title: Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.
abstract: Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.
authors.affiliation: Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Feeney
authors.forename: Veronica
authors.initials: V
authors.lastname: Carvalhal
authors.forename: X Christopher
authors.initials: XC
authors.lastname: Yu
authors.forename: Betty
authors.initials: B
authors.lastname: Chan
authors.forename: David A
authors.initials: DA
authors.lastname: Michels
authors.forename: Yajun Jennifer
authors.initials: YJ
authors.lastname: Wang
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
authors.forename: Jan
authors.initials: J
authors.lastname: Ressl
authors.forename: Brendon
authors.initials: B
authors.lastname: Dusel
authors.forename: Michael W
authors.initials: MW
authors.lastname: Laird
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006639:Histidine / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine