title: Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody.
abstract: Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of the sequence variant was detected by an imaged capillary isoelectric focusing (ICIEF) assay, showing a new basic species in mAb charge variant profile. The new basic variant was fractionated and enriched by ion-exchange chromatography, analyzed by reduced light and heavy chain mass determination, and characterized by HPLC-UV/MS/MS of tryptic and endoproteinase Lys-C peptide maps. A Serine to Arginine sequence variant was identified at the heavy chain 441 position (S441R), and confirmed by using synthetic peptides. The relative level of the S441R variant was estimated to be in the range of 0.3-0.6% for several mAb batches analyzed via extracted ion chromatogram (EIC). This work demonstrates the effectiveness of using integrated analytical methods to detect and identify protein heterogeneity and the importance of monitoring product quality during mAb bioprocess development.
authors: Oceanside Pharma Technical Development, Genentech, Oceanside, CA 92056, United States. ren.diya@gene.com
authors: Diya
authors: D
authors: Ren
authors: Jian
authors: J
authors: Zhang
authors: Ross
authors: R
authors: Pritchett
authors: Hongbin
authors: H
authors: Liu
authors: Jennifer
authors: J
authors: Kyauk
authors: Jun
authors: J
authors: Luo
authors: Ashraf
authors: A
authors: Amanullah
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001120:Arginine / Q000032:analysis* / Q000737:chemistry; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D020539:Sequence Analysis, Protein; D012694:Serine / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D012694:Serine; D001120:Arginine; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase