title: Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses.
abstract: Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle (C(t) ) value, a measure of population heterogeneity; (iii) mean C(t) changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications.
authors.affiliation: Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. he_luhong@lilly.com
authors.forename: Luhong
authors.initials: L
authors.lastname: He
authors.forename: Christal
authors.initials: C
authors.lastname: Winterrowd
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Kadura
authors.forename: Christopher
authors.initials: C
authors.lastname: Frye
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D018628:Gene Dosage; D016133:Polymerase Chain Reaction; D059010:Single-Cell Analysis / Q000379:methods*; D019076:Transgenes