Impact of protein fouling on nanoparticle capture within the Viresolve® Pro and Viresolve® NFP virus removal membranes

Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters.     

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.     

Qualification of a novel inline spiking method for virus filter validation

Virus filters are widely used in bioprocessing to reduce the risk of virus contamination in therapeutics. The small pores required to retain viruses are sensitive to plugging by trace contaminants and frequently require inline adsorptive prefiltration. Virus spiking studies are required to demonstrate virus removal capabilities of the virus filter using scale down filters. If prefiltration removes viruses and interferes with the measurement of virus filter LRV, the standard approach is to batch prefilter the protein solution, spike with virus, and then virus filter. For a number of proteins, batch prefiltration leads to increased plugging and significantly lower throughputs than inline prefiltration. A novel inline spiking method was developed to overcome this problem. This method allows the use of inline prefiltration with direct measurement of virus filter removal capabilities. The equipment and its operation are described. The method was tested with three different protein feeds, two different parvovirus filters, two virus injection rates; a salt spike, a bacteriophage spike, and two mammalian virus spikes: MMV and xMuLV. The novel inline method can reliably measure LRV at throughputs representative of the manufacturing process. It is recommended for applications where prefiltration is needed to improve throughput, prefiltration significantly reduces virus titer, and virus filter throughput is significantly reduced using batch vs. inline prefiltration. It can even help for the case where the virus preparation causes premature plugging.     

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.     

Virus removal from factor IX by filtration: Validation of the integrity test and effect of manufacturing process conditions

Virus removal from a high purity factor IX, Replenine^®-VF, by filtration using a Planova 15N filter has been investigated. A wide range of relevant and model enveloped and non-enveloped viruses, of various sizes, were effectively removed by this procedure. Virus removal was confirmed to be effective when different batches of filter were challenged with poliovirus-1. It was confirmed that intentionally modified filters that failed the leakage test had completely lost the ability to remove virus, thus confirming that this test demonstrates gross filter failure. In the case of the more sensitive integrity test based on gold particle removal, it was found that a pre-wash step was not essential. Planova filters that had been modified by sodium hydroxide treatment to make them more permeable, and filters manufactured with varying pore-sizes over the range of 15–35 nm, were tested. The integrity test value that resulted in the removal of >4 log₁₀ of poliovirus-1 from the product correlated with that recommended by the filter manufacturer. Virus removal from the product was not influenced by filter load mass, flow-rate or pressure. These studies confirm the robustness of this filtration procedure and allow suitable process limits to be set for this manufacturing step.     

Achieving high mass‐throughput of therapeutic proteins through parvovirus retentive filters

Parvovirus retentive filters that assure removal of viruses and virus‐like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size‐based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass‐throughput values of 7.3, 26.4, and 76.2 kg/m² were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass‐throughput values of 73, 137, and 192 kg/m² were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large‐scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small‐scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large‐scale filter capacity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Protein fouling during constant-flux virus filtration: Mechanisms and modeling

As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5–100 L/m²/h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20–30 L/m²/h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.     

Mechanistic evaluation of virus clearance by depth filtration

Virus clearance by depth filtration has not been well‐understood mechanistically due to lack of quantitative data on filter charge characteristics and absence of systematic studies. It is generally believed that both electrostatic interactions and sized based mechanical entrapment contribute to virus clearance by depth filtration. In order to establish whether the effectiveness of virus clearance correlates with the charge characteristics of a given depth filter, a counter‐ion displacement technique was employed to determine the ionic capacity for several depth filters. Two depth filters (Millipore B1HC and X0HC) with significant differences in ionic capacities were selected and evaluated for their ability to eliminate viruses. The high ionic capacity X0HC filter showed complete porcine parvovirus (PPV) clearance (eliminating the spiked viruses to below the limit of detection) under low conductivity conditions (≤2.5 mS/cm), achieving a log₁₀ reduction factor (LRF) of > 4.8. On the other hand, the low ionic capacity B1HC filter achieved only ～2.1–3.0 LRF of PPV clearance under the same conditions. These results indicate that parvovirus clearance by these two depth filters are mainly achieved via electrostatic interactions between the filters and PPV. When much larger xenotropic murine leukemia virus (XMuLV) was used as the model virus, complete retrovirus clearance was obtained under all conditions evaluated for both depth filters, suggesting the involvement of mechanisms other than just electrostatic interactions in XMuLV clearance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:431–437, 2015     

Evaluation and validation of virus removal by ultrafiltration during the production of diaspirin crosslinked haemoglobin (DCLHb).

Virus retention during ultrafiltration through A/G Technology filter cartridges was investigated to characterize the removal process and validate the degree of virus titre reduction during the filtration of red blood cell haemolysates performed as part of the production of diaspirin crosslinked haemoglobin (DCLHb). When viruses were suspended in phosphate buffered saline solution, retention was greater with larger sized viruses and smaller filter pore size. Virus titre was maintained at starting levels in the filter retentate circuit during the course of filtration, suggesting that the virus removal mechanism is predominantly size exclusion. Evaluation of specific processing variables indicated that the retention of phiX174 virus was increased in the presence of red blood cell haemolysate or at high membrane crossflow rates and transmembrane pressures, while the retention of EMC virus was less sensitive to variations in these parameters. Using these results to design a validation protocol, log reduction values of >7.9 were demonstrated for the retention of human immunodeficiency virus, pseudorabies virus and bovine viral diarrhoea viruses, 7.6 for hepatitis A virus, and 4.2 for porcine parvovirus. It was also shown that the retention of viruses was maintained during repetitive use of the same filter cartridge.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova? BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0–49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294–1302, 2017     

