The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress

Journal of Pharmaceutical Sciences
Joon H HuhMasazumi Matsumura

Abstract

Human immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) antibodies contain multiple disulfide bonds, which are an integral part of the structure and stability of the protein. Open disulfide bonds have been detected in a number of therapeutic and serum derived antibodies. This report details a method that fluorescently labels free cysteine residues, quantifies, and identifies the proteolytic fragments by liquid chromatography coupled to online mass spectrometry. The majority of the open disulfide bonds in recombinant and serum derived IgG1 and IgG2 antibodies were in the constant domains. This method was applied to the identification of cysteines in an IgG2 antibody that are involved in the formation of covalent intermolecular bonds because of the application of a severe agitation stress. The free cysteine in the CH 1 domain of the IgG2 decreased upon application of the stress and implicates open disulfide bonds in this domain as the likely source of free cysteines involved in the formation of intermolecular disulfide bonds. The presence of comparable levels of open disulfide bonds in recombinant and endogenous antibodies suggests that open disulfide bonds are an inherent feature of antibodies and that the susceptibility of ...Continue Reading

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Citations

Feb 19, 2016·Pharmaceutical Research·Saradha ChandrasekharElizabeth M Topp
May 18, 2016·Journal of Immunology Research·Roy Jefferis
Oct 15, 2014·Journal of Pharmaceutical Sciences·Xue XiaMichael Blaber
Mar 8, 2018·MAbs·Alexandre AmbrogellyHongcheng Liu
Dec 17, 2016·Electrophoresis·Bernd Moritz, Jan Olaf Stracke
Feb 9, 2020·Scientific Reports·Youngsil SeoMyung-Hee Kwon
Jul 28, 2016·Biotechnology Progress·Hongcheng LiuAlyssa Neill
Sep 24, 2019·Antibodies·Alain Beck, Hongcheng Liu
Nov 1, 2020·Protein Engineering, Design & Selection : PEDS·Michael UlitzkaHarald Kolmar
Feb 23, 2021·Journal of Pharmaceutical Sciences·Michael T KimDavid D Weis
Oct 19, 2013·Journal of Pharmaceutical Sciences·Roxana E IacobDamian Houde
May 14, 2021·Journal of Pharmaceutical and Biomedical Analysis·Yuling Zhang, Pei Qi

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