title: Identifying low-level sequence variants via next generation sequencing to aid stable CHO cell line screening.
abstract: Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here, for the first time, we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify, and eliminate cell lines containing low-level point mutations in recombinant coding sequence. Among the top eleven mAb producers chosen from transfectant, clone or subclone stages, three of the cell lines contained either missense or nonsense point mutations at a low level of less than 2%. Subsequent LC/MS/MS characterization detected ∼3% sequence variants with an amino acid change from Ser to Leu at residue 117 in the heavy chain of transfectants 11 and 27. This substitution is consistent with the RNAseq finding of a C/T mutation located at 407 base pair (TCA→TTA) in the heavy chain coding sequence. Here we demonstrate that RNAseq is a rapid and highly sensitive method to identify low-level genetic mutation de novo corresponding to the amino acid substitution that elicits sequence variant(s). Its implementation in CLD constitutes an early and effective step in identifying desired CHO expression cell lines.
authors.affiliation: Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bartkowiak
authors.affiliation: Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Bernard
authors.initials: B
authors.lastname: Nabiswa
authors.affiliation: Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Pratibha
authors.initials: P
authors.lastname: Mishra
authors.affiliation: Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: John
authors.initials: J
authors.lastname: Fann
authors.affiliation: Process Sciences Analytics, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: David
authors.initials: D
authors.lastname: Ouellette
authors.affiliation: Process Sciences Analytics, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Ivan
authors.initials: I
authors.lastname: Correia
authors.affiliation: Protein Science, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Dean
authors.initials: D
authors.lastname: Regier
authors.affiliation: Protein Science, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
authors.forename: Junjian
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D011994:Recombinant Proteins
keywords: CHO cell line; next generation sequencing; point mutation; recombinant monoclonal antibody; sequence variants