title: Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1.
abstract: We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC-MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.
authors.affiliation: Protein Analytical Chemistry, Genentech, South San Francisco, CA, USA. yonghuaz@gene.com
authors.forename: Taylor
authors.initials: T
authors.lastname: Zhang
authors.forename: Yungfu
authors.initials: Y
authors.lastname: Huang
authors.forename: Scott
authors.initials: S
authors.lastname: Chamberlain
authors.forename: Tony
authors.initials: T
authors.lastname: Romeo
authors.forename: Judith
authors.initials: J
authors.lastname: Zhu-Shimoni
authors.forename: Daniel
authors.initials: D
authors.lastname: Hewitt
authors.forename: Mary
authors.initials: M
authors.lastname: Zhu
authors.forename: Viswanatham
authors.initials: V
authors.lastname: Katta
authors.forename: Brad
authors.initials: B
authors.lastname: Mauger
authors.forename: Yung-Hsiang
authors.initials: YH
authors.lastname: Kao
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D016466:CHO Cells; D018388:Codon, Terminator / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D005973:Glutamine / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics
chemical_list: D018388:Codon, Terminator; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D020033:Protein Isoforms; D005973:Glutamine