Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing

Murine hybridoma cells used in the production of monoclonal antibodies (mAb 's) produce endogenous type C retrovirus particles. Regulatory agencies req uire a demonstration that mAb's intended for human use are free of retrovir us with an adequate margin of safety. This is usually achieved by validatio n studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viru ses, including a murine retrovirus. In this report, we assess the utility o f the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell- culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10( 13) pU/ mL) were substantially above the detection limit of the TM-PERT ass ay (similar to 10(6) pU/ mL). The nature of the RT activity from cell cultu re was complex, but the bulk of RT activity in clarified mAb harvests appea rs to be contained in large molecular weight viral particles. In laboratory -scale chromatographic runs, sufficient RT activity was present in mAb-cont aining eluates to accurately calculate its log(10) reduction value (LRV), t ypically between 2 and 4 log(10) per step. Monoclonal antibody purified usi ng a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate t hat the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is idea lly suited to investigate and optimize retrovirus clearance in purification processes.