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10¹⁰ PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4‐5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3‐101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79‐85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic‐Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V₇₅). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less‐pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log₁₀. A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V₇₅ for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:135–144, 2015     

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (～20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (～100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co‐spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in‐house viral validation studies and the results from the co‐spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log₁₀ reduction factors (LRF) to support clinical trial applications in both USA and Europe. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:79–85, 2014     

Virus removal capacity at varying ionic strength during nanofiltration of AlphaNine® SD

Nanofiltration is incorporated into the manufacturing processes of many protein biopharmaceuticals to enhance safety by providing the capacity to retain pathogens while allowing protein drugs to pass through the filter. Retention is mainly a function of size; however, the shape of the pathogen may also influence retention. The ability of the Viresolve^® Pro nanofilter to remove different sized viruses during the manufacture of a Coagulation Factor IX (Alphanine^® SD) was studied at varying ionic strength, a process condition with the potential to affect virus shape and, hence, virus retention. Eight viruses were tested in a scale-down of the nanofiltration process. Five of the viruses (EMCV, Reo, BVDV, HIV, PRV) were nanofiltered at normal sodium processing conditions and three (PPV, HAV and WNV) were nanofiltered at higher and lower sodium. Representative Reduction Factors for all viruses were ≥4.50 logs and removal was consistent over a wide range of ionic strength.     

Porcine circovirus (PCV) removal by Q sepharose fast flow chromatography

The recently discovered contamination of oral rotavirus vaccines led to exposure of millions of infants to porcine circovirus (PCV). PCV was not detected by conventional virus screening tests. Regulatory agencies expect exclusion of adventitious viruses from biological products. Therefore, methods for inactivation/removal of viruses have to be implemented as an additional safety barrier whenever feasible. However, inactivation or removal of PCV is difficult. PCV is highly resistant to widely used physicochemical inactivation procedures. Circoviruses such as PCV are the smallest viruses known and are not expected to be effectively removed by currently‐used virus filters due to the small size of the circovirus particles. Anion exchange chromatography such as Q Sepharose^® Fast Flow (QSFF) has been shown to effectively remove a range of viruses including parvoviruses. In this study, we investigated PCV1 removal by virus filtration and by QSFF chromatography. As expected, PCV1 could not be effectively removed by virus filtration. However, PCV1 could be effectively removed by QSFF as used during the purification of monoclonal antibodies (mAbs) and a log₁₀ reduction value (LRV) of 4.12 was obtained. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1464–1471, 2013     

Filter bed systems treating domestic wastewater in the Nordic countries – Performance and reuse of filter media

Nine filter beds have been constructed in the Nordic countries, Denmark, Finland, Norway and Sweden. Filter beds consist of a septic tank followed by an aerobic pre-treatment biofilter and a subsequent saturated flow grass-covered filter. Thus, filter beds are similar to subsurface flow constructed wetlands with pre-treatment biofilters, but do not have wetland plants with roots submerged into the saturated filter. All saturated filters contain Filtralite^®P, a light-weight expanded clay aggregate possessing high phosphorus sorption capacity. The filter bed systems showed stable and consistent performance during the testing period of 3 years. Removal of organic matter measured as biochemical oxygen demand (BOD) was >80%, total phosphorus (TP) >94% and total nitrogen (TN) ranged from 32 to 66%. Effluent concentrations of fecal indicator bacteria met the European bathing water quality criteria in all systems. One system was investigated for virus removal and somatic viruses were not detected in the effluent. The investigations revealed that the majority of the BOD and nitrogen removal occurred in the pre-treatment filters and the phosphorus and bacteria removal was more prominent in the saturated filters. The saturated filters could be built substantially smaller than the current design guidelines without sacrificing treatment performance. The used filter material met the Norwegian regulations for reuse in agriculture with respect to heavy metals, bacteria and parasites. When saturated with phosphorus, the light-weight aggregate, Filtralite^®P used in the saturated bed is a suitable phosphorus fertilizer and additionally has a liming effect.     

Improvement of virus safety of an antihemophilc factor IX by virus filtration process

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized productionscale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were (i)v.12 for HAV, (i)t.28 for PPV, (i)u.33 for EMCV, (i)u.51 for HIV, (i)u.17 for BVDV, and (i)u.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.     

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.     

Multi‐round virus filter integrity test sensitivity

Virus filtration is becoming increasingly prominent in biopharmaceutical recovery processes as a robust method to remove a broad range of virus types. Increasing batch sizes will require large numbers of individual virus filter elements operating in parallel. Before adopting a more complex strategy for managing the integrity testing of large assemblies of virus filters, it is important to understand the sensitivity of the forward flow diffusion test for a single filter and for multiple filters in a single housing. An approach has been developed to estimate the largest hole that could consistently go undetected in a single filter within a larger assembly of virus filters. The integrity test limited minimum log reduction value (LRV) is determined based on the size of the hole as a function of the number of filters in the housing. This minimum LRV is shown to be largely insensitive to the number of filters within the housing. The likelihood of such damage occurring is expected to be very low. This analysis suggests there is minimal benefit to placing filters in individual housings or to adjusting the test specification to compensate for larger numbers of filters in a given housing. Biotechnol. Bioeng. 2009;103: 574–581. © 2009 Wiley Periodicals, Inc.     

Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.