Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. Because the assays currently used in these studies (e.g. TEM, in vitro infectivity assays) are expensive, time consuming and insensitive, improvements in the way viral measurement and clearance/inactivation are measured are highly desirable. To examine the potential of PCR based assays as next generation assays for viral safety evaluation, we assessed the utility of TaqMan fluorogenic 5'nuclease PCR-Enhanced Reverse Transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. The levels of RT activity contained in cell-culture harvests (108-1013 pU/mL) were substantially above the detection limit of the TM-PERT assay (106 pU/mL). The nature of the RT activity from cell-culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight virions. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log10 reduction value (LRV), typically between 2 and 4 log10 per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicated that the TM-PERT assay, because it is quantitative, highly sensitive and can be used to analyze a large number of samples in a short period of time, is ideally suited to investigate mechanisms of retrovirus clearance and/or to optimize retrovirus removal/inactivation in purification processes. A manuscript describing results from these experiments has been submitted to Biotechnology Progress. To critically examine the performance of the TM-PERT assay in viral safety evaluation, we evaluated the specificity, accuracy, range, precision and robustness of TM-PERT. We found that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g., transmission electron microscopy, viral sequence specific TaqMan). Cell culture derived DNA polymerases appeared to contribute only modestly to the assay background. TM-PERT was linear and precise between 107 and 1013 pU/mL, establishing the assay range. The assay was robust in that storage of test articles for 1 week at room temperature or multiple freeze/thaw cycles had little effect on subsequent RT quantification and no interference of the assay by protein or DNA concentrations predicted to be present in cell culture samples was evident. Sporadic background amplification signals present in some assays appeared to correlate with MS2 template quality. A manuscript describing data from these experiments is in preparation for submission to Biologicals. In collaboration with Genentech, we are extending these studies by examining the impact of process changes on retrovirus expression and LRV by robust virus inactivation steps. Preliminary results from these studies have shown that cell culture process changes such as fermentation scale-up, pH shifts and media changes have only modest impacts on retrovirus expression. Experiments examining the robustness of low pH retrovirus inactivation are on going.
