol. Prog. 30(1) 2014: 79–85; doi:10.1002/btpr.1820.

3 Sofer G, et al. PDA Technical Report No. 41: Virus Filtration. PDA J. Pharm. Sci. Technol. 59(S-2) 2005: 1–42.

4 CPMP BWP 268/95. Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses. European Medicines Agency: London, UK, 14 February 1996.

5 ICH Q5A. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin. US Fed. Reg. 63(185) 1998: 51074.

6 Victoria JG, et al. Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus. J. Virology 84(12) 2010: 6033–6040; doi:10.1128/JVI.02690-09.

7 Skrine J. A Biotech Production Facility Contamination Case Study — Minute Mouse Virus. PDA J. Pharm. Sci. Technol. 65(6) 2011: 599–611; doi:10.5731/pdajpst.2011.00823.

8 Moody M, et al. Mouse Minute Virus (MMV) Contamination — A Case Study: Detection, Root Cause Determination, and Corrective Actions. PDA J. Pharm. Sci. Technol. 65(6) 2011: 580–588; doi:10.5731/pdajpst.2011.00824.

9 Aranha H, Forbes S. Viral Clearance Strategies for Biopharmaceutical Safety, Part 2: A Multifaceted Approach to Process Validation. BioPharm 14(5) 2001: 43–54, 90.

10 Yamamoto A, et al. Effect of Hydrodynamic Forces on Virus Removal Capability of Planova Filters. AIChE J. 60(6) 2014: 2286–2297 ; doi:10.1002/aic.14392.

11 Hongo-Hirasaki T, Komuro M, Ide S. Effect of Antibody Solution Conditions on Filter Performance for Virus Removal Filter Planova 20N. Biotechnol. Prog. 26(4) 2010: 1080–1087; doi:10.1002/btpr.415.

12 Asher D, et al. PDA Technical Report No. 47: Preparation of Virus Spikes Used for Virus Clearance Studies. Parenteral Drug Association: Bethesda, MD, 2010.

13 Chen D, Chen Q. Virus Retentive Filtration in Biopharmaceutical Manufacturing. PDA Letters 15 April 2016: www.pda.org/pda-letter-portal/archives/full-article/virus-retentive-filtration-in-biopharmaceutical-manufacturing Accessed on 21FEB2016.

14 Hongo-Hirasaki T, et al. Effects of Varying Virus-Spiking Conditions on a VirusRemoval Filter Planova 20N in a Virus Validation Study of Antibody Solutions. Biotechnol. Prog. 27(1) 2011: 162–169; doi:10.1002/btpr.533.

15 Stauss DM, et al. Removal of Endogenous Retrovirus-Like Particles from CHO-Cell Derived Products Using Q Sepharose Fast Flow Chromatography. Biotechnol. Prog. 25(4) 2009: 1194–1197; doi:10.1002/btpr.249.

16 Slocum A, et al. Impact of Virus Preparation Quality on Parvovirus Filter Performance. Biotechnol. Bioeng. 110(1) 2013: 229–239; doi:10.1002/bit.24600.

17 Roush D, et al. Limits in Virus Filtration Capability? Impact of Virus Quality and Spike Level on Virus Removal with Xenotropic Murine Leukemia Virus. Biotechnol. Prog. 31(1) 2015:135–144; doi:10.1002/btpr.2020.

18 Gombold J, et al. Systematic Evaluation of In Vitro and In Vivo Adventitious Virus Assays for the Detection of Viral Contamination of Cell Banks and Biological Products. Vaccine 32(24) 2014: 2916–2926; doi:10.1016/j.vaccine.2014.02.021.

19 US Food and Drug Administration. Guidance for Industry: Nucleic Acid Testing (NAT) to Reduce the Possible Risk of Human Parvovirus B19 Transmission by Plasma-Derived Products. US Fed. Reg. 74(143) 2009: 37231–37232.

20 Lute S, et al. Phage Passage After Extended Processing in Small Virus Retentive Filters. Biotechnol. Appl. Biochem. 47(Part 3) 2007: 141–151; doi:10.1042/BA20060254.

Corresponding author Michael Burnham is a senior principal scientist in process development and commercialization, Alexander Schwartz is a viral clearance scientist, and Joseph Hughes is vice president of biologics testing at WuXi AppTec, Inc., 4751 League Island Boulevard, Philadelphia, PA 19112; 1-215-218-7100 x5542; mike. burnham@wuxiapptec.com. Esha Vyas is field applications manager, Nanna Takahashi is an account manager, Pauline Nemitz was field applications manager (now with Sartorius Stedim Biotech), Daniel Strauss is a principal scientist, and Naokatsu Hirotomi is executive vice president and general manager of Asahi Kasei Bioprocess America, Inc., 1855 Elmdale Avenue, Glenview, IL 60026.

The post Advanced Viral Clearance Study Design: A Total Viral Challenge Approach to Virus Filtration appeared first on BioProcess International.

Figure 1: Fouling of chromatography particle surfaces by compound contaminant associations

One of the early disappointments in development of immunoglobulin G (IgG) purification technology was ultrafiltration on membranes with 50–100 kDa cutoffs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that most host cell proteins were smaller than that. IgG was retained. Parallel concentration and buffer exchange could be performed going into a follow-on polishing step. These features made it an obvious candidate for initial capture, but it did not perform as hoped. Membrane fouling sabotaged its concentration–diafiltration potential, and prohibitive levels of host contaminants remained in the IgG fraction. Protein A affinity chromatography became established as the preferred capture method. Ultrafiltration was consigned to the supporting role of concentration and diafiltration after purification was essentially complete.

Protein A has since proven to be consistent and highly competent, but like ultrafiltration, it should be capable of much better performance than it generally delivers. In its prechromatography days, it was hailed for its exquisite specificity, binding only IgG. The expectation was that the affinity chromatography version would achieve essentially complete purification in a single step. In practice, host cell protein (HCP) contamination often is observed in the range of 1,000–10,000 ppm. DNA also persists. It does not seem reasonable that protein A should have affinity for either, and it doesn’t. Their persistence points instead to a nonspecific contamination pathway.

Depressed protein A performance is now understood to result mainly from chemical fouling of chromatography media during sample application. Chemical fouling is distinct from physical fouling. Physical fouling involves cells and debris of a size sufficient to clog filters and columns. Chemical fouling involves nonspecific chemical interactions of contaminants with the surfaces of purification media; 20–40% of host contaminants in cell culture harvests are associated in compound assemblages that include hundreds of species (1–3). Some are weakly associated, some strongly. Some of their components bind more strongly to protein A than IgG does and act as anchors for associated species that individually lack affinity for the chromatography surface.

Figure 2: Dissociation of contaminant subsets coincident with IgG elution

Accumulation of such assemblages on chromatography surfaces interferes with performance in two major ways. Aggregates of 50–400 nm block IgG access to the diffusive pores in chromatography particles. That reduces dynamic capacity on protein A as much as 20% compared with loading purified IgG (Figure 1). IgG elution conditions subsequently dissociate contaminant subsets from still-anchored components, and the dissociated subsets cooccupy the IgG fraction (Figure 2). 99% of host contaminants in IgG eluted from protein A derive from this pathway (1).

The effects of chemical fouling are not limited to protein A. They compromise all chromatography methods. They reduce capacity on traditional and multimodal cation exchangers by 50–65% (4, 5). Chemical fouling burdens even size-exclusion chromatography (SEC), in which the chemical surfaces of chromatography particles are generally assumed to be inert. In fact, SEC interacts so strongly with some contaminant heteroassociations that they smear all the way across the elution profile instead of being restricted to the aggregate fractions corresponding to their actual size class (6).

Removing chemical foulants before column loading enables dramatic improvement: HCP removal by protein A improves more than 100-fold and DNA removal more than 1,000-fold. IgG binding capacity is elevated to the same levels obtained when protein A is loaded with purified IgG (3). Improvements with ion-exchange and multimodal capture are even greater (4, 5).

Given that chemical fouling depresses performance of all chromatography methods and that advance removal of chemical foulants enables them to fulfill their fractionation potential, could it also enable ultrafiltration to deliver the potential envisioned in the 1980s? This article addresses that question and goes a step further. It coordinates capture by ultrafiltration with an in-line polishing chromatography step that takes advantage of ultrafiltration’s abilities to perform parallel concentration and diafiltration.

Removing Chemical Foulants from Cell Culture Harvests
Advance removal of chemical foulants from cell culture harvests targets the most reactive species: the most negatively charged, the most positively charged, the most hydrophobic, largest and least soluble. This is accomplished by adding a combination of flocculating agents to cell-free or cell-containing harvests. Allantoin crystals bind aggregates, viruses, and endotoxins by hydrogen bonding (7–9). Octanoic (caprylic) acid precipitates a variety of HCP and viruses (3–5). Electropositive polymers, particles, or depth filters target DNA, virus, and acidic HCP. Removal of solids eliminates 40–70% of HCP, 99% of DNA, 2–3 logs of endotoxin, 5–9 logs of virus, and 75–95% of aggregates (1–5). Typical IgG losses of about 10% mostly represent misfolded product associated with aggregates. Other approaches are discussed in the literature (10–12).

Figure 3: Size distribution of host cell protein (HCP) before and after foulant removal; note the different scales on the left and right frames.

Figure 3 illustrates SEC profiles before and after flocculation of a Chinese hamster ovary (CHO) cell culture harvest. The aggregate population — which has nearly the same cumulative mass as the IgG peak — contains misfolded IgG, but the dominant species are HCP associated with chromatin nucleation centers (1–5). This illustrates why ultrafiltration initially failed to meet expectations as an IgG capture method. The aggregates would have been coretained with the IgG where they interfered with pore flux and remained in the retentate with the IgG after processing. The right-hand frame in Figure 3 shows why advance foulant removal should enable ultrafiltration to deliver outstanding performance.

Figure 4: Flow diagrams through an apparatus configured to support ultrafiltration with a single adsorbent channel

Ultrafiltration-Adsorption for Integrated IgG Capture–Polishing
Figure 4 illustrates a basic apparatus for coordinating ultrafiltration with in-line adsorption (13–15). The first frame shows the system with only an ultrafiltration unit in line. The second frame illustrates the flow path with an adsorptive chromatography unit also in line. Additional adsorbent channels can be added, and any given adsorbent can be operated in either bind–elute or flow-through mode. Figure 5 shows an early prototype with two adsorbent channels.

Figure 5: An early laboratory prototype ultrafiltration-adsorption system has two adsorbent channels, each equipped with a hollow-fiber membrane adsorber. Changes in flow path were controlled on this unit by manual three-way valves.

Equilibration: Processing begins by equilibrating the system. The equilibration buffer is formulated to the loading conditions for the chromatography adsorbent. The adsorbent is put off line. Ultrafiltration remains on line during the entire process. Concentration and diafiltration begin coincident with sample introduction. Most small contaminants are eliminated through the permeate during this phase. The chromatography adsorbent goes on line before the retentate is fully equilibrated, and the retentate is recycled through the adsorbent while buffer exchange and concentration continue. The process is complete when buffer conditions in the system match the equilibration buffer.

Monoliths and Membrane Adsorbers: The chromatography adsorbent can be a monolith, a membrane adsorber, or a packed column. Monoliths and membranes have lower protein-binding capacity per unit volume than columns with similar selectivity, but the ultrafiltration step reduces that capacity requirement. Monoliths and membranes, meanwhile, maintain their binding efficiency at flow rates over 10× higher than columns. That has value when integrating ultrafiltration with solid-phase adsorption because the filter area is relatively large and requires high volumetric flow rates to support reasonable process times.

