Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization – A case study
Introduction
Biologicals produced in mammalian cell cultures (MCC) become increasingly important for public health and the pharmaceutical industry. Contamination by adventitious agents is a major safety concern for biologicals produced in MCC. To ensure safety, government regulations require manufacturers to demonstrate that biologicals produced in MCC are free of adventitious agents. The overall safety assurance is accomplished by the combination of raw material control/testing, in-process control/testing, and virus clearance studies. Master cell bank (MCB) testing is an essential part of the overall safety strategy for biologicals produced in MCC. The purpose of MCB testing is to demonstrate that MCB is free of detectable adventitious agents. Tests that comply with 9 CFR part 113 sections 53 [1], 46 [2], and 47 [3] for bovine viruses are routinely performed as part of the in vitro viral screening for MCBs that have been exposed to bovine-derived raw materials such as fetal bovine serum (FBS). The panel of bovine viruses screened includes bovine viral diarrhea virus, bovine adenovirus, bovine parvovirus, bluetongue virus, bovine respiratory syncytial virus, reovirus, and rabies virus [3]. For compliance with European Agency for the Evaluation of Medicinal Products regulations [4], infectious bovine rhinotracheitis virus and bovine parainfluenza type 3 are also added to the panel. Two cell lines, a bovine cell line and a primate cell line, are used in the 9 CFR bovine virus testing for detection of viruses with potentially different cell tropisms [1]. Sometimes, fetal horse serum (FHS) instead of FBS is used as supplement in the culture medium to minimize the possibility of false positives as FBS may be contaminated by bovine viruses as well as to avoid potential false negatives caused by the presence of antibodies against bovine viruses in FBS.
Section snippets
Cell lines and reagents
An MCB derived from Chinese Hamster Ovary (CHO) cells that had been genetically engineered to produce therapeutic protein was screened for various adventitious agents including bovine viruses. The detector cells used in the 9 CFR bovines test were Vero (African green monkey kidney cells; ATCC CCL 81) and BT (bovine turbinate; ATCC CRL 1390). Additional cell lines used during the investigation included CHO-K1 (ATCC CCL 61), MRC-5 (human diploid lung cells; ATCC CCL 171) and PT-1 (porcine
Isolation of the virus
On day 19 of the 9 CFR test, all three 75-cm2 flasks and both 6-well plates of Vero cells derived from the original flask of Vero cells inoculated with the test article (Fig. 1) displayed severe CPE in the form of nearly total cell lysis. The growth medium from the three 75-cm2 flasks was collected, aliquoted, and stored at −60 °C or below. In the mean time, none of the flasks or 6-well plates of the negative control Vero cells showed any abnormality. The BT cell portion of the test was taken to
Discussion
MCB testing is a critical component of the overall safety strategy for biologicals produced in MCC, which involves various in vitro and in vivo assays to screen for adventitious agents (ICH Q5A guideline and FDA PTC). CHO cells are commonly used for industrial scale production of therapeutic biologicals such as recombinant monoclonal antibodies. Unlike mouse-derived production cell lines such as NS0 and Sp2/0Ag14, CHO cells do not harbor infectious endogenous retroviruses [8], [9], [10]. In
Acknowledgments
We gratefully acknowledge the skilled technical assistance of Suzanne Dieringer-Boyer, Richard Grier, Andrew Glenn, and Vagdevi Srikrishna. We also thank Kathryn Struck, Jeanette Bulkwalter and Ryan Lybarger for their assistance in preparation of the manuscript.
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