title: Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies.
abstract: This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS(E) peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.
authors.affiliation: Waters Corporation.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Xie
authors.affiliation: Waters Corporation.
authors.forename: Asish
authors.initials: A
authors.lastname: Chakraborty
authors.affiliation: Waters Corporation.
authors.forename: Joomi
authors.initials: J
authors.lastname: Ahn
authors.affiliation: Waters Corporation.
authors.forename: Ying Qing
authors.initials: YQ
authors.lastname: Yu
authors.affiliation: Waters India Pvt. Ltd.
authors.forename: Deepalakshmi P
authors.initials: DP
authors.lastname: Dakshinamoorthy
authors.affiliation: Waters Corporation.
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.affiliation: Waters Corporation.
authors.forename: Weibin
authors.initials: W
authors.lastname: Chen
authors.affiliation: Waters Corporation.
authors.forename: St John
authors.initials: SJ
authors.lastname: Skilton
authors.affiliation: Waters Corporation.
authors.forename: Jeffery R
authors.initials: JR
authors.lastname: Mazzeo
mesh_terms: D000595:Amino Acid Sequence; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics*; D059451:Biosimilar Pharmaceuticals / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D055808:Drug Discovery / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D007167:Immunotherapy / Q000379:methods*; D062069:Inventions; D013058:Mass Spectrometry; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry; D012984:Software; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D059451:Biosimilar Pharmaceuticals; D007074:Immunoglobulin G; D011134:Polysaccharides; D000068878:Trastuzumab
references.citation: Eur J Biochem. 1979 Sep;99(2):323-31
references.pmid: 387413
references.citation: J Am Soc Mass Spectrom. 2008 Nov;19(11):1643-54
references.pmid: 18707900
references.citation: Protein Sci. 1993 Mar;2(3):331-8
references.pmid: 8453372
references.citation: Eur J Biochem. 1977 Feb;72(3):443-52
references.pmid: 320001
references.citation: Anal Chem. 2006 Sep 15;78(18):6645-50
references.pmid: 16970346
references.citation: Nat Biotechnol. 2007 Feb;25(2):145-6
references.pmid: 17287732
references.citation: Rapid Commun Mass Spectrom. 2005;19(16):2331-6
references.pmid: 16041822
references.citation: Mol Cell Proteomics. 2006 Jan;5(1):144-56
references.pmid: 16219938
references.citation: Proteomics. 2009 Mar;9(6):1683-95
references.pmid: 19294628
references.citation: Nat Rev Drug Discov. 2009 Mar;8(3):226-34
references.pmid: 19247305
references.citation: J Am Soc Mass Spectrom. 2009 Nov;20(11):2021-33
references.pmid: 19744865
references.citation: Trends Pharmacol Sci. 2009 Jul;30(7):356-62
references.pmid: 19552968
references.citation: J Chromatogr B Biomed Sci Appl. 2001 Mar 10;752(2):233-45
references.pmid: 11270864
references.citation: Anal Chem. 2005 Sep 15;77(18):6004-11
references.pmid: 16159134
references.citation: Curr Pharm Biotechnol. 2008 Dec;9(6):482-501
references.pmid: 19075687
references.citation: J Biol Chem. 1982 Mar 25;257(6):3018-25
references.pmid: 6277922
references.citation: Curr Pharm Biotechnol. 2008 Dec;9(6):423-30
references.pmid: 19075682
references.citation: Mass Spectrom Rev. 2007 May-Jun;26(3):370-88
references.pmid: 17410555
references.citation: Pharm Res. 1989 Nov;6(11):903-18
references.pmid: 2687836
references.citation: Curr Pharm Biotechnol. 2008 Dec;9(6):421-2
references.pmid: 19075681
references.citation: Anal Chem. 2008 Dec 15;80(24):9491-8
references.pmid: 19072263
references.citation: N Engl J Med. 2008 Mar 13;358(11):1109-17
references.pmid: 18337601
references.citation: J Pept Res. 2004 May;63(5):437-48
references.pmid: 15140161
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2005 May 25;819(2):203-18
references.pmid: 15833284
references.citation: MAbs. 2009 Sep-Oct;1(5):394-416
references.pmid: 20065643
references.citation: Anal Chem. 2005 Apr 1;77(7):2187-200
references.pmid: 15801753
references.citation: Anal Chem. 2009 Jul 15;81(14):5699-708
references.pmid: 19518054
references.citation: Nat Biotechnol. 2008 Sep;26(9):962-3
references.pmid: 18779793
references.citation: Rapid Commun Mass Spectrom. 2007;21(5):730-44
references.pmid: 17279597
references.citation: Protein Sci. 1999 Nov;8(11):2519-23
references.pmid: 10595558
references.citation: Pharm Res. 1994 May;11(5):764-71
references.pmid: 8058650
references.citation: J Proteome Res. 2006 Apr;5(4):1003-9
references.pmid: 16602709
references.citation: Anal Biochem. 2009 Sep 15;392(2):145-54
references.pmid: 19497295
references.citation: J Biopharm Stat. 2010 Jan;20(1):46-61
references.pmid: 20077248
references.citation: Science. 2006 Aug 4;313(5787):670-3
references.pmid: 16888140
references.citation: Biochemistry. 1989 Feb 7;28(3):1377-84
references.pmid: 2713370
references.citation: MAbs. 2009 Jul-Aug;1(4):332-8
references.pmid: 20073133
references.citation: Pharmacogenet Genomics. 2009 May;19(5):383-7
references.pmid: 19319024
references.citation: Pharm Res. 1992 Nov;9(11):1386-93
references.pmid: 1475223
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 1;878(3-4):403-8
references.pmid: 20036624
references.citation: Anal Biochem. 2009 Sep 1;392(1):12-21
references.pmid: 19457431
references.citation: Nat Rev Drug Discov. 2009 Mar;8(3):181
references.pmid: 19247295