title: O-linked glucosylation of a therapeutic recombinant humanised monoclonal antibody produced in CHO cells.
abstract: An unpredictable modification of a therapeutic recombinant humanised monoclonal antibody (rh-mAbX) produced using CHO cells was found. LC/MS analysis of rh-mAbX indicated the presence of heterogeneity in the light chain with a corresponding mass shift of 162 Da compared to the theoretical mass. To characterise the heterogeneity, that is, the attached moiety, several analyses were performed. Peptide mapping of rh-mAbX indicated that the attached moiety was located in the amino acid sequence from Leu20 to Lys45, which is a part of the variable region of the light chain. The peptide was efficiently purified in two-steps by RP-HPLC by utilising two different types of RP columns. N-terminal sequencing and LC/MS/MS analysis of the peptide suggested that Ser29 of the light chain was the modification site, and that the attached moiety was a single O-linked hexose. HPAEC-PAD analysis following β-elimination indicated the presence of an O-linked glucose in the modified peptide. Monosaccharide composition analysis after acid hydrolysis supported this result. The content of antibodies containing this species was determined to be approximately 10% by Lys-C peptide mapping detected at 280 nm. Thus, this study demonstrated the formation of a unique O-linked glucosylation posttranslational modification in a recombinant humanised monoclonal antibody produced in CHO cells.
authors.affiliation: Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan. Electronic address: tanakamgm@chugai-pharm.co.jp.
authors.forename: Megumi
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koga
authors.affiliation: Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
authors.forename: Atsuto
authors.initials: A
authors.lastname: Kobe
authors.affiliation: Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Sekimori
authors.affiliation: Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Aso
authors.affiliation: Department of Pharmaceutics, Toho University, Tokyo, Japan.
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Terada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003412:Cricetulus; D006031:Glycosylation; D006868:Hydrolysis; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D011994:Recombinant Proteins