Figure 6: Efficiency of media use by different chromatography formats; arrow shows the direction of flow. Monoliths and membrane adsorbers both achieve virtually complete saturation with only a single pass because mass transport is convective. Columns packed with porous particles seldom achieve much better than 50% saturation in a single pass because of the inefficiency of diffusive mass transport.

Monoliths and membrane adsorbers are also preferred because they support much better media use. Saturation can be achieved with only a single pass because both types of adsorbers rely on convective mass transport. Convective mass transport efficiency is independent of both flow rate and solute size. Columns packed with porous particles rely on diffusion for product to enter the pores, and diffusive efficiency is dependent on both flow rate and solute size. This explains why single-pass loading fails to saturate packed columns in a single pass (Figure 6) and why they benefit from multiple-pass loading.

Porous-particle columns remain an important option despite their slowness because they come with a higher diversity of ligands than monoliths or membrane adsorbers, especially including multimodal adsorbents. Their linear flow-rate restrictions can be compensated for with short beds in either axial or radial-flow formats. Another benefit of a porous-particle column is that its bed volume can be customized to match the capacity requirements of a separation.

Figure 7: Experimental results from foulant removal, followed by ultrafiltration-adsorption with a single adsorbent channel in flow-through mode; the tangential-flow filtration (TFF) cartridge was a regenerated cellulose membrane with a 30-kDa cutoff. The adsorbent was a strong anion-exchange monolith.

Figure 7 illustrates results from an experiment with a prospective Herceptin (trastuzumab) biosimilar. Chemical foulants were extracted in advance with allantoin, octanoic acid, and electropositive particles or depth filters as described elsewhere (1–5, 14). The buffer was 50 mM Tris, pH 8.0. Antibody was concentrated to 20 g/L over the course of the experiment. The adsorbent was put in line after 2.5 diavolume (DV) of concentration/ buffer exchange, and the experiment was complete at 5 DV. Process time was 4–6 h. HCP were reduced to <37 ppm, DNA to <1 ppb, and aggregates to about 0.1%. Process recovery was 86%.

Table 1: Ultrafiltration with a single adsorbent channel in flow-through mode

Table 1 shows results from different harvests of the same cell line using different foulant removal methods and different ultrafiltration media and adsorbents. In brief, 30-kDa cellulose ultrafiltration membranes gave the same results as 50-kDa PES membranes. Membranes with 100-kDa ratings caused antibody losses with this antibody but might be suitable for other antibodies. Monoliths, membrane adsorbers, and porous-particle columns were interchangeable. Strong and weak anion-exchange adsorbents were interchangeable. A multimodal cation exchanger (Capto MMC) and a phenyl membrane adsorber gave results comparable to those of anion-exchangers.

This is not to suggest that the technique will work with any adsorbent for any antibody. Conditions were optimized for each adsorbent. With other antibodies, performance among adsorbents varied more substantially. In all cases, however, performance was affected most dramatically by the efficiency of foulant removal. Omitting foulant removal resulted in universal failure, with HCP contamination commonly persisting at 10,000–30,000 ppm.

Figure 8: Experimental results from foulant removal followed by capture with protein A, then polishing ultrafiltration-adsorption with a single adsorbent channel in flow-through mode; the TFF cartridge was a PES membrane with a 50-kDa cutoff. The adsorbent was a weak anion exchange monolith (CIM EDA).

Ultrafiltration–Adsorption for Polishing After Stand-Alone Capture
Figure 8 summarizes a purification in which a protein A eluate still at pH 3.5 was introduced to the system. It was concentrated to 25 g/L and buffer-exchanged to 50 mM MES, pH 5.5, while it recycled through a weak anion-exchange monolith. A 0.22-µm membrane filter was placed in line ahead of the monolith to remove turbidity that developed during pH adjustment. This particular antibody was a rare outlier that still contained >2,000 ppm HCP after the protein A step despite having been treated in advance to remove foulants. Coordinated ultrafiltration–adsorption still reduced HCP to <10 ppm, DNA to <1 ppb, and aggregates to <0.1% (13–15).

The ability of the method to reduce HCP from 2,000 ppm to less than 10 ppm highlights two important points: First, by suspending the mechanisms that compromise purification performance, advance foulant removal enables purification methods to achieve the results they should be able to deliver. Second, keeping the ultrafiltration channel in line eliminates small contaminants throughout the entire process cycle. This is an especially important enhancement for flow-through applications because it removes nonadsorbed small contaminants from the system. By contrast, nonadsorbed contaminants in conventional flow-through chromatography applications stay with the antibody.

Coordination of Multistep Chromatography Processes
Flow-through steps with a single adsorbent channel are most compelling: They involve fewer buffers and less process time, but additional adsorbent channels can be added to enable complete multistep purification processes on a single instrument.

For two flow-through channels, the process begins with sample equilibration/concentration. The first adsorbent is put in line and kept there until the sample is equilibrated. Then that adsorbent is put off-line, and the second is brought in-line. The system is equilibrated with a buffer designed to optimize contaminant removal with the second adsorbent. Then rinsing the system with clean buffer displaces product from the internal flow path, after which the system is sanitized with 1 M NaOH.

Using adsorbents in bind–elute mode involves more buffer inputs and takes more time but still conserves the benefits of keeping sample concentrations high throughout the process. It also suspends the difficulty of buffer exchange between process steps. Once your protein is in the system, you can buffer-exchange it in conjunction with adsorption. Large non-IgG proteins such IgM, factor VIII, and von Willebrand factor particularly suggest themselves as natural subjects for ultrafiltration-adsorption because they could accommodate membranes with larger pore-size distributions, potentially removing an even broader spectrum of contaminants.

Figure 9: Experimental size-exclusion chromatography (SEC) results showing purification of bacteriophage M13 with a two-channel ultrafiltrationadsorption (UA) system; baseline triangles mark the point at which sample salts elute from the column; ELISA = enzyme-linked immunosorbent assay, qPCR = quantitative polymerase chain reaction, QA = quaternary amine

Virus particles for vaccines, gene therapy vectors, or antibiotic replacement also are obvious candidates. Figure 9 summarizes purification of a bacteriophage with a two-channel apparatus (13–15). The steps were concentration and elimination of small contaminants through the membrane in parallel with equilibration to 50 mM MES, pH 6, then binding to a cation-exchange monolith. The cation exchanger was eluted and put off-line, and an anion-exchange monolith put in-line. The buffer was exchanged to 50 mM HEPES, pH 7.0, causing the virus to bind; then it was eluted.

Future Directions
The biopharmaceutical industry’s evolution toward continuous processing raises the obvious question of whether coordinated ultrafiltration-adsorption can fulfill this ideal. It can, but in a fundamentally different way than approaches such as simulated moving-bed (SMB) chromatography. SMB represents genuine continuous processing: It processes feed continuously at the rate produced and continuously delivers processed product. SMB, however, supports only a single chromatography method per instrument. Multiple instruments are required to conduct multistep processes. Each instrument contains dozens of moving parts and coordinates thousands of mechanical events per day. Method development requires sophisticated simulation software to model the
nonintuitive retrograde order of chromatography events across multiple columns.

Processing with ultrafiltration-adsorption occurs in batch mode, but it can support a complete multistep purification process on a single instrument. All the system’s components can be sanitized ahead of use in a single step and at the end of a process in a single step. A simple surge tank to accumulate harvest between cycles enables round-the-clock production. Even systems configured for multiple adsorption steps involve only a fraction of the moving parts and monitoring devices required by SMB and thousands of fewer mechanical events per process cycle. Development involves established intuitive concepts and familiar guidelines.

Equipment access could be considered a limitation for ultrafiltration-adsorption systems to the extent that they are not presently available commercially. But the components are. Data discussed in this article were obtained with commercially available integrated tangential-flow filtration units, modified to include UV, pH, and conductivity sensors for in-line process monitoring. Those components have been available at a number of process scales for decades, as have the chromatography media, so scalability seems unlikely to be an issue. Experienced chromatographers can assemble a functioning unit within a few hours.

It is impossible to predict whether or when the industry will be ready to embrace another new instrument technology, but several key points are already clear: Ultrafiltration is absolutely capable of providing the processing benefits envisioned by the industry’s early founders, and we can certainly use it more effectively than tradition has taught us. The evolution of chemistry and instrumentation since those days has put us in better-than-ever position to do so. The increasing productivity and economic demands being placed on the industry meanwhile demand that we not overlook opportunities that lie easily within our reach.

Acknowledgments
Thanks to the Bioprocessing Technology Institute in Singapore, where the foulant removal and ultrafiltration-adsorption technology discussed in this article were developed under a grant from the Singaporean Agency for Science, Technology and Research (A*STAR).

References
1 Gagnon P, et al. Nonspecific Interactions of Chromatin with Immunoglobulin G and Protein A, and Their Impact on Purification Performance. J. Chromatogr. A 1340, 2014: 68–78; doi:10.1016/j.chroma.2014.03.010.

2 Gagnon P, et al. Non-Immunospecific Association of Immunoglobulin G with Chromatin During Elution from Protein A Inflates Host Contamination, Aggregate Content, and Antibody Loss. J. Chromatogr. A 1408, 2015: 151–160; https://doi.org/10.1016/j.chroma.2015.07.017.

3 Nian R, et al. Advance Chromatin Extraction Improves Capture Performance of Protein A Affinity Chromatography. J. Chromatogr. A 1431, 2016: 1–7; doi:10.1016/j.chroma.2015.12.044.

4 Nian R, Gagnon P. Advance Chromatin Extraction Enhances Performance and Productivity of Cation Exchange Chromatography-Based Capture of Immunoglobulin G Monoclonal Antibodies. J. Chromatogr. A 1453, 2016: 54–61; doi:10.1016/j.chroma.2016.05.029.

5 Gagnon P, et al. Chromatin-Mediated Depression of Fractionation Performance on Electronegative Multimodal Chromatography Media, Its Prevention, and Ramification for Purification of Immunoglobulin G. J. Chromatogr. A 1374, 2016: 145–155; doi:10.1016/j.chroma.2014.11.052.

6 Tan L, et al. Characterization of DNA in Cell Culture Supernatant By Fluorescence-Detection Size-Exclusion Chromatography. Anal. Bioanal. Chem. 407, 2015: 4173–4181; doi:10.1007/s00216-015-8639-9.

7 Vagenende V, et al. Amide-Mediated Hydrogen Bonding at Organic Crystal Interfaces Enables Selective Endotoxin Binding with Picomolar Affinity. ACS Appl. Mat. Interfaces 5(10) 2013: 4472–4478; doi:10.1021/am401018q.

8 Vagenende V. et al. Self-Assembly of Lipopolysaccharide Layers on Allantoin Crystals. Colloids Surf. B Biointerfaces 120, 2014: 8–14; doi: 0.1016/j.colsurfb.2014.04.008.

9 Vagenende V. et al. Allantoin As a Solid Phase Adsorbent for Removing Endotoxins. J. Chromatogr. A 1310, 2013: 15–20; doi:10.1016/j.chroma.2013.08.043.

10 Singh N, et al. Clarification Technologies for Antibody Manufacturing Processes: Current State and Future Perspectives. Biotechnol. Bioeng. 113(4) 2016: 698–716; doi:10.1002/bit.25810.

