Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.     

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     

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.     

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.     

Viral clearance in end-to-end integrated continuous process for mAb purification: Total flow-through integrated polishing on two columns connected to virus filtration

There are few reports of the adoption of continuous processes in bioproduction, particularly the implementation of end-to-end continuous or integrated processes, due to difficulties such as feed adjustment and incorporating virus filtration. Here, we propose an end-to-end integrated continuous process for a monoclonal antibody (mAb) with three integrated process segments: upstream production processes with pool-less direct connection, pooled low pH virus inactivation with pH control and a total flow-through integrated polishing process in which two columns were directly connected with a virus filter. The pooled virus inactivation step defines the batch, and high impurities reduction and mAb recovery were achieved for batches conducted in succession. Viral clearance tests also confirmed robust virus reduction for the flow-through two-column chromatography and the virus filtration steps. Additionally, viral clearance tests with two different hollow fiber virus filters operated at flux ranging from 1.5 to 40 LMH (liters per effective surface area of filter in square meters per hour) confirmed robust virus reduction over these ranges. Complete clearance with virus logarithmic reduction value ≥4 was achieved even with a process pause at the lowest flux. The end-to-end integrated continuous process proposed in this study is amenable to production processes, and the investigated virus filters have excellent applicability to continuous processes conducted at constant flux.     

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.     

Cellulose-based virus-retentive filters: a review

Viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. Viral filters are also essential protection elements against airborne viral particles. The present review first focuses on cellulose-based filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. Data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption–elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. Viral analysis of field water samples by the viradel technique is also surveyed. This review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. Some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review.     

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.     

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.     

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.     

Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification.

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.     

Adapting virus filtration to enable intensified and continuous monoclonal antibody processing

Ongoing efforts in the biopharmaceutical industry to enhance productivity and reduce manufacturing costs include development of intensified, linked, and/or continuous processes. One approach to improve productivity and process economics of the polishing step (i.e., anion exchange chromatography) is to preconcentrate the product intermediate using a single-pass tangential flow filtration step before loading on the resin. This intensification of the polishing step consequently leads to changes in product intermediate concentration for subsequent virus filtration operations, potentially impacting filter performance and methods for evaluating viral clearance. The filtrate flux performance of a virus filtration operation was evaluated with monoclonal antibody (mAb) solutions of varying concentrations. These data were used to evaluate the effect on filter sizing for a hypothetical mAb perfusion process. The optimum mAb concentration to minimize the area of the virus filter was a function of the filtration step duration and reflected the competing effects of increasing concentration and decreasing volumetric flux on the membrane productivity. mAb solutions at high and low concentrations were used to evaluate viral clearance with extended filtration times (e.g., 24–72 h) simulating continuous processing conditions. Modifications to more traditional filtration viral clearance study methods were required to avoid experimental artifacts associated with the extended filtration time. No virus passage through the filter was observed under these conditions, similar to previous results for batch processes. These data demonstrate the feasibility of obtaining effective virus removal even when mAb concentration and filtrations times are increased by up to an order of magnitude from current common practices.     

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.     

Effect of solution pH on protein transmission and membrane capacity during virus filtration

Although virus filtration is now an integral part of the overall viral clearance strategy for the purification of many commercial therapeutic proteins, there is currently little understanding of the factors controlling the performance of the virus filters. The objective of this study was to examine the effects of solution pH on protein transmission and capacity during virus filtration. Data were obtained using bovine serum albumin as a model protein with Viresolve 180 membranes oriented with both the skin-side up and skin-side down. Membranes were also characterized using dextran sieving measurements both before and after protein filtration. Membrane capacity and protein yield were minimal at the protein isoelectric point, which was due to the greater degree of concentration polarization associated with the smaller protein diffusion coefficient at this pH. In contrast, the actual protein sieving coefficient was maximum at the protein isoelectric point due to the absence of any strong electrostatic exclusion under these conditions. The yield and capacity were both significantly greater when the membrane was oriented with the skin-side down. These results provide important insights into the effects of solution conditions on the performance of virus filtration membranes for protein purification.     

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.     

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     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

The removal of viruses during the purification of equine antisera using filtration aids Hyflo Super-Cel and Fulmon Super A.

The manufacturing process in Australia for equine antisera against various venoms/toxins is based primarily on ammonium sulphate precipitation of pepsin-digested IgG, whereby Fc and F(ab')(2)fragments are separated. The capacity of the process to remove non-enveloped and enveloped model viruses was assessed using a scaled-down process. Each virus was added to mid-process samples from equine plasma before the material was applied to Hyflo Super-Celtrade mark filtration followed by Fulmonttrade mark Super A filtration. Samples were analysed pre- and post-filtration and the log clearance of the viruses calculated. The mean clearance factors for viral load of canine adenovirus type II (CAV(2)), poliovirus type 1 (PV1), infectious bovine rhinotracheitis virus (IBR) and canine distemper virus (CDV) were 5.3 logs, 4.2 logs, 5.7 logs and 4. 0 logs respectively. Clearance results as virus is adsorbed to the filtration aids which are removed from the process, thereby demonstrating improved viral safety of equine antisera produced by CSL.     

Influence of salts on virus adsorption to microporous filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.