Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody - PubMed

Affiliations

• PMID: 21900054
• DOI: 10.1016/j.jchromb.2011.08.015

Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody

Diya Ren et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2011.

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.

Similar articles

• Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines.

  Fu J, Bongers J, Tao L, Huang D, Ludwig R, Huang Y, Qian Y, Basch J, Goldstein J, Krishnan R, You L, Li ZJ, Russell RJ. Fu J, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Nov 1;908:1-8. doi: 10.1016/j.jchromb.2012.09.023. Epub 2012 Sep 26. J Chromatogr B Analyt Technol Biomed Life Sci. 2012. PMID: 23122394

• Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry.

  Scott RA, Rogers R, Balland A, Brady LJ. Scott RA, et al. MAbs. 2014;6(6):1453-63. doi: 10.4161/mabs.36222. MAbs. 2014. PMID: 25484040 Free PMC article.

• Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells.

  Guo D, Gao A, Michels DA, Feeney L, Eng M, Chan B, Laird MW, Zhang B, Yu XC, Joly J, Snedecor B, Shen A. Guo D, et al. Biotechnol Bioeng. 2010 Sep 1;107(1):163-71. doi: 10.1002/bit.22780. Biotechnol Bioeng. 2010. PMID: 20506532

• Amino acid misincorporation in recombinant proteins.

  Wong HE, Huang CJ, Zhang Z. Wong HE, et al. Biotechnol Adv. 2018 Jan-Feb;36(1):168-181. doi: 10.1016/j.biotechadv.2017.10.006. Epub 2017 Oct 26. Biotechnol Adv. 2018. PMID: 29107148 Review.

• Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS.

  Ruppen I, Verscheure L, Vandenheede I, Ortiz A, de Melo IS, Liebig T, Sandra P, Beydon ME, Sandra K. Ruppen I, et al. J Pharm Biomed Anal. 2023 Nov 30;236:115743. doi: 10.1016/j.jpba.2023.115743. Epub 2023 Sep 22. J Pharm Biomed Anal. 2023. PMID: 37757547 Review.

Cited by

• Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry.

  Griaud F, Winter A, Denefeld B, Lang M, Hensinger H, Straube F, Sackewitz M, Berg M. Griaud F, et al. MAbs. 2017 Nov/Dec;9(8):1337-1348. doi: 10.1080/19420862.2017.1366395. Epub 2017 Aug 28. MAbs. 2017. PMID: 28846476 Free PMC article.

• Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis.

  Harris C, Xu W, Grassi L, Wang C, Markle A, Hardman C, Stevens R, Miro-Quesada G, Hatton D, Wang J. Harris C, et al. MAbs. 2019 Nov-Dec;11(8):1452-1463. doi: 10.1080/19420862.2019.1667740. Epub 2019 Oct 1. MAbs. 2019. PMID: 31570042 Free PMC article.

• Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies.

  Beck A, Liu H. Beck A, et al. Antibodies (Basel). 2019 Feb 19;8(1):18. doi: 10.3390/antib8010018. Antibodies (Basel). 2019. PMID: 31544824 Free PMC article. Review.

• Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm.

  Boyd D, Ebrahimi A, Ronan S, Mickus B, Schenauer M, Wang J, Brown D, Ambrogelly A. Boyd D, et al. MAbs. 2018 Apr;10(3):346-353. doi: 10.1080/19420862.2018.1438795. Epub 2018 Mar 20. MAbs. 2018. PMID: 29537936 Free PMC article.

• Influence of glycosylation pattern on the molecular properties of monoclonal antibodies.

  Zheng K, Yarmarkovich M, Bantog C, Bayer R, Patapoff TW. Zheng K, et al. MAbs. 2014 May-Jun;6(3):649-58. doi: 10.4161/mabs.28588. Epub 2014 Mar 24. MAbs. 2014. PMID: 24662970 Free PMC article.

MeSH terms

Substances