Investigation of virus retention by size exclusion membranes under different flow regimes

Virus removal by filter membranes is regarded as a robust and efficient unit operation, which is frequently applied in the downstream processing of biopharmaceuticals. The retention of viruses by virus filtration membranes is predominantly based on size exclusion. However, recent results using model membranes and bacteriophage PP7 point to the fact that virus retention can also significantly be influenced by adsorptive interactions between virus, product molecules, and membranes. Furthermore, the impact of flow rate and flow interruptions on virus retention have been studied and responsible mechanisms discussed. The aim of this investigation was to gain a holistic understanding of the underlying mechanisms for virus retention in size exclusion membranes as a function of membrane structure and membrane surface properties, as well as flow and solution conditions. The results of this study contribute to the differentiation between size exclusion and adsorptive effects during virus filtration and broaden the current understanding of mechanisms related to virus breakthroughs after temporary flow interruptions. Within the frame of a Design of Experiments approach it was found that the level of retention of virus filtration membranes was mostly influenced by the membrane structure during typical process-related flow conditions. The retention performance after a flow interruption was also significantly influenced by membrane surface properties and solution conditions. While size exclusion was confirmed as main retention mechanism, the analysis of all results suggests that especially after a flow interruption virus retention can be influenced by adsorptive effects between the virus and the membrane surface. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2747, 2019.     

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles

Virus filtration with nanometer size exclusion membranes (“nanofiltration”) is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18–26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.     

Removal of Viruses from Human Intravenous Immune Globulin by 35 nm Nanofiltration

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.     

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.     

Effects of solution conditions on virus retention by the Viresolve® NFP filter

Virus filtration can provide a robust method for removal of adventitious parvoviruses in the production of biotherapeutics. Although virus filtration is typically thought to function by a purely size‐based removal mechanism, there is limited data in the literature indicating that virus retention is a function of solution conditions. The objective of this work was to examine the effect of solution pH and ionic strength on virus retention by the Viresolve^® NFP membrane. Data were obtained using the bacteriophage ?X174 as a model virus, with retention data complemented by the use of confocal microscopy to directly visualize capture of fluorescently labeled ?X174 within the filter. Virus retention was greatest at low pH and low ionic strength, conditions under which there was an attractive electrostatic interaction between the negatively charged membrane and the positively charged phage. In addition, the transient increase in virus transmission seen in response to a pressure disruption at pH 7.8 and 10 was completely absent at pH 4.9, suggesting that the trapped virus are unable to overcome the electrostatic attraction and diffuse out of the pores when the pressure is released. Further confirmation of this physical picture was provided by confocal microscopy. Images obtained at pH 10 showed the migration of previously captured phage; this phenomenon was absent at pH 4.9. These results provide important new insights into the factors governing virus retention using virus filtration membranes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1280–1286, 2015     

Impact of virus preparation quality on parvovirus filter performance

Virus‐removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus‐removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010 ). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations. Biotechnol. Bioeng. 2013; 110: 229–239. © 2012 Wiley Periodicals, Inc.     

Development and qualification of a novel virus removal filter for cell culture applications

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.     

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.     

Pathogen safety profile of a 10% IgG preparation manufactured using a depth filtration-modified process

Gamunex^®-C is a highly purified liquid 10% IgG preparation manufactured by a process that includes caprylate precipitation and incubation, and chromatography steps. In the original process, caprylate precipitation was followed by cloth filtration to remove impurities. The highly porous cloth filter has since been replaced with a tight depth filter. The impact of this process modification on pathogen reduction and product is presented.Virus and prion reduction was determined under set-point conditions using scaled-down models of the manufacturing process, and at or outside operating limits to determine robustness. Product protein compositions before and after the process modification were compared directly using manufacturing data.Filtration through a tight depth filter substantially increased nonenveloped virus reduction, and virus reduction was maintained even when a compromised depth filter was used. In addition, prion reduction was improved by about three logs. The product IgG content, purity, and IgG subclass distribution remained comparable to the original cloth filtration process.The replacement of cloth filtration with depth filtration increased the pathogen safety margin of the manufacturing process without impacting the product composition.     

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.     

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     

A three plus three parameters mechanistic model for viral filtration

Viral filtration is an expensive regulatory requirement in downstream processing of monoclonal antibodies (mAbs). This process step is typically operated with an overdesigned filter in order to account for any batch to batch variability in the filter, as well as the feed characteristics. Here, we propose a simple, six‐parameter mechanistic model for viral filtration where three parameters are membrane‐specific while the other three depend on feed characteristics and membrane‐feed interactions. Viruses are considered as passive particles which are retained by the membrane on the basis of size exclusion. The model envisages that the viral filter contains two kind of pores: virus‐retentive, small‐sized pores and non‐retentive, large‐sized pores. The small‐sized pores get blocked during filtration resulting in decrease in active membrane area, while the large‐sized pores get constricted during filtration. The length of constricted part increases during filtration and contributes to increase in hydraulic resistance of the filter. Rate of these processes (blocking and constriction) are assumed to be proportional to the instantaneous rate of retention of the viral particles. The general nature of the model is validated with the experimental data on viral filtration for four different commercial membranes used in biotech industries as well as different model viruses. The proposed model has been demonstrated to describe the behavior of filters with very good accuracy. The best‐fit model parameter values indicate about the various phenomena that are responsible for differences in the behavior of the membranes as well as change in retention and flux with feed concentration. The proposed model can be used for improving design of virus filters as well as in appropriate sizing of the filters during processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1538–1547, 2017     

Investigations of prion and virus safety of a new liquid IVIG product.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

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.     

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process

Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log₁₀). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.     

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.     

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     

Long-term survival of human rotavirus in raw and treated river water

This study was aimed at assessing the role of water as a vehicle for rotavirus spread by determining how well these viruses survive in the water environment. A cell culture adapted strain of human rotavirus subgroup 2, grown in MA-104 cells, was used as a model. Virus survival was tested in the following types of water samples, derived from the Ottawa River, at two different times of the year: (i) raw water (RW), (ii) muncipally treated tap water (TW), and (iii) raw water that had been filtered (FW) through a membrane (0.22 micron). The water samples, with approximately 5.0 X 10(4) plaque-forming units (PFU) of the virus, were held at either 4 or 20 degrees C and tested for infectious virus over a period of 64 days. The TW samples had a total and free chlorine content of 0.05 and less than 0.05 mg/L, respectively. The chlorine in these samples was not neutralized before virus contamination. Irrespective of the holding temperature, the virus titre in FW remained essentially unaltered throughout the test period. In TW held at 4 degrees C, there was no significant drop in the virus titre even after 64 days, whereas at 20 degrees C the titre in TW was reduced by about 2 log10 over the same period. Even though the loss of virus infectivity was most rapid in RW held at 20 degrees C, it took about 10 days for a 99.0% reduction in the plaque titre of the virus. These findings, therefore, indicate that rotaviruses can survive for several days in raw and treated river water thus making recreational and potable waters potential vehicles for the transmission of rotavirus infections.