title: Evaluation of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus in mAb cell-culture.
abstract: 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 evaluation 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. In a previous report, we demonstrated the utility of TaqMan fluorogenic 5'-nuclease product-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. In this report, we evaluate the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We find 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 derived DNA polymerases appear to contribute only modestly to the assay background and RT activity in clarified cell culture harvests is contained largely in Type C particles. TM-PERT is linear and precise between 10(7)and 10(13) pU/ml, establishing the assay range. The assay is robust in that test article storage condition and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples.
authors.affiliation: Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA. brorson@cber.fda.gov
authors.forename: Kurt
authors.initials: K
authors.lastname: Brorson
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Swann
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hamilton
authors.forename: Mehnaz
authors.initials: M
authors.lastname: Mustafa
authors.forename: Christina
authors.initials: C
authors.lastname: de Wit
authors.forename: Lenore A
authors.initials: LA
authors.lastname: Norling
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Stein
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004330:Drosophila; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008854:Microscopy, Electron; D008979:Moloney murine leukemia virus / Q000235:genetics; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D051381:Rats; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D013395:Sucrose; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase