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1 SUPPLIER Side Advanced Viral Clearance Study Design A Total Viral Challenge Approach to Virus Filtration Michael Burnham, Alexander Schwartz, Esha Vyas, Nanna Takahashi, Pauline Nemitz, Daniel Strauss, Naokatsu Hirotomi, and Joseph Hughes 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 Product Focus: Biologicals Process Focus: Downstream processing Who Should Read: Process development, analytical, and manufacturing Keywords: Model viruses, virus filters, nanofiltration, plasma products, recombinant proteins, large-volume testing Level: Intermediate 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 Process Step X-MuLV (retrovirus) (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 virusretentive filtration. Multiple approaches Anticipated Virus Logarithmic Reduction Value (LRV) Traditional Approach Total Viral Challenge Approach Low-pH hold >4 to >5 log 10 >6 to >7 log 10 Virus-removal filter >4 to >5 log 10 >6 to >7 log 10 Anion-exchange column 2 to >4 log 10 2 to >6 log 10 Capture column 1 to 4 log 10 1 to 4 log 10 * MVM (parvovirus) Virus-removal filter 4 to >5 log 10 4 to >6 log 10 Anion-exchange column 2 to >4 log 10 2 to >6 log 10 Other (capture column) 1 to 4 log 10 1 to 4 log 10 * * may be unnecessary 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 wellcharacterized 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 difficultto-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 REPRINT WITH PERMISSION ONLY 52 BioProcess International 16(3) March 2018 Advertorial

2 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 largevolume testing over the traditional percentage-spiking method without largevolume testing (Table 1). In our virusremoval filtration studies, we used protein solutions spiked with ultrapurified minute virus of mice (MVM, nm) or xenotropic murine leukemia virus (X-MuLV, 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 Contract Manufacturing Excellence Comprehensive biologics and pharmaceuticals service offering, with multi-functional and experienced project teams nurturing customers products from pre-clinical through to clinical and commercial manufacture within three GMP approved facilities. Visit our website

3 DENARASE Highly active endonuclease from Serratia marcenscens Ask us for A free sample Full cgmp compliance Animal product free production Endotoxin free production strain High purity & activity Cleaves all forms of DNA and RNA The patent protected production process is based on the recombinant expression in a Bacillus sp., combining high product yields with the advantages of an endotoxin-free production strain. DENARASE has a high purity of > 99 % and is manufactured in full compliance with cgmp requirements. DENARASE is widely used in biopharmaceutical manufacturing to quantitatively eliminate host cell DNA and RNA during the production of biologicals and vaccines, as well as for viscosity reduction in biotechnology applications. DENARASE breaks DNA and RNA chains into fragments of 2 5 bases, making it an efficient DNA removal technology. DNA can bind to target products such as monoclonal antibodies and viruses. Blocking of functional groups needed for affinity may result in lower recovery rates after chromatography steps. DENARASE reduces the DNA content at an early stage of the process, thus increasing product yields. High DNA concentrations cause high viscosities, rendering filtration and chromatography difficult. DENARASE helps to reduce viscosity and to improve process economics. DENARASE can prevent cell clumping in bioreactors. The protein consists of two subunits with a calculated molecular weight of 27 kda each. DENARASE unspecifically cleaves all forms of DNA and RNA very efficiently. It hydrolyses phosphodiester bonds between nucleotides (single-stranded, double stranded, linear, and circular, sequence-independent) leaving short fragments with a length of 2-5 bases with a 5 -monophosphate end. DENARASE is active and stable in all tested commonly used buffers, even in the presence of ionic detergents. c-lecta GmbH Perlickstrasse 5 D Leipzig, Germany DENARASE@c-LEcta.com c-lecta GmbH is a German biotechnology company specializing in the development of customized enzymes and production strains for industrial applications. Patented technologies are employed for discovery and optimization of tailor-made enzymes and microbial strains. c-lecta s know-how in microbiology and strain engineering allows efficient development of production strains for protein and small-molecule products. Proprietary technologies are used for the identification of new enzymes from biodiversity and for effective enzyme engineering.

4 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 log 10 or more (16, 17). Modern parvovirus preparations are roughly log 10 (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 log 10 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. For all run conditions, we applied the feed material in the same manner to each filter and collected filtrate in two 100-L/m 2 fractions followed by a 10-minute complete system depressurization, then collected a single 15-L/m 2 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. 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 Equation 1 Virus LRV = log 10 Starting material virus load Filtrate virus load Figure 1: Flux curves for Planova 20N filtrations at high pressure (14 psi) Flux (L/m/h) MVM Run 1 MVM Run 2 30 X-MuLV Run 1 Mock Run Throughput (L/m 2 ) Table 2: Viral clearance data for minute virus of mice (MVM) sorted by total viral challenge Study ID Total MVM Challenge (PFU per filter) MVM Comment A1 9.2 log LRV Inconsistent results A2 9.3 log LRV B1 9.2 log LRV B2 9.1 log LRV C1 9.4 log LRV Nonrobust clearance C2 9.5 log LRV D1 9.0 log LRV D2 8.9 log LRV E1 7.5 log * LRV Consistent, robust viral clearance F * * Different pool volumes were used in tests, so both runs showed complete clearance but had different MVM log reduction values. Figure 2: Flux curves for Planova 20N filtrations at low pressure (10 psi) Flux (L/m/h) MVM Run 1 MVM Run 2 X-MuLV Run 1 Mock Run Throughput (L/m 2 ) Figure 3: Flux curves for Planova BioEX filtrations at high pressure (45 psi) Flux (L/m/h) MVM Run 1 50 MVM Run 2 X-MuLV Run 1 25 Mock Run Throughput (L/m 2 ) Figure 4: Flux curves for Planova BioEX filtrations at low pressure (30 psi) 200 MVM Run MVM Run 2 X-MuLV Run Mock Run Throughput (L/m 2 ) Flux (L/m/h) 54 BioProcess International 16(3) March 2018 Advertorial