11 McNerney T, et al. PDADMAC Flocculation of Chinese Hamster Ovary Cells: Enabling a Centrifuge-Less Harvest Process for Monoclonal Antibodies. mAbs 7(2) 2015: 413–427; doi:10.1080/19420862.2015.1007824.

12 Kang Y, et al. Development of a Novel and Efficient Cell Culture Flocculation Process Using a Stimulus Responsive Polymer to Streamline Antibody Purification. Biotechnol. Bioeng. 110(11) 2013: 2928–2937; doi:10.1002/bit.24969.

13 Gagnon P, et al. Flow-Through. Turbocharged. Oral presentation, Prep XVIII, Philadelphia, July 26–29, 2015.

14 Gagnon P. Apparatus and Methods for Fractionation of Biological Products. United States Patent Application 20170173537, 2017.

15 Gagnon P, et al. The Nokia Syndrome. Can Protein A Survive? Oral presentation, IBC Bioprocess Development and Production Week, Huntington Beach, March 30–April 2, 2015.

Pete Gagnon is CSO at BIA Separations and a member of BPI’s Editorial Advisory Board; pete.gagnon@biaseparations.com.

The post IgG Purification By Ultrafiltration: Time for Another Look appeared first on BioProcess International.

Figure 1: Usual clarification options for viral vaccines and vectors

Viral vaccines rely on the antigen properties of a virus or virus-like entity to trigger an immune response and induce immune protection against a forthcoming viral infection. Through development of recombinant viral vaccines, developers can reduce risks associated with the presence of live and inactivated viruses. Instead, recombinant vaccines induce immunity against a pathogen by relying on the capacity of one or more antigens delivered by means of viral vectors or the baculovirus/plasmid system (1). Viral vaccines are formulated with or without adjuvants and categorized as shown in Table 1.

Table 1: Classification of viral vaccines

Viral vaccine manufacturing processes can be templated. They follow a general scheme, starting with production in either an embryonated egg or mammalian/insect cell culture. After production, the bulk harvest material is processed to purify a vaccine of interest. Following upstream production and lysis (optional), a clarification step typically is introduced to start the purification process by either centrifugation or filtration. This is a critical unit operation because it strongly affects product recovery and subsequent downstream purification.

Selection of the clarification methodology depends on the type of cells involved, the nature of the virus, and properties of the process fluids. Filtration-based technologies have gained prominence in vaccine clarification following the increasing implementation of single-use technologies upstream. Filtration methods include membrane technology (microfiltration operated in normal-flow filtration (NFF) mode, tangential-flow filtration (TFF), or depth filters operated as NFF.

Here we provide a comprehensive overview of different filtration technologies and their application in viral vaccine clarification. We also outline challenges and present current best practices.

Considerations for Clarification of Viral Vaccines: Type of Substrate
Composition, type, and level of contaminants to be removed during clarification mainly depend on the upstream process and expression system used for production. In most cases, the virus particles must be kept integral during the clarification step.

For decades, embryonated chicken eggs have been used to produce both human and animal vaccines. However, the resulting allantoic fluid harvest (rich in virus particles and cellular debris) is a challenging feed for clarification. This fluid has a high solids content (>25%, which increases with embryo age) and high mineral and protein content, hence its highly viscous consistency. It also contains rudimentary tissue compounds from chicken embryos such as feathers, beaks, blood vessels, and/or blood cells.

Several viral vaccines have moved away from an egg-based process toward the use of live cells (e.g., plant, microbial, avian, or mammalian). The composition of these feed streams can vary significantly. Based on cell viability  — and lysis method selected (e.g., chemical or mechanical), if applicable — the impurity profile in the fluid to be clarified can differ considerably. For example, low cell viability can indicate high levels of contaminants released into a feed stream from cell lysis. Typical contaminants such as host cell DNA, host cell proteins (HCPs), lipids, and bigger particles such as cell debris can be identified. The proportion of solids in the feed usually is an indicator of purifying challenges ahead (~6–8% mammalian cells or up to 40% in yeast). Compared with allantoic fluid, however, cell culture harvests are considerably cleaner in terms of solids load and soluble content.

Another expression method is the baculovirus expression vector system (BEVS) used with insect cells. This is gaining interest particularly for producing viral vectors and virus-like particles (VLPs). Other expression systems such as bacteria, yeast, and plant cells also can be used to produce viral particles.

Considerations on Key Product Quality Criteria and Control Strategies
Yield: In most cases, yield is an off-line measurement conducted at the end of a number of process steps. Depending on the success criteria for each step, yield can be the main parameter to consider when selecting one option over another for a given step. Based on the size and properties of viral particles, yield could be affected by the clarification method used. For example, whereas positive charges increase nucleic acids and HCP capture, diatomaceous earth can retain viruses by adsorption.

Some viruses are shear sensitive and can be damaged by high shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages through TFF. Due to their large size, viruses larger than 100 nm also can be retained simply by tight filters. Thus, companies should take such factors into consideration when selecting depth filters because some depth filter devices include a 0.1-μm membrane that could cause retention-driven product loss. Other process elements such as aggregation and an excess of impurities can compromise viral particle recovery.

Final Product Purity Levels: Regulatory agencies provide recommendations and requirements regarding acceptable residual amounts of contaminants in final drug products. For reasons of patient safety and tolerance, host cell DNA in a final product must be reduced to appropriate levels. In 1998, the World Health Organization (WHO) specified the maximum residual DNA content in a vaccine to be <10 ng/dose. Since then, the European Medicines Agency (EMA) proposed more stringent conditions based on the type of cell line (tumorigenic origin) used in vaccine manufacturing. The US Food and Drug Administration (FDA) follows a case-by-case evaluation approach and recommends that manufacturers reduce both the size (~200 bp) and amount of DNA per dose.

To date, a final DNA content of <10 ng/ dose commonly is accepted for most biologics. Similarly, a recombinant monoclonal antibody (MAb) product must reach clearance of impurities to <100 ppm of HCP, ≤10 ng/dose of DNA, and <5% of immunogenic aggregates (Table 2).

Table 2: Acceptable remaining impurities in vaccine products

Feed Quality Evaluation Criteria and Processing Parameters: Several parameters can be used to assess the clarity or quality of a product, either during process development or after each manufacturing process step. Turbidity is an easy parameter to monitor and provides an immediate assessment of feed quality. For example, it enables the detection of depth filter breakthrough.

The two primary methods of ascertaining the effectiveness of the clarification step are centrifugation and filtration. Turbidity can be monitored simply by absorbance/scatter in the visible range, providing an immediate assessment of particle load within the filtrate or centrate. Turbidity also relates to filter capacity, which is the volume of feed a depth filter can process before the pressure drop breaches specifications. Capacity relating to both pressure drop and turbidity breakthrough are linked, and specifications for both should be set during process development. For some processes (particularly those with a smaller average particle size in the feed), turbidity breakthrough is the limiting factor in sizing a filtration train. Often, capacity limit is attributed to pressure drop. But because high pressures or flow rates can cause premature turbidity breakthrough, both mechanisms can be related.

Technology Options for Clarification of Virus Vaccines: Because of the extreme diversity of viral vaccines in terms of size, structure, shape, and expression system, no unified template exists for their production and purification. Those processes can be divided into four different phases: upstream/production, clarification, purification, and formulation.

To reduce burden on downstream purification steps, the main objective of a clarification process is to remove undesirable materials, including whole cells, cell debris, colloids, and large aggregates. As the first downstream process, clarification should be optimized to maximize product yield and purity. Several serial operational steps often are required to achieve a desired level of clarification. The first operation (often referred to as primary clarification) removes larger particles, and the second (often referred to as secondary clarification) removes colloids and other submicron particles (Figure 1).

In theory, all available technologies (low-speed centrifugation, microfiltration TFF, NFF) can be selected and potentially combined to clarify viruses. As with other manufacturing process steps, a clarification process should have predictable scalability, be manufacturing-friendly (e.g., be easy to use, reduce holdup volume, provide operator safety), and have a low cost of goods (CoG). However, the clarification process of viral vaccines has two unique characteristics that can require more tailored solutions:

• low solids content with a high nucleic acid and colloid content, requiring higher retention capacity
• high feed variability and cell culture enhancements, requiring more robustness (2).

Although centrifugation can handle a high solids load and traditionally has been used in batch and continuous modes, it requires large capital investment and high maintenance costs. More important, centrifugation scale-up can be problematic because of unreliable scale-down models with nonlinear scalability and high-shear operation for shear-sensitive vaccines. But NFF and TFF have gained interest for vaccine clarification because they are significantly easier to scale-up and implement.

NFF: Primary clarification using NFF typically involves depth filters that often contain positively charged material and filter aids (e.g., diatomaceous earth) that improve retention of cell debris, colloids, and negatively charged contaminants. NFF relies on two main mechanisms for particle retention: size exclusion and adsorption.

NFF membrane filters can be used in secondary filtration because they retain particles by size exclusion. Certain grades of depth filters have a tighter pore-size distribution (which offers greater colloidal particle retention) but do not have high holding capacity. Noncharged depth filters also can be used for clarification while offering higher cost-effectiveness for small batches (≤1,000 L). They typically use three media types:

• melt-blown media fabricated in a pleated format to achieve higher flow rates and holding capacities
• graded density of concentrically wrapped media to allow the filter to remove finer contaminants progressively
• membranes to provide higher retention efficiency.

TFF membranes with retention ratings in the range of 0.1–0.65 μm have been used to retain cells, cell debris, and other large contaminants. Most TFF devices are linearly scalable and reusable after cleaning, and hence greatly reduce consumable costs. However, certain viruses (e.g., extracellularly produced enveloped virus-like particles) can be damaged by high-shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages in TFF processes. Open-channel TFF devices (cassette format without screen) are preferred to minimize shear.

Case Studies
Egg-Based Vaccines: In influenza vaccine processes, a typical allantoic fluid harvest is rich in proteins (e.g., ovalbumin, lysozyme, ovomucin) and contains 45 μg hemagglutinin antigen per egg (~3–4 μg HA and 10⁸–10⁹ infectious units/mL of allantoic fluid). The typical gravity-settled turbidity of a virus-containing allantoic fluid (VCAF) generally is 46–132 NTU. Low-speed zonal continuous centrifugation around 4,000–5,000g often is the preferred option to remove large particles and thus gets used for primary clarification, typically providing a recovery yield of 70% (3, 4). Many vaccine manufacturers use sucrose gradient zonal centrifugation to purify and concentrate viruses. However, polypropylene and cellulose-based depth filters also can be implemented for filtration. Fair capacities of 150–210 L/m² and up to 3× reduction of feed stream turbidity can be achieved with those allantoic fluid harvests. That option is appropriate for influenza vaccines, which are prone to adsorption loss during clarification on charged filters.

NFF also can be used for secondary clarification. Combinations of polypropylene, cellulose, and glass-fiber materials generally demonstrate good efficiency (5). Using a salt solution can reduce association between a virus vaccine and solid debris, resulting in a yield increase of about twofold without compromising viral particle integrity.

One study demonstrated that higher ionic strength on allantoic debris increased influenza virus yields (6). In that case, 1.5 M NaCl was applied to pooled allantoic fluids of various influenza strains, and the sample was centrifuged for different durations to understand the amount of virus partitioned in the supernatant and pellet. Control samples were kept at 0.15M NaCl. This test scope was expanded to include other influenza strains — A/New Caledonia (H1N1), A/Texas (H3N2), B/Jiangsu and B/Hong Kong — to demonstrate an average twofold yield increase. Further work verified the integrity of the purified virus in control and higher ionic strengths. Data showed that higher ionic strength did not adversely affect the live titer of the tested influenza virus strains (2). Specifically, studies have reported use of a 1.2-μm cellulose nitrate (CN) filter polypropylene media followed by 0.45-μm filter polyvinylidene fluoride (PVDF) membrane for clarification of cell-based influenza with a loading of 111 L/m² and 105 L/m², respectively (7).

