Monoclonal antibody (mAb) cell lines produce endogenous retroviruses (ERV), thus validation of retrovirus removal by a mAb manufacturing process is a requirement for licensure and clinical IND use for mAb products. Validation of retrovirus removal typically involves spike/removal studies of chromatography and other steps of the manufacturing process. The amount of retrovirus in antibody bulk harvest samples is typically quantitated by transmission electron microscopy (TEM) and in samples from validation studies of processing steps by infectivity assays (Mus dunni). Although many steps in downstream processing have been shown repeatedly to be robust (protein A and anion exchange chromatography, solvent/detergent inactivation, viresolve filtration), current policy requires measurement of ERV for each antibody-producing cell line at the bulk harvest stage and clearance validation studies for multiple downstream processing steps. Currently employed clearance validation studies are limited, however, by the feasible load of spiking infectious ERV and by assay sensitivity. Given the cost and review time expended, improvements in the way viral clearance/inactivation is measured are highly desirable. Recently developed, PCR-based reverse transcriptase (PBRT, PERT) assays, may be able to improve these studies. PBRT assays are about one million fold more sensitive than conventional reverse trascriptase (RT) assays and because they measure RT activity, not infectious viruses, PBRT has a detection limit 4 to 6 orders of magnitude lower than TEM and Mus dunni, and are more rapid than infectivity assays. In theory, PBRT assays could replace the current assays used in validation studies, possibly lowering the number of steps that need to be validated or even allowing final product testing only. We propose to perform a small scale production of a model mAb, using steps commonly employed by industry in mAb manufacture and measure virus clearance of these steps using a modified PBRT assay. Several PERT assays are currently in place at DVP/OVRR, including a fluorescent probe PBRT assay. Our strategy is to produce the model mAb using an FPLC system currently in place in DMA, purifying the mAb using protein G, anion exchange and hydrophobic interaction columns. RT levels in column loads, washes and eluates will be measured by PBRT to determine ERV clearance. It is possible that we may be able to demonstrate total clearance by individual steps. If it is the case that all or almost all of the ERVs are removed by the first step (protein G), spike/removal studies can be performed on subsequent steps. PBRT will be compared to TEM and infectivity assays to demonstrate the relative advantage of PBRT assays to measure ERV in bulk harvests and validation studies.