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6 Table 3: Viral clearance data resulting from filtrations spiked with minute virus of mice (MVM); indicates complete clearance Filter Type Planova 20N Planova BioEX Filtration Pressure Run Load Titer (PFU/mL) 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. 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 log 10 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. 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, Total Challenge (PFU/filter) Log Reduction Values Fraction* Pool** 14 psi log 7.4 log 4.8 LRV 6.1 LRV log 4.8 LRV 6.2 LRV 10 psi log 7.2 log 4.6 LRV 5.9 LRV log 4.6 LRV 5.9 LRV 45 psi log 7.3 log 4.7 LRV 6.0 LRV log 4.7 LRV 6.0 LRV 30 psi log 7.3 log 4.7 LRV 6.1 LRV log 4.7 LRV 6.1 LRV * without large-volume testing ** with large-volume testing Table 4: Viral clearance data from filtrations spiked with xenotropic murine leukemia virus (X-MuLV); indicates complete clearance Filter Type Planova 20N Planova BioEX Filtration Pressure Run Load Titer (PFU/mL) Total Challenge (PFU/filter) Log Reduction Values Fraction* Pool** 14 psi log 7.3 log 4.7 LRV 6.0 LRV 10 psi log 4.7 LRV 6.0 LRV 45 psi log 7.2 log 4.6 LRV 6.0 LRV 30 psi log 4.6 LRV 6.0 LRV * without large-volume testing ** with large-volume testing 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 log 10 PFU/run could mitigate the risk of such artifacts in viral clearance studies, as observed in studies E1 and F1. Process Flux: All filtrations were executed successfully and demonstrated minimal impact of virus spikes on process performance (Figure 1 4). For highpressure 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 lowpressure 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. 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. To provide context for setting virusspiking 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 log 10 PFU/ ml (18). 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 log 10 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 log 10, resulting in a worst-case MVM titer of 4 log 10 copies/ml at the virusfiltration step. Spiking virus at around 5 log 10 PFU/mL thus still provides a greater challenge than the worst-case level for that step. Understanding relevant viral challenge situations during plasma-product 56 BioProcess International 16(3) March 2018 Advertorial

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8 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 log 10 copies/ml (19). Without any additional virus-removal steps, a 5 log 10 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 log 10 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: Stuckey J, et al. A Novel Approach to Achieving Modular Retrovirus Clearance for a Parvovirus Filter. Biotechnol. Prog. 30(1) 2014: 79 85; doi: /btpr Sofer G, et al. PDA Technical Report No. 41: Virus Filtration. PDA J. Pharm. Sci. Technol. 59(S-2) 2005: 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 ICH Q5A. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin. US Fed. Reg. 63(185) 1998: Victoria JG, et al. Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus. J. Virology 84(12) 2010: ; doi: / JVI Skrine J. A Biotech Production Facility Contamination Case Study Minute Mouse Virus. PDA J. Pharm. Sci. Technol. 65(6) 2011: ; doi: /pdajpst 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: ; doi: / pdajpst Aranha H, Forbes S. Viral Clearance Strategies for Biopharmaceutical Safety, Part 2: A Multifaceted Approach to Process Validation. BioPharm 14(5) 2001: 43 54, Yamamoto A, et al. Effect of Hydrodynamic Forces on Virus Removal Capability of Planova Filters. AIChE J. 60(6) 2014: ; doi: /aic 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: ; doi: /btpr Asher D, et al. PDA Technical Report No. 47: Preparation of Virus Spikes Used for Virus Clearance Studies. Parenteral Drug Association: Bethesda, MD, Chen D, Chen Q. Virus Retentive Filtration in Biopharmaceutical Manufacturing. PDA Letters 15 April 2016: Accessed on 21FEB Hongo-Hirasaki T, et al. Effects of Varying Virus-Spiking Conditions on a Virus- Removal Filter Planova 20N in a Virus Validation Study of Antibody Solutions. Biotechnol. Prog. 27(1) 2011: ; doi: /btpr 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: ; doi: /btpr Slocum A, et al. Impact of Virus Preparation Quality on Parvovirus Filter Performance. Biotechnol. Bioeng. 110(1) 2013: ; doi: /bit 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: ; doi: /btpr 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: ; doi: /j. vaccine 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: Lute S, et al. Phage Passage After Extended Processing in Small Virus Retentive Filters. Biotechnol. Appl. Biochem. 47(Part 3) 2007: ; doi: /ba 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; 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 To share this in PDF or professionally printed format, contact Jill Kaletha: jkaletha@ mossbergco. com, Advertorial March (3) BioProcess International 57