Another option is use of TFF with a 0.65-μm or 0.45-μm microfiltration membrane device operated with permeate flux control (8). Using a “two-pump process” with a permeate pump in addition to the standard TFF feed pump allows for permeate flux control to manage/reduce polarization and fouling. That provides better characterization of the “critical” flux — the limiting flux above which a process becomes unstable (9).

Moreover, researchers conducted primary clarification of allantoic fluid using a 40-μm bag filter followed by an open-channel microfiltration device (Prostak 0.65 μm; data not shown). Using a crossflow of 3 LPM/channel, with transmembrane pressure (TMP) at 0.2 bar and dP at 0.4 bar, a capacity of 33 L/m² was achieved. The MF process was designed for 10× concentration and 5× diafiltration, and the Prostak allowed for 60× reuse.

Viruses in Adherent Cells on Microcarriers: Microcarriers can be used as a support matrix for the growth of adherent cells such as Vero cells. Primary clarification of these cells grown on microcarriers can be performed using a 75-μm stainless steel sieve to remove microcarriers from harvest. A Millistak+ C0HC depth filter medium then can be used for secondary clarification and directly followed by a sterilizing-grade filter (10). This study was reported for a cell density of 0.78 x 10⁶ cells/mL. Results showed varying filter capacity based on cell density or cell viability of harvest.

Viral Vectors: Trial results on lentiviruses from the supernatant of HEK293T cells show positive performance (data not shown). The negative charge of lentiviruses is known to be responsible for poor recoveries with positively charged depth filters. Therefore, despite the fact that no sign of plugging was observed using a Millistak+ C0HC depth filter, very low viral vector recovery was recorded. However, use of a 1.0/0.5 μm Polysep II filter led to both a high capacity and high 84% recovery. The Polygard CN filter, on the other hand, behaved quite well in terms of recovery (75%) but showed more signs of plugging. An extra 10–20% lower virus recovery should be considered for the following 0.45 μm or 0.22μm (sterile) filtration step.

Two other studies on lentivirus feed streams confirm the positive recovery performance using the Polygard CN filters (CN25, CN12, CN10, and CN06 with more than 80% recovery). Other studies report an interesting result with recoveries reaching up to 90% for the Millistak+ CE50 filter that does not contain inorganic filter aid (e.g., diatomaceous earth; data not shown).

Adenoviruses also can be prone to adsorption, but divergent results have been reported. In some cases, good adenovirus recovery is observed even when a positively charged depth filter medium containing diatomaceous earth such as the Millistak+ HC medium is used (11). If adenovirus is lost, Polygard and Clarigard filters can be used instead, but a secondary clarification might be needed to reduce turbidity (12).

Clarification Options
Different approaches are used to produce viral vaccines, making designing a typical template for their clarification difficult. Indeed, these products can be produced by different expression systems and possess a range of physicochemical properties. The clarification method selected should take those factors into account to ensure that yield and contaminants removal are sufficient.

NFF and microfiltration TFF technologies are increasingly preferred to centrifugation because of their more predictable scalability and robustness. Low CoG also can be achieved by tailoring the choice of media chemistry and porosity to gain high recovery and productivity with satisfactory impurity removal. Continuing innovation in this field by suppliers of purification and filtration systems will help vaccine manufacturers meet their current and future challenges on the road to developing novel and efficient therapies.

References
1 Nascimento IP, Leite LCC. Recombinant Vaccines and the Development of New Vaccine Strategies. Braz. J. Med. Biol. Res. 45(12) 2012: 1102–1111.

2 Besnard L, et al. Clarification of Vaccines: An Overview of Filter-Based Technology Trends and Best Practices. Biotechnol. Advances 34(1) 2016: 1–13.

3 Hendriks J, et al. An International Technology Platform for Influenza Vaccines. Vaccine 29 Suppl 1, 2011: A8–11.

4 Eichhorn U. Influenza Vaccine Composition. US Patent US7316813 B2, 2008.

5 Lampson GP, Machlowitz RA. Process for Producing Purified Concentrated Influenza Virus. US Patents US3547779 A, 1970.

6 Hughes K, et al. Yield Increases in Intact Influenza Vaccine Virus from Chicken Allantoic Fluid through isolation from Insoluble Allantoic Debris. Vaccine 25(22) 2007: 4456–4463.

7 Thompson M, Wee J, Nagpal A. Methods for Purification of Viruses. European Patent EP2334328 A4, 2012.

8 Lau SY, et al. Impact of Process Loading on Optimization and Scale-Up of TFF Microfiltration. BioProcess J. 13(2) 2014: 46–55; doi:10.12665/J132.Pattna.

9 Raghunath B, et al. Best Practices for Optimization and Scale-Up of Microfiltration TFF Processes. Bioprocess. J. 11(1) 2012: 30–40.

10 Thomassen YE, et al. Scale-Down of the Inactivated Polio Vaccine Production Process. Biotechnol. Bioeng. 110(5) 2013: 1354–1365.

11 Namatovu HH, et al. Evaluation of Filtration Products in the Production of Adenovirus Candidates Used in Vaccine Production: Overview and Case Study. BioProcess J. 5(3) 2006: 67–74.

12 Weggeman M, van Corven EJJM. Virus Purification Methods. US Patent: US8124106 B2, 2012.

Corresponding author Anissa Boumlic (anissa.boumlic@merckgroup.com) is associate director of the vaccine segment at Millipore S.A.S.

The post Filter-Based Clarification of Viral Vaccines and Vectors appeared first on BioProcess International.

Photo 1: Five-station tangential-flow filtration (TFF) screening system design agreed upon by the collaborators

Ultrafiltration and diafiltration (UF–DF) of therapeutic proteins are performed in either tangential or crossflow mode using membrane filters. UF–DF plays a critical role in both downstream and upstream processes for the biopharmaceutical industry (1). In upstream production processes, classical tangential-flow filtration (TFF) or alternating tangential-flow (ATF) systems are used in high–cell-density perfusion for protein expression by cell culture (2). TFF is used in downstream processing for UF–DF and concentration of therapeutic proteins. TFF unit operations are common in protein purification because of their scalability and amenability to continuous processing (3–5). Typical TFF process development involves optimization of transmembrane pressure (TMP), permeate flux, feed flowrate, buffer compositions, and the interdependence of all those parameters. Optimization requires a broadly defined design space and many experiments needing significant time and resource investments.

Table 1: Key objectives and system requirements for UF–DF; GUI = graphical user interface

With speed to first-in-human initiatives leading to shrinking timelines in early process development, the demand for enhanced throughput has increased significantly in recent years. Expedited timelines and minimal material availability in early phases complicate process development of TFF. For increased throughput on chromatography-based unit operations, options are available such as high-throughput chromatography and robotic screening tools such as TECAN systems. However, few such tools are available commercially for filtration unit operations. The few systems on the market require users to adapt to device requirements, leaving very little flexibility to work. It is important that such tools provide as smooth a flow as possible, with accurate information for scaling up TFF processes to intermediate (for further process development/verification) and then manufacturing scales (Table 1).

Early phase process development verifies initial platform fit assessment of a downstream process and helps companies determine whether downstream process modifications are needed. Early phases of process development often require rapid decisions made regarding choice of membrane types and UF–DF process conditions. Although the objectives for early phase development primarily focus on determining platform suitability of process conditions for a given molecule, late-stage UF–DF development focuses on more detailed process characterization and risk assessment. That includes improving process yield, throughput, and consistency as well as performance validation (reproducibility, sensitivity, and lot-to-lot variations) with a representative scale-down model. For a suitable scale-down model, it is critical to distinguish between system- and process-related variations in UF–DF. For process development or characterization, superior control of system parameters helps developers differentiate between system- and process-related responses.

Here we report the design and development of a fully automated TFF multistation consisting of five independently operated TFF stations with advanced system controls that can run in parallel for screening UF–DF process conditions (Photo 1).

Benefits of Laboratory-Scale Process Automation
A typical laboratory-scale TFF process is semiautomated with a few pumps, some dial pressure gauges, a scale, and manual valves. Integrating system parts from multiple suppliers poses a challenge. Data logging requires an operator to monitor and export process data manually to third-party software for analysis, often with the challenge of merging data from different sources that have uncoordinated time stamps. In a process that can have a run-time of several hours, varying a range of conditions with multiple runs can consume many personnel resources, especially if run manually. Therefore, automation is critical to enable process optimization while minimizing key personnel resources.

We believe that to create a more efficient development operation, we need a system with automated process steps that have defined endpoints, control of key process-independent variables throughout a process, calculation of key process values (e.g., ∆P, TMP, and flux), alarm settings that shut down the pump, data-logging and real-time trend viewing. By its nature, such an automated system would provide benefits of minimal user interaction. With automated data-logging, user focus can be placed on analysis rather than data collection.

Photo 2: Five-station TFF screening system’s graphical user interface (GUI) with process schematic and system control ability

Creating the TFF Screening System
In discovery and early phases of biologics process development, low expression titers typically limit availability of therapeutic protein. Bristol-Myers Squibb (BMS) wanted an automated TFF system for screening at these early phases when only small amounts of proteins are available. We believed it should have the ability to generate data from small-scale processes that could provide reliable information for scaling up. To address these challenges and add increased throughput capability for fast and efficient process development, BMS collaborated with PendoTECH (an industry supplier of downstream process development systems) to develop a multistation medium-throughput TFF screening system.

Table 2: Designed process capabilities

Here we report on development of a fully automated system with features as listed above. A parallel TFF screening system consisting of five independently operated stations was designed and built based on BMS requirements for typical early phase TFF process-development goals. The multistation parallel TFF system described herein can provide ≤70% reduction in material requirements, speed up design-space development for nonplatform molecules, and potentially reduce development times by 50–80% (Photo 2).

Table 3: Feed pump and permeate scale sensitivity

Defining User Requirements: Before establishing the requirements of a new TFF screening tool, we conducted a thorough market survey of available options and their capabilities and weighed their relative merits and limitations. Most systems did not support parallel screening and required too many manual interventions because they lacked high-level automation. To address the disadvantages of existing equipment, several requirements shaped development of the parallel TFF screening system.

Along with the process capabilities outlined in Table 2 and 3, the following characteristics were desired for this new laboratory-scale system to efficiently perform TFF process development experiments:

Photo 3: Ability to real-time trend all process values and quickly export trends

It should control five independent TFF trains, monitoring and recording process parameters from each one (Photo 3). It should enable “walk-away automation,” automatically stopping a TFF train when a set UF–DF goal is reached. We needed flexibility for processing volumes and high-precision diaphragm pumps to minimize shear stress on proteins. Operating pressure should extend up to 40 psi and flow rate range 1.0–100 mL/min. A simple graphical user interface (GUI) and user-friendly data retrieval and manipulation would facilitate operator efficiencies, as would real-time trending of process values. And the system would need a small footprint to minimize operational cost by making the most efficient use of laboratory bench space.

