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.     

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     

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.     

Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification

Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.     

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.     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow‐rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log₁₀ of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance During the Purification Of Mammalian Cell Substrate Derived Biotherapeutics

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

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.     

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.     

Salt tolerant membrane adsorbers for robust impurity clearance

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50–150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ?X174 (pI ～ 6.7) at pH 7.5 and phage PR772 (pI ～ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host‐cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of pH,conductivity, host cell protein,and DNA size distribution on DNA clearance in anion exchange chromatography media

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018     

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.     

Defining the mechanistic binding of viral particles to a multi‐modal anion exchange resin

A multi‐tiered approach to determine the binding mechanism of viral clearance utilizing a multi‐modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X‐MuLV, MVM, REO3, and PrV). First, virus spiked buffer‐only experiments were conducted to evaluate the virus's affinity for single mode and multi‐modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups. This mechanistic view agreed with the general surface characteristics of the different virus species in terms of isoelectric point and relative hydrophobicity values. This hypothesized mechanistic binding was then tested with commercially relevant, in‐process materials, in which competitive binding occurred between the load components (e.g., viruses, target product, and impurities) and the resin. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1019–1026, 2018     

Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production

Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log₁₀ clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6 log₁₀ dynamic range with a limit of detection (LOD) of ≈1 genome copy/μL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.     

Virus clearance validation across continuous capture chromatography

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.     

Development of adsorptive hybrid filters to enable two-step purification of biologics

Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process- and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze? AEX) and salt-tolerant biomimetic (Emphaze? ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus. The potential to remove soluble aggregates was also examined. Our aim was to develop a mechanistic understanding of the interactions governing adsorptive removal of impurities during filtration by evaluating the effect of various filter types, feed streams, and process conditions on impurity removal. The ionic capacity of these filters was measured and correlated with their ability to remove impurities for multiple molecules. The ionic capacity of AHF significantly exceeded that of traditional adsorptive depth filters (ADF) by 40% for the Emphaze? AEX and by 700% for the Emphaze? ST-AEX, providing substantially higher reduction of soluble anionic impurities, including DNA, HCPs and model virus. Nevertheless, we determined that ADF with filter aid provided additional hydrophobic functionality that resulted in removal of higher molecular weight species than AHF. Implementing AHF demonstrated improved process-related impurity removal and viral clearance after Protein A chromatography and enabled a two-step purification process. The consequences of enhanced process performance are far reaching because it allows the downstream polishing train to be restructured and simplified, and chromatographic purity standards to be met with a reduced number of chromatographic steps.