Premium
A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography
Author(s) -
Zhang Min,
Lute Scott,
Norling Lenore,
Hong Connie,
Safta Aurelia,
O'Connor Deborah,
Bernstein Lisa J.,
Wang Hua,
Blank Greg,
Brorson Kurt,
Chen Qi
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22172
Subject(s) - retrovirus , chinese hamster ovary cell , virus , biology , reverse transcriptase , agarose gel electrophoresis , virology , chromatography , computational biology , cell culture , chemistry , polymerase chain reaction , biochemistry , dna , gene , genetics
Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time‐consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q‐PCR) assay that quantifies the endogenous type C retrovirus‐like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model‐virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q‐PCR were confirmed by an orthogonal quantitative product‐enhanced reverse transcriptase (Q‐PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q‐PCR assays. Biotechnol. Bioeng. 2009;102: 1438–1447. © 2008 Wiley Periodicals, Inc.