A User–Supplier Collaboration: BMS and PendoTECH had a history of about eight years working closely together in a customer–supplier relationship involving both new and existing products, so we had an established working relationship with key personnel in place. As required for a successful collaboration, we first established timelines and milestones, defining and aligning our strategic interests. Device development began with a feasibility evaluation to develop a system that must meet at minimum these key features: work with small volumes of product and use both existing and new smaller area filters in development; be fully automated and able to perform parallel processing in a small footprint; and include safety alarms and check parameters. All product development programs involve risks and unknowns. So we scheduled technical reviews and decision points throughout our program for project refinements and deciding next steps based on information gained.

Feasibility evaluations were performed to investigate key technical aspects of the project. One of those was whether PendoTECH’s microcontroller platform could handle the complex processing required to keep a TFF process running without errors at the speeds required. At the heart of many of the company’s electronic products is a microcontroller platform and associated expertise for programming complex tasks in the “C” language. Process-control systems often are defined partly by the number of inputs and outputs they can handle. For this system, each station has 11 inputs/outputs (I/Os), and a multistation multiplies that by five for 55, with two data ports to communicate with a GUI) running on a personal computer, thus yielding a total of 57 I/Os. The Freescale Semiconductor (now part of NXP Semiconductors) microcontroller at the heart of PendoTECH’s platform has been proven in many critical control applications such as factory monitoring systems and automobiles. Thus, selecting this control platform takes advantage of an I/O-rich architecture supported by a fast microcontroller processor, all in an industrial and compact form. A multitasking real-time operating system programmed in native “C” language provides the necessary flexibility to code complex and robust control algorithms for concurrently supporting five independent TFF processes. Even though the popular programmable logic controller (PLC) is used for many industrial control projects and has an advantage of nearly unlimited I/O capabilities, the microcontroller’s ideal features in factors of size, cost, and programming flexibility made it the superior choice for this project.

After feasibility testing of the microcontroller platform was successful, the project could move into its next phase. Microcontroller programming was completed and the control hardware configured. We tested a crude breadboard set-up with one station connected to observe its control performance — the other four stations had their processing running with no equipment interfaced. That testing was successful, so the formal user requirements could be defined (as listed in the “User-Requirement Specifications” box).

The project was budgeted and resources committed to finish system building, and it was decided for multiple reasons to limit the system to five stations at least initially. Further requirements were added to the detailed design:

• To meet the challenge of fitting in 3 ft² of bench space, a vertical design layout was required for each station.
• To eliminate the need for a diafiltration pump, gravity feed with a valve would be the ideal approach.
• Technical points included confirming the number and type of I/Os required, using a flow restrictor to control diafiltration feed without a pump, having two feed bags for fed batch operation, designing the GUI and load cells, liquid-level sensors, and interfacing with different vessels.

Performance of Key Components 
Programming and Software Development: After software and GUI development and shakedown/debugging were completed (Photo 2),system use could begin. PendoTECH implemented changes and/or corrections based on feedback provided by BMS after initial use.

Microcontroller: With fine-tuning of the final programming, the microcontroller was highly efficient for reading inputs, sending outputs, monitoring alarms, and controlling recipe steps.

Pumps: For high-pressure capability, low-flow design, and a low-pulsation pump stroke, the final pump selected for use with this system was KNF Neuberger’s SIMDOS10 diaphragm pump. Because only a console model was available, PendoTECH worked with KNF engineering staff to develop an original-equipment manufacturer (OEM) panel-mounted model for use with the system that would fit within the compact-form design constraint.

Diaphragm pumps have check valves that control pump-chamber inlet and outlet flow during their pump strokes. It is important that those check valves seal during each pump stroke because performance can degrade with a drop in flow rate from the actual set point. To prevent particulates or foreign matter from interfering with check-valve sealing, the pump inlet tubes are outfitted with a 35-μm filter that can be replaced periodically. Even though that is the standard pump option, a modular design with pump controls and power delivered to the pumps from the base of the system enables easy integration of other pumps (e.g., peristaltic) for lower pressure processes.

Throttle Valve: UF–DF typically operates with one of three control strategies: constant retentate pressure, constant transmembrane pressure, or constant filtrate flux. A throttle valve controls TMP and retentate pressure to a user-entered set point. This is a key independent variable for process design and replaces a manual pinch clamp.

The throttle valve enables automation. As process conditions change, such as increased product viscosity during concentration, the valve can adjust automatically to control TMP. This prevents process upsets or alarms and enables processes to run automatically at the higher concentrations desired for many modern final formulations.

Another key feature of this throttle valve is that it prevents users from struggling to set it manually in the correct position that will meet desired pressure set points. With a 0.125-in. (3.2-mm) inner diameter (size 16) targeted for use with this TFF system, the travel distance for a valve to affect flow is about 20% of the tube diameter (~0.64 mm). The valve has a very complex “stepper motor” that can move in very small increments and thus enable precise control to user-entered set points. The valve runs an autocalibration procedure when the system is turned on to reset the home position of its motor positioner.

Load Cells for Permeate Weight: Load-cell performance is critical for measurement of permeate weight, which is used to estimate flow and measure diafiltration volumes. A basic industrial load cell was outfitted to a compact platform that minimizes bench space and cost. This load cell is connected to the system and tared to zero as needed through the GUI. In addition, a simple calibration wizard can be executed by users at any time based on a 500-g weight. That ensures accuracy of data collected during the experiments that follow.

System Design, Hardware Expandability, and Future Enhancements: There is large convergence of technology on this TFF system: solenoid valves, ultrasonic air detectors, capacitance-level sensors, a Wheatstone bridge load cell, compact Luer pressure sensors, a miniature diaphragm pump, and a valve with stepper-motor control. One objective in the system design was to minimize the amount of external wiring required. With so many external components required to control and monitor TFF processes, either panel-mount receptacles or wires running through water-tight grommets would be placed as close as possible to their point of use to streamline the length and organization of external cabling. Future enhancements should include addition of a possible on-line means of conductivity measurement and a low-flow flowmeter (0.1–15 mL/min) to enable permeate recycling with flow measurement for flux excursions. Photo 1 shows the final hardware design and a representative real time trends of the UFDF process parameters.

Table 4: Comparing UF–DF system capabilities at different scales

Performance of the TFF Screening System
Over the past two years, many scientists and engineers in BMS’s early phase purification development group have used this system extensively. As complex as the system is, its GUI has been described as easy to use. Mechanically, the level sensors are easy to adjust on their slide bars to desired concentration points. During this time, the five-station TFF screening system has demonstrated robust reproducibility. For three different BMS molecules, we have conducted 70 UF–DF runs in fed-batch mode, 45 concentrations to 60–75 g/L, and 45 concentration/diafiltration studies over a period of 45–50 days without any technical errors or loss of information. All 160 runs yielded complete recovery of all process material. The system was cleaned in place and sanitized with 0.2-N sodium hydroxide after each run. UF–DF studies were performed with maximum inlet feed pressure of 25 psi and maximum feed flow rate of 480 L/m/h. Tubing and the inline 35-μm filter were replaced with each change of molecule, and the permeate scale calibration was confirmed every 10 runs. The throttle-valve’s home position sensor was calibrated while replacing the system tubing according to its auto-calibration procedure.

Photo 4: Intuitive GUI for process/pilot development system for scale-up

Scale-Up to Platform, Pilot, and Manufacturing
Table 4 compares typical UF–DF system capabilities. The multistation UF–DF screening system has shown excellent process scalability to intermediate and pilot scale. The UF–DF process parameters obtained from the multistation TFF system were scaled up successfully to a bench-scale operation with a membrane of 0.1–0.5 m². The larger scale TFF skid used a PSG Quattroflow Q150 diaphragm pump that can operate ≤3 L/min for bench-scale operation, and a larger model can be used for process operations ≤99 L/min. A PendoTECH TFF process-control system that has been on the market for years operates only one independent UF/DF process at a time (Figure 1, a cart version of this system; Photo 4, the system GUI). It offers the same automation and control features as the screening system while adding features such as on-line pH, conductivity, and temperature sensing; retentate flow-control capability; options for on-line UV monitoring; and pump and process-scale flexibility.

Figure 1: Process development system in cart for robust process development

With a new feature added to the TFF process control system, a robust design of experiments (DoE) can be conducted. To determine optimal flux and operational conditions, the GUI’s Process Excursion tab allows users to enter up to 10 different process conditions for feed flow and TMP. That can be set for up to four different concentrations, thus fully automating a matrix of 40 different process conditions. That allows for executing a range of UF–DF process conditions required to perform statistical DoE in a relatively short time. Each stage in Photo 5 would take place with the system in 100% recycle mode, with permeate redirected back to the retentate vessel and creating a roughly steady-state condition.

Photo 5: Process development system — excursions of ≤40 conditions in flow, transmembrane pressure (TMP), and concentration

A Pivotal Advancement
The multistation UF–DF tool developed through collaboration between BMS and PendoTECH is being used extensively by the BMS biologics process development team. The system has been pivotal particularly in accelerating early phase UF–DF process development and characterization activities. The capability to execute a completely automated statistical DoE study with minimal manual intervention is a first on the bioprocessing market (Photo 5). As new biopharmaceutical products work their way through the development pipeline, such novel process development tools will enable this industry to meet its goals of speed, flexibility, quality, and cost as laid out by the BioPhorum Operations Group (BPOG) in a number of publications (6–8).

Acknowledgments
We acknowledge Gregory Barker, Sibylle Herzer, and Matthew Conover of BMS for their contributions during this work. We also acknowledge John Benson (product design engineer) and Richard Holowczak (microcontroller programmer and integration expert) at PendoTECH for their technical contributions. This article is exclusively sponsored by PendoTECH.

References
1 Liu HF, et al. Recovery and Purification Process Development for Monoclonal Antibody Production. MAbs 2(5) 2010: 480–499.

2 Clincke M-F, et al. Very High Density of CHO Cells in Perfusion by ATF or TFF in WAVE Bioreactor, Part 1: Effect of the Cell Density on the Process. Biotechnol. Progr. 29(3) 2013: 754–767.

3 Pollock J, et al. Integrated Continuous Bioprocessing: Economic, Operational, and Environmental Feasibility for Clinical and Commercial Antibody Manufacture. Biotechnol. Progr. 33(4) 2017: 854–866.

4 Raghunath B, et al. Best Practices for Optimization and Scale-Up of Microfiltration TFF Processes. Bioprocessing J. 11(1) 2012: 30–40.

5 van Reis R, et al. Linear Scale Ultrafiltration. Biotechnol. Bioeng. 55(5) 2000: 737–746.

6 Ebner CG. Facilities of the Future Conference: Innovating the Future of Manufacturing. Facilities of the Future Conference: Innovating the Future of
Manufacturing. ISPE 20–22 February 2018, Bethesda, MD.

7 Jones S. BioPhorum Operations Group Technology Roadmapping, Part 4: Efficiency, Modularity, and Flexibility As Hallmarks for Future Key Technologies. BioProcess Int. 15(2) 2017: 14–19.

8 Aeby T, Munk M. Meeting the Demand for a New Generation of Flexible and Agile Manufacturing Facilities: An Engineering Challenge. BioProcess Int. 13(11) 2015: S16–S23.

Corresponding author Atul Bhangale is a scientist II, Yan Chen is an associate director, and Anurag Khetan is site director of biologics process development at Bristol Myers Squibb in Hopewell NJ; 1-609-818-7205; atul.bhangale@bms.com. James Furey is general manager of PendoTECH LLC.

The post A UF–DF Screening System for Bioprocess Development: Efficient and Cost-Effective Process Fit and Scale-Up to Manufacturing appeared first on BioProcess International.

