title: Impact of cell culture process changes on endogenous retrovirus expression.
abstract: Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.
authors.affiliation: Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, USA.
authors.forename: Kurt
authors.initials: K
authors.lastname: Brorson
authors.forename: Christina
authors.initials: C
authors.lastname: De Wit
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hamilton
authors.forename: Mehnaz
authors.initials: M
authors.lastname: Mustafa
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Swann
authors.forename: Robert
authors.initials: R
authors.lastname: Kiss
authors.forename: Ron
authors.initials: R
authors.lastname: Taticek
authors.forename: Gian
authors.initials: G
authors.lastname: Polastri
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Stein
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D002087:Butyrates / Q000494:pharmacology; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D006224:Cricetinae; D020077:Endogenous Retroviruses / Q000235:genetics* / Q000302:isolation & purification*; D015967:Gene Expression Regulation, Viral; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D002087:Butyrates; D011994:Recombinant Proteins; D010100:Oxygen