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

Abstract

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.

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

“…Extraction of viral genomic material was then performed using either the EZ1 Virus Mini Kit v2.0 on a BioRobot EZ1 (Qiagen, Inc., Valencia, CA) or the MagAttract Virus Mini Kit v1.3 on a BioRobot M48 (Qiagen, Inc.). The QPCR assays used to quantify X-MuLV, SV40, and MMV VP were performed as previously described (Shi et al, 1999a(Shi et al, , 2004Zhan et al, 2002). For viral clearance studies, sample interference was determined by comparing 1:10 diluted samples with undiluted samples.…”

“…In addition to the virus purification literature, there are also published reports describing viral clearance using QSFF resin (Curtis et al, 2003;Norling et al, 2005;Shi et al, 1999bShi et al, , 2004Strauss et al, 2009;Valera et al, 2003). Although most of these do not describe much characterization of the processes used, a few have demonstrated that salt concentration (or conductivity), salt composition, and pH are important factors for viral clearance (Curtis et al, 2003;Strauss et al, 2009).…”

“…One important function of the AEX step is its ability to remove putative viral contaminants and impurities. Virus reduction studies investigating AEX conditions have shown the step to be highly effective at removing both enveloped and non-enveloped viruses, consistently achieving log 10 reduction values (LRVs) greater than 4 (Curtis et al, 2003;Norling et al, 2005;Shi et al, 1999bShi et al, , 2004Strauss et al, 2009;Tayot et al, 1987;Valera et al, 2003;Zolton and Padvelskis, 1984). In order to ensure that this unit operation maintains its ability to remove viruses while allowing for further development of the step, it is important to gain an understanding of the mechanisms by which AEX processes remove viruses from mAb feedstocks.…”

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

“…The validation studies are typically scaled-down spiking studies using model viruses and production scale in-process intermediates. The virus clearance of individual purification unit operations, such as chromatography or virus filtration, is measured and expressed as a log 10 reduction value, or LRV (Brorson et al, 2004;Shi et al, 2004). The LRV of orthogonal steps (modules where clearance mechanisms differ) are added together to compute the overall process LRV for comparison to the quantity of endogenous retrovirus in the cell culture harvests.…”

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

“…[37][38][39][40][41] Current regulations require that the manufacturing process demonstrate the ability to clear model viruses to ensure the safety of these cell-linederived products prior to approval, with additional built-in safety for robustness. [42][43][44] AEX chromatography has been frequently applied as a viral clearance step during downstream processing, in addition to impurity clearance of molecular species such as HCP and DNA.…”