Steady improvements in batch-fed cell culture have led to bottlenecks in downstream processing. Filter suppliers are working to improve available tools for purifying therapeutic proteins, to wring every possible efficiency out of those tools, and to make them operate together harmoniously. The combination of high titers and high-value products places a premium on preventing yield loss. Bioprocessors want to optimize filtration primarily for cost reasons.

In this eBook, author Angelo DePalma discusses financial aspects, clarification/harvest and virus filtration options, and innovation with experts from supplier companies, academic institutions, biopharmaceutical companies. They highlight how each stakeholder can work toward solving the case of upstream–downstream capacity mismatch.

Just fill out the form below to download the eBook now.

The post eBook: Making Filtration Work appeared first on BioProcess International.

Current biomanufacturing is driven to pursue continuous processing for cost reduction and increased productivity, especially for monoclonal antibody (MAb) production and manufacturing. Although many technologies are now available and have been implemented in biodevelopment, implementation for large-scale production is still in its infancy.

RENTSCHLER BIOPHARMA (WWW.RENTSCHLER-BIOPHARMA.COM)

In a lively roundtable discussion at the BPI West conference in Santa Clara, CA (11 March 2019), participants touched on a number of important issues still to be resolved and technologies that are still in need of implementation at large scale. Below, moderator Peter Satzer (senior scientist with the Austrian Center of Industrial Biotechnology, Vienna) summarizes key points raised in that session. Based on his highlights, BPI asked a number of industry representatives to comment on those points further, and their responses follow.

Requirements for Continuous Downstream Applications
by Peter Satzer

Minimizing Buffer and Hold Tanks: One critical parameter is integration of unit operations on manufacturing shop floors using minimum numbers of buffer tanks and hold tanks. The benefit of continuous manufacturing can be realized fully only if auxiliary equipment for buffer preparation and hold tanks are minimized between downstream operations. Although continuous buffer preparation and reduction of buffer hold tanks are being investigated (and also were the subjects of BPI West presentations), direct integration without the need for hold tanks between unit operations requires further exploration.

Chromatography Operations: Some unit operations such as flow-through chromatography and single-pass, tangential-flow filtration (SPTFF) can be integrated easily into a continuous downstream process scheme at any point because they offer constant inflow and outflow of material. Such operations can be called truly or fully continuous.

Other unit operations — especially bind–elute chromatography — are much harder to implement because they don’t provide truly continuous operation, but rather produce individual elution peaks with fluctuating product and impurity concentrations. Usually the unit operation following such a step cannot handle such changes in flow and concentration directly, requiring inclusion of surge vessels and hold tanks that interrupt the process. This makes continuous periodic countercurrent chromatography (PCC) a unit operation that is harder to implement than other operations such as flow-through chromatography and new, alternative purification methods, including continuous precipitation.

Viral Inactivation: Current implementation of continuous processing in the MAb world could make use of viral inactivation steps as surge vessels. Biomanufacturers could fill parallel vessels periodically and empty them to provide constant flow and product concentration for subsequent unit operations. This might not be possible for all antibody and nonantibody products, however. Alternative purification techniques such as continuous precipitation offer a constant inflow and outflow of material and prevent integration issues.

Residence-Time: Another important point to consider when thinking about continuous integrated unit operations is the residence-time distribution through all unit operations. Understanding of this concept is limited, but it is critical to ensure batch definition and ensuring process robustness. Although a narrow residence-time distribution is preferred for batch definition and to minimize product loss in case of contamination, a broad residence-time distribution smooths process irregularities and leads to a more robust process. The trade-off between those two approaches will need to be discussed in the future.

PCC Chromatography: PCC can be detrimental to our understanding of residence-time distribution because it can split it during an operation. A PCC operation will produce one or two residence-time distribution peaks depending on the state of the operation, and no easy model can be applied. If bind–elute polishing also is implemented using PCC, the resulting residence-time distribution can be split into up to four individual distributions traveling through the system, which complicates decisions about when to discard material. Currently, this issue is too poorly understood and modeled. It either is not addressed, is addressed with wide safety margins for discarding material, or is addressed by implementing a hybrid process that uses batch manufacturing after a PCC unit operation.

Alternative technologies to bind–elute chromatography such as flowthrough chromatography, precipitation, crystallization, and flocculation can offer solutions to those issues in the future to preventing integration problems. Developers of future technologies should aim for a constant mass flow through all unit operations, enabling integration and a seamless downstream process train.

The Roundtable Discussion

Based on the discussion highlights above, BPI invited further insights from several advisors, end users, and supplier companies. Included below are additional comments from Satzer along with responses from participants listed in the box at the left. BPI invites further comments and welcomes submitted manuscripts detailing how your organization approaches these issues.

Capacity Mismatch
We’ve heard recent discussion about a “capacity mismatch” between upstream and downstream aspects of biopharmaceutical drug-substance manufacturing. A couple decades of production improvements have created challenging process streams for separation and purification. Can continuous downstream processes offer a partial solution to this problem?

Satzer: Continuous downstream processes can offer solutions in two separate ways. The first way is to increase downstream equipment use by using equipment 24–7 and therefore increasing downstream productivity. The second way is through a change in mindset, in which up- and downstream operations are integrated directly rather than remaining distinct and separate — which forces them into direct communication. That opens the possibility of solving problems earlier (upstream), where solutions might be easier to achieve.

Holzer: Great improvements have been made in cell-specific productivity, media development, and process design during recent decades, resulting in some cases up to 100× increased product concentration in fermentation broth. The product output of the same upstream installation could be increased proportionally.

However, improvements in chromatography media capacity and membrane performance have not given the same range of productivity increase, instead doubling or tripling it. Therefore, work is needed on process design to overcome the downstream bottleneck. Related design studies easily show the great potential of continuous processing.

Monge: Recent advances in upstream processing have led to processes in which expression titers have increased beyond 10 g/L or even 20 g/L. Availability of concentrated fed-batch options on a pilot and commercial scale has resulted in DSP handling product amounts ranging from 5 kg to 40 kg per batch. On one hand, process intensification efforts have reduced overall footprint and time in the upstream train by eliminating intermediary seeding steps. But this has led to an increased footprint in downstream processing (DSP) that has required some biopharmaceutical companies to switch from single-use to hybrid/stainless steel technologies.

One way to address the challenge is to implement multicolumn chromatography at the capture step to ease the burden or to have continuous DSP with perfusion-based upstream processing (USP) — enabling biopharmaceutical companies to debottleneck this “capacity mismatch” because the amount of product to be processed is distributed daily. The approach also provides high flexibility in product output, reduces the overall DSP footprint, and enables closed processing and cleanroom declassification. With the same scale for clinical and commercial production, the technology transfer process is seamless.

Daumke: Different options can help solve the mismatch. One option would be a single-use centrifuge, and another would be filtration. In the latter case, alluvial filtration can be developed and built as a continuous skid working similarly to a chromatography system. When capacity is reached in the first filter, the system switches automatically to the next filter, and so on.

Faude: Continuous processing contributes to facing DSP challenges in two ways. First, it usually goes in hand with increased automation compared with conventional batch processing. For example, using more sophisticated chromatographic devices allows manufacturing to run efficiently over 24 hours. Second, the continuous processing mode enables higher resin use, resulting in lower buffer amounts needed for processing. Both can have significant impact on costs. Moreover, continuous processing can trigger optimization of simple process steps such as flow-through applications and should push forward the development of resins, membranes, and other technologies that can deliver fast mass transfer.

Buffer Preparation
Whereas continuous upstream processing uses more buffers and media, continuous downstream processing should reduce the number of buffers required for separation and purification operations. Does the end result balance out? And does it make more sense to buy and store premade, ready-to-use buffers for continuous processing — or to make them on demand based on powders and/or concentrates?

Satzer: The final goal has to be preparation of buffers and media based on powders or concentrates. For concentrated buffers (and reduction of different buffers used in the complete downstream stream), efforts have been made already and the first prototypes created (some presented at BPI West). Media tanks especially occupy a significant portion of a shop floor and either take up significant space or have to be refilled regularly (requiring personnel, analytics, maybe cleaning, and so on). Buffer preparation is an essential part of DSP, so development of continuous buffer preparation from powders is fundamental to a fully continuous process with minimal shop-space requirements.

Holzer: Perfusion or chemostat cell culture processing typically need more media while significantly improving plant productivity. Depending on the process design of unit operations in DSP, the amount of necessary buffers and solutions may be reducible.

For example, continuous downstream processing (cDSP) could be achieved by connecting staggered batch operations, but that would not reduce buffer consumption. Yet in the case of countercurrent multicolumn chromatography processes or single-pass TFF, typically more product can be processed with less buffer. It is important to analyze an individual product, plant, and strategy to come up with an adapted, balanced, and cost-effective processing platform.

Cost for buffer preparation, analysis, and storage are significant for recovery and purification steps and can become an operational bottleneck for some plants. That might be not seen during clinical phases, but it becomes evident during commercial production. Cost studies and risk assessments should support decision making. Results often bring about interest in working with concentrated solutions using in-line dilution systems. Currently, technologies that allow buffer or media preparations starting from powders are under development.

Monge: Performing chromatography in multicycle, sequential batches with smaller columns/adsorbers that require a high degree of saturation has led to a considerable decrease in downstream buffer demand. Choosing the right buffer-management strategy would require a company not only to evaluate the buffer demand on a unit operation and process level, but also to evaluate the impact of each proposed preparation and distribution concepts on mobility, adjacency, and room classification.

As far as continuous processing is concerned, choosing ready-made buffers connected at the point-of-use, along with in-line dilution and stream conditioning for steps with low buffer-demand could be an option. So could choosing a buffer-on-demand system with powder/concentrate that eventually will be formulated into buffers and released in real time for steps with moderate to high buffer demand.

Adapting that approach would mean that buffer needs/demands in DSP could be addressed continuously by a modular, intelligent buffer skid that is adjacent (e.g., in close vicinity) to the DSP skid but in a different room. That would eliminate the need for high-volume single-use mixers for buffer preparation and significantly reduce the area required for buffer preparation and distribution.

Faude: Buffer reduction in continuous downstream processing seems unable to adjust to increased media amounts used in USP. Using buffer concentrates is a very interesting possibility for reducing downstream buffer volume substantially, especially when DSP occurs in facilities that are not designed for continuous processing.

Filtration
Continuous filtration can involve problems related to low flux. Does this — or do other challenges — limit its potential in downstream processing? What kinds of technological solutions are needed?

Faude: Prepared backup filters placed in parallel might solve the problem. Switching to a parallel filter could be automatic depending on the flux course. Doing so might make filtration a simpler and better-controllable process step compared with continuous column chromatography.

Satzer: Filtration can be made continuous in two different ways: by exchanging filters or by using membranes in TFF mode (either single pass or not) with regular cleaning and regeneration or. In the end, all filters tend to foul over time in a continuous process and will cease to function at some point. The exchange of filters and continuous filtration can be used (and must be used in the case of dead-end filtration) and has been demonstrated by our group and others for viral filtration. In my opinion, the technology exists and can be used, but large-scale implementation is still missing.

Holzer: Many different filtration principles (e.g., micro-, ultra-, and nanodepth filtration and membrane adsorption) are applied during downstream processing. Most filters are used in a frontal-filtration mode batch process, in which concentrations change over time and reach the maximal limit of filtration capacity. Bioburden reduction, depth, or sterile filters are examples.

Redesign of those filters and unit operations into a tangential continuous process mode often is not of any process and economical interest. Therefore, the most efficient way is to work with staggered-batch or parallel-batch configurations. The flux is proportional to the membrane surface area or the number of filters used.

TFF processes allowing for continuously run unit operations are applied typically to cell separation, buffer exchange, and product concentration. Because the flow of a continuously run operation usually is lower than in a batch operation, technology is available or can be designed properly (for example, in the case of single-pass TFF).

Reducing the number of unit operations during downstream processing — such as by buffer exchanges or concentration between steps — is a great help for reducing cost and improving productivity. This is facilitated by the availability of chromatography media that can accommodate higher salt concentrations and flow rates with more targeted buffer choices.

The integration of viral filtration steps into continuously run DSP lines continues to be challenging. Process models and software could help us further characterize this unit operation, capitalize on historical data, and assist in process design and control. Use of process analytical technologies (PAT) to reveal rapid information about filter integrity or the amount of filtered product are of utmost importance as well.

Monge: Continuous processing created an additional driver to understand further the technical and operational aspects of establishing filtration steps toward manufacturing implementation. The worst-case criteria for batch-mode filtration do not translate to newer process scenarios. Besides a lower volumetric flow and prolonged exposure, significant challenges that need addressing include filter installation, integrity testing, and the impact of process variations and physicochemical feedstock variations.

The challenges mostly involve virus filtration because the number of other filtration steps is decreased in an integrated processing approach. Small, smart surge tanks are one way to reduce many concerns. They can serve to bridge downtime and compensate for process perturbations from other unit operations.

Daumke: In my opinion, the whole liquid flow needs to be optimized to reach the flow needed. Clarification the use of (for example) depth or alluvial filtration requires a certain flow to become effective. Therefore, other options need to be investigated (e.g., continuous centrifuges or TFF).

Chromatography
Continuous bind–elute chromatography involves concerns over residence time and loading capacities. Do you see this as a chance for more flow-through operations to succeed? What about alternative purification technologies such as precipitation/flocculation?

Satzer: Bind–elute chromatography cannot be run in fully continuous mode. Flow-through operations would be preferred, but in many cases they cannot offer the same purification efficiency as bind–elute chromatography. We demonstrated that precipitation/flocculation can yield similar purities to affinity chromatography in bind–elute mode while offering a constant mass flow. So I believe that those technologies (along with flow-through chromatography for polishing) will solve the issues of bind–elute chromatography.

That said, I think that bind–elute chromatography is here to stay. There might be separation and purification tasks for which no suitable precipitation technology exists, and the biopharmaceutical world is conservative when integrating new methods. It will tend to implement more familiar methods (such as PCC), at least in the near future.

Holzer: Control of residence time and loading capacity is important for batch and continuous (countercurrent multicolumn chromatography) bind–elute as well as for flow-through chromatography (where impurities are bound and can break through if residence times and process conditions are not controlled). Such applications require tighter control and more process understanding for continuous bind–elute chromatography because robustness is reduced.

Flocculation and precipitation steps for impurities and target products are applied in several industrial production processes (e.g., impurity precipitation/ flocculation mainly for products expressed in microorganisms and product precipitation in Cohn plasma fractionation). Based on the performance of protein A capture steps for antibodies, it might be difficult in this case to compete with bind–elute chromatography. Such alternative technologies need to be evaluated case by case, taking into account precipitation behavior, solubilization conditions, processing times, product quality, yields, and so on.

Monge: Bind–elute chromatographic operations are by nature discontinuous processes and can become a bottleneck. Other techniques such as multicolumn and simulated moving-bed chromatography can bring resin-based systems closer to a continuous mode. However, the approach requires sophisticated hardware systems with tedious automation and validation needs.

On the other hand, membrane-based flowthrough polishing provides a cost-effective and highly productive alternative to resin-based bind–elute chromatography. Many experts anticipate that eventually all polishing flowthrough steps could be membraneadsorber–based. Current concerns about larger volumes could be addressed with new process schemes. Given that chromatography steps comprise most of the process bottlenecks and complexity in DSP, alternative approaches such as precipitation or flocculation that increase product purity without compromising product quality/activity before chromatographic steps will help streamline the process.

Faude: Independent and continuous processing flowthrough operations are used wherever possible to shorten process times and achieve more economic separations. Even for chromatographies that traditionally were applied in bind–elute mode for MAb purification (e.g., cation exchange), suppliers are beginning to provide new resins designed especially for flowthrough applications.

Alternative technologies are under consideration, but platform processes are still in development. Technologies that can be used more generically will attract greater interest. An example is the recent trend toward considering flocculation technologies to optimize harvest-filter capacity and precipitation for optimizing impurity reduction.

Virus Safety
Low-pH and detergent treatments are difficult to automate, which is necessary for continuous processing. Viral safety is not a single unit operation, but rather is expressed as the cumulative effects of many operations. Does continuous processing bring new challenges to achieving such results? And which viral safety operations will be most difficult to adapt to a continuous approach?

Faude: Low-pH treatment especially needs equipment and technology for appropriate process control. Detergent treatment can be implemented easily from a technical point of view, but detergent removal, potential side reactions with the detergents, and the potential presence of trace impurities increase development and analytical costs. Additional challenges in virus clearance studies for continuous processes are to acquire representative starting materials and addressing ramp-up and shut-down phases as well as transient operation ranges of steady-state unit operations.

Satzer: The first parameter to assess when moving to continuous operation is a new definition for exposure time. Typically in batch processes this is defined as a certain time period, but for continuous operations there is a residence-time distribution (meaning that some molecules stay longer, some not as long in the low-pH environment). It has to be ensured that even molecules with shorter residence times will have adequate viral inactivation. This issue can be prevented by implementing larger safety margins or by using hybrid processes in which viral inactivation remains a batch operation.

Another approach is to redefine the viral inactivation step itself. Historically, this is set (for antibodies) to be around one hour. New research shows that a few minutes are sufficient for total viral inactivation. So in comparison with the current approach, there already is a very large safety margin. I think the greater challenges in viral clearance has to do with filtration, because filters have to be exchanged, and exchanges have to be validated. In general, technologies for implementation of continuous viral safety have been shown at laboratory scale, so I think implementation is rather straightforward for this step.

Holzer: New technologies such the BioSC pilot system (Novasep) allow for perfect control of unit operations such as low pH- or detergent-based viral inactivation, definition of different unit operations (such as staggered-batch, parallel-batch, or continuous multicolumn chromatography), integration of several different unit operations to achieve cDSP, and the possibility for different process scheduling of simultaneously run unit operations. Control and integration of viral filtration into cDSP needs more development.

Monge: Virus inactivation (VI) is considered one of the two orthogonal steps required for virus safety in a biotherapeutic production process. Making the process automated and continuous poses several challenges. Various industry groups have adopted different approaches — including a tubular plug-flow reactor, a continuous stirred-tank reactor, and column-based reactor — and successfully demonstrated the implementation.

The challenge currently is a lack of standardized and validated viral inactivation strategy at scale or in a proven scale-down model. With continuous processing, the significant problem is dividing an integrated operation into discrete unit operations for virus clearance testing. Virus filtration is a robust orthogonal method in downstream applications that is amenable for use in a continuous process. Issues that need addressing here are process variation/ perturbations and physicochemical feedstock changes. However, in both cases, validation-scale challenges are real and need to be understood before implementing a full-scale continuous process.

Residence Time
Can you describe the distribution of residence time across a downstream process and how it affects products and the bottom line? Is this a problem for continuous operations, or is there a way to address it?

Satzer: Residence-time distribution through complete processes is poorly understood. Research has been limited because batch processing does not have or need a description of residence-time distributions. This question is unique to continuous processing and therefore relatively new in the biopharmaceutical world. Models are available for some unit operations and for some molecules, but to fully understand all residence-time distributions of all unit operations and impurities and products, we still need a lot of research. To assess the question for product quality, we have to define what components to track, because not all have the same residence-time distribution. For instance, aggregates tend to elute at the front and back of a protein-A elution peak, creating different residence-time distributions for the product and for the impurities. Both have to be known before you can make any decision on product quality at the end of your process.

Only the generation of true “digital twins” with process models for all involved molecular species (such as product, impurities) can solve this completely. But discussion has only just started on what parameters have to be tracked and what might be omitted from those models.

Monge: Processing time ranges need to be characterized for unit operations and integrated downstream processes to ensure product stability and control of impurities. Based on the criticality of product residence time at a specific unit operation, additional studies might be necessary to define control strategies (e.g., tight control of pH and time for low-pH virus inactivation). The characterization of residence-time distribution becomes even more important in cDSP because it serves also as information for material traceability and helps during impact analyses in case of investigations.

Timing/Transition
It’s a general belief that the transition from batch to continuous processing should be made as early as possible in product development. If you consider the challenges that come with transitioning too early and too late, where does the “sweet spot” typically come between them?

Satzer: At the moment we face some issues in process development. In my opinion, the switch starts with the cell line. Current cell lines are adapted for fed-batch high production, so they might not be at peak performance for use in perfusion cultures. We experience quite drastic changes in impurity patterns and therefore downstream development when switching from batch-produced material to perfusion material.

I think the earlier that transition is made, the better, and one limiting factor (availability of small-scale equipment for running perfusion) has been lifted recently with combining the ambr system (from Sartorius Stedim Biotech) with automated screening of chromatography performance using Robocolumns technology. Additionally, this might depend a great deal on the product produced.

For antibodies, we know that protein A will perform, regardless of what material you use, and minimal adjustments are necessary when the feedstock changes. But for products that do not have a high-performance affinity capture option, providing material that adequately resembles that produced in pilot/large-scale DSP as early as possible is crucial for process development.

Holzer: Typically, process design of countercurrent chromatography or membrane steps, plug–flow reactions, in-line dilution (ILD), and so on need additional developments and dimensioning studies. Therefore, this development stage would be perfect for implementation. The results of these studies allow also for specifying processing equipment requirements. However, changes that mainly concern process scheduling and not development of the unit operation to achieve cDSP could be implemented later in product/ process development. In some cases, facility-fit challenges might be addressed with implementation of in-line buffer dilution or continuous multicolumn chromatography for one single unit operation even for commercialized products.

Faude: The development of continuous processing seems to be more time consuming unless you have a robust platform for some types of molecules. But that could conflict with the importance of time to [reach] toxicology testing and time-to-market considerations in early phases. The transition to continuous processing might be during late-stage development with a stronger focus on robustness and economical aspects.

Monge: Speed to clinic and market are significant drivers for biopharmaceutical companies. In this context, it would be better first to develop a batch-based platform process and manufacture product for clinical, process validation, preapproval/registration, and commercial scale. That would enable companies to enter the market and cater to its needs quickly without having to worry about uncertainties that continuous processing could bring from quality, manufacturing, automation, and regulatory perspectives. A parallel work stream needs to be in place where the same product is developed using a continuous process along with evaluation of the transition from batch to continuous manufacturing and identifying a bridging strategy. Once that is completed, then a postapproval change to a continuous process can be made by filing a prior approval supplement (PAS) as outlined by the US Food and Drug Administration (FDA) in its latest draft guidance covering quality considerations for continuous manufacturing.

S. Anne Montgomery is cofounder and editor in chief of BioProcess International. Peter Satzer, PhD, is a senior scientist with the Austrian Center of Industrial Biotechnology (ACIB), Muthgasse 18, A-1190 Vienna AUT; +43 1 47654 79114; petersatzer@ acib.at; and lecturer at the University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna AUT, email peter.satzer@boku.ac.at.

The post Making Downstream Processing Continuous and Robust: A Virtual Roundtable appeared first on BioProcess International.

Antibody production platform processes have been widely adopted in biomanufacturing, but many unit operations are not suitable for integration and automation. Here we describe the work of integrating unit operations by transforming a column operation to a more robust cassette format. We have selected a biomolecule-friendly buffer (phosphate) to eliminate, or delay, the performance of a circulating tangential flow ultrafiltration/diafiltration (UF/DF) operation, so the harvest-to-purified-bulk process can be integrated, resulting in a single, direct-flow operation, that reduces the batch process time from about two weeks to six hours.

Figure 1: Antibody production platform

Antibody production platform processes were established in the late 1990s. In the past 20 years, biomanufacturers have been using engineered Chinese hamster ovary (CHO) and NS0 cells to produce antibodies, bispecific biomolecules, and other fusion proteins. Figure 1 shows the current platform process.

Such processes are associated with the following problems:

• They are labor intensive because they typically comprise many unit operations from harvest to formulation.
• They are inefficient because each batch requires about two weeks to process from harvest to formulated bulk drug substance.
• Chromatography unit operations are not robust because a simple introduction of air to a column can compromise the integrity of the packed resin bed and risk loss of a multimillion-dollar batch.

To overcome these challenges, we conducted studies to integrate and consolidate the current platform process from many independent unit operations to one. Doing so significantly reduced the processing time and enables future automated continuous processing without operators on the manufacturing floors.

Table 1: MabSelect SuRe LX protein A process steps and buffers

Material and Methods
Materials: The antibody starting material is an IgG1 produced by a Chinese hamster ovary (CHO) cell culture fed-batch process. Affinity chromatography is operated using MabSelect SuRe LX resin from GE Healthcare and operated using a peristaltic pump or ÄKTA chromatography workstation, also from GE Healthcare. Buffers were prepared in OED CMC Biologics Bioprocessing group at AbbVie. The depth filters were from Millipore, the Mustang Q anionexchange membrane filter was from Pall, the Virosart Max 0.1 μm prefilter was from Sartorius Stedim Biotech, and the Viresolve Pro virus filter was from Millipore. JSR Life Sciences and SPF Technologies provided the Chromassette chromatography platform.

Table 2: Buffer reduction from 15 to three stock solutions

Methods: Size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC) was used to assess the level of aggregates, and the NanoDrop2000 system from Thermo Fisher Scientific was used to assay IgG concentration by absorbance at 280 nm. The QuantiChrom phosphate assay kit (DIPI-500) from BioAssay Systems was used to assay phosphate concentration, and an enzyme-linked immunosorbent assay (ELISA) to assay host-cell protein (HCP) content.

Results
Buffer Simplification: To integrate the process, the buffers used in the platform had to simplified and reduced. Our current platform uses ~15 buffers consisting of Tris, acetate, and phosphate buffers as well as NaOH and HCl. The following experiments demonstrated that Tris and acetate buffers can be eliminated.

Figure 2: Product profile of flowthrough purification before ultrafiltration and diafiltration

Table 1 shows that the protein A chromatography methods and the transition from Tris and acetate buffers to the phosphate buffer scheme. The typical platform elution was ~2 CV at 20–30 g/L. 10 mM phosphoric elution buffer eluted a broader peak leading to larger eluate volume. If the equilibration wash is lowered to pH 6.0, the elution volume can be reduced to ~2.5 CV using the 10 mM phosphoric acid (H₃PO₄) eluent.

The protein A eluate pool eluted using 10 mM H₃PO₄ was titrated to pH 3.5 using 0.2 N hydrochloric acid (HCl) and held for 30 minutes for viral inactivation (VI), then titrated to pH 6.8 using 0.1 N sodium hydroxide (NaOH). The neutralized pool was then processed through the remainder of the purification process (depth filter + Q anion-exchange filter + virus filtration) using 10 mM phosphate buffer at pH 6.8 as a single direct-flow process step. The flow through was collected in 14 fractions.

Figure 3: Increase use of protein A capacity

Results in Figure 2 show that the middle fractions reached concentrations of 19-20 mg/mL. Aggregates and HCP were very low. More important, the mean phosphate concentration was 10 mM. Therefore, if the formulation is in 10 mM phosphate at pH 6.8, the final UF/DF unit operations can be eliminated, and the total number of buffers can decrease by half or even more with three stock solutions of sodium chloride (NaCl), NaOH, and H₃PO₄ (Table 2).

Figure 4: Higher loading capacity with two columns

Multicolumn Protein A Chromatography: To increase the final purified bulk concentration, without the UF/DF step at the end of the process, it is necessary to increase protein concentration in protein A eluate pool. This can be achieved by loading the protein A column to a much higher mass capacity. Figure 3 shows that loading too much protein will have significant product breakthrough and reduce the step yield. That problem can be prevented by using two columns in series.

Figure 5: Scaffold support and HETP tests (HETP = height equivalent to the theoretical plate)

Figure 4 shows that the first column can be loaded to >80 g/L on MabSelect Sure LX resin at a 1.5-min residence time. This can reduce resin usage by 38% and increase the protein A eluate concentration. Newer protein A resins, such as the PrismA resin from GE Healthcare and Amsphere A3 resin from JSR Life Sciences, can further increase protein A eluate concentration.

Column to Cassette Transformation: To overcome process robustness issue of a column operation, the new Chromassette platform was introduced to replace the column. This platform has an integrated scaffold support every ~700 μm that prevents bead compression even at high flow rates and restricts the movement of packed resin bed. In this way, air cannot break up a packed bed and create channels. Figure 5 shows that air intentionally introduced into a Chromassette platform packed with MabSelect SuRe LX resin had virtually no impact on the height equivalent to the theoretical plate (HETP) and peak asymmetry results.

Figure 6: Comparing protein A column with Chromassette unit shows nearly identical results.

Figure 6 shows test results comparing protein A chromatography performances using a traditional column and the Chromassette system. The chromatograms and process performances are nearly identical.

Figure 7: Platform process can be simplified

Integrated Process Concept: With the introduction of the Chromassette platform to replace columns and the development of a protein A membrane, the current platform process from harvest to purified bulk can be converted to a cassette format. A new concept is proposed that eight independent unit operations in Figure 7 are integrated into a one-unit operation as shown in Figure 8. This results in a uniform integrated recovery and purification (IRP) system. The IRP platform flows in series through the process train starting with harvest depth filters followed by a protein A Chromassette system, a virus inactivation chamber, postvirus inactivation depth filters, Q filter, a polishing Chromassette unit, and finally, the virus filter. The purified bulk can then be filtered through a 0.2 μm filter into a bag for storage.

Figure 8: Concept of integrated bioprocess

Figures 9 and 10 show a proof of concept of the IRP platform. In Figure 9, an ambr250 bioreactor (from Sartorius Stedim Biotech) harvest of 250 mL was processed through two depth filters, 0.2-m filter, and a protein A Chromassette unit. After virus inactivation, material was further processed through a depth filter, 0.2‑μm Q filter, 0.1-μm filter, 20-nm virus filter, and finally another 0.2-μm filter into a bag of purified bulk drug substance.

Figure 9: Proof of concept for integrated
bioprocess

Figure 10 shows chromatograms of the harvest and protein A as well as the subsequent purification steps following virus inactivation. The gradual increase observed in the UV trace is not a result of product breakthrough, but rather a result of buffer dilution from depth filters. The purified bulk produced was 20 mg/mL concentration at pH 5.9 and contained 0.6% aggregate. More important, the entire IRP process was performed in six hours instead of the two weeks needed to complete the current manufacturing platform process.

Figure 10: Results of integrated bioprocess (Agg% = percent of aggregates)

Future Bioprocessing Without Operators
Because the IRP platform is using robust filters and the Chromassette platform, and replacing disposable filter units is simple, robots could be programmed for future bioprocesses (Figure 11). Rather than on the manufacturing floor, operators could be in control rooms monitoring operation. The paradigm shift reduces the chance of contamination and operator errors introduced by human operators. In addition, because an IRP platform is compact, it can be housed in a small space, and the environment can be kept clean to prevent contamination. That could reduce the manufacturing footprint by at least 80% and shorten the time and expense for facility construction. Ultimately, such new systems have the potential to reduce manufacturing costs and ensure product safety.

Figure 11: Future bioprocess without operators

Advantages of an Integrated Bioprocess
The number of monoclonal antibody platform purification process buffers can be reduced significantly by using only phosphate buffers. With the introduction of the Chromassette platform to replace column chromatography, the unit operation is more robust. In addition, after transitioning a column into cassette format, the entire process from harvest to purified bulk can be integrated into one unit operation and completed in six hours. In the future, such integrated bioprocesses may be operated continuously at large scale by robots, producing approximately one ton of antibody per year within a small manufacturing footprint and with few required equipment systems.

Acknowledgments
Many scientists in Bioprocess Development and Analytical Development from the AbbVie OED CMC Biologics teams contributed to this article. We acknowledge Richard Wu for performing a number of downstream experiments and Dan Rush for many analytical assays. Thanks to Barry Wolf and Nikki Nguyen and others in BioProcess group for producing the cell culture harvest.

Corresponding author Ping Y. Huang is the director and head of bioprocess development. Yekaterina Lin is a senior scientist of bioprocess purification development. Robert J. Duffy is a principal scientist of bioprocess purification development. Amit Varma is senior director and head of CMC biologics, at CMC Biologics, Oncology Early Development, at AbbVie; ping.huang@abbvie.com. This article is adapted from the authors’ poster at the 2019 BPI West conference.

The post An Integrated Bioprocess for Antibodies: From Harvest to Purified Bulk in Six Hours appeared first on BioProcess International.

Filter integrity is a fundamental element of sterility assurance during production of biopharmaceutical and vaccine products. Integrity test results are a key foundation for drug lot release, so any external element that could affect their reliability must be viewed as a critical issue. But when should a filter integrity test be performed? This article highlights the Sartocheck 5 Plus filter integrity tester as a means to address regulatory requirements. Please fill out the form below to read the full article…

The post Addressing Regulatory Requirements for Filter Integrity Testing appeared first on BioProcess International.

Viral filtration (VF) using nanofilters removes endogenous and/or adventitious viruses from biologic drug-substance manufacturing processes (1). The gold particle test (GPT) is performed as part of postuse integrity testing — to complement postuse leakage testing — for cellulose filters such as Planova 20N filters from Asahi Kasei Corporation. First, a proprietary gold-colloid solution matched to the filter type (e.g., 20N) is filtered through the test article. That filter’s pore-size distribution can be assessed using spectrophotometric absorbance readings of the integrity-test…

The post Viral Nanofilter Integrity: Using Variable-Pathlength UV-Vis Spectroscopy for the Gold Nanoparticle Test appeared first on BioProcess International.

The production of increasingly higher cell densities has stressed the already limited solids-handling capabilities for traditional intermittent ejection centrifuge systems. By contrast, a single-use disc-stack centrifuge based on the solids-flow principle offers distinct advantages for cell culture harvesting. Such benefits include solids handling of high-density cell culture processes and elimination of the separation disruption and aerosol generation associated with the intermittent solids ejection. A single-use system also provides well-established benefits of disposable components — such as removal of steam- and…

The post Development of a Single-Use Hermetic Centrifuge System for Mammalian Harvest with Moderate to High Cell Content appeared first on BioProcess International.