Multistep denaturation and hierarchy of disulfide bond cleavage of a monoclonal antibody

Analytical Biochemistry
T Brody

Abstract

A humanized, monoclonal antibody of the IgG4 class was treated with SDS at room temperature. Analysis using SDS-PAGE revealed the progressive formation of a series of denaturation intermediates, of increasing apparent molecular weights. The detectable species migrated with apparent molecular weights of 137, 152, 162, 182, 196, and 210 kDa. Antibody not incubated in SDS had an apparent molecular weight of 137 kDa, while boiling in SDS provoked the immediate conversion of all antibody to the slowest migrating form (210 kDa). A study designed to test if all rungs of the ladder were equally cleavable by mild treatment with mercaptoethanol revealed small amounts of a novel form, and conditions were devised to generate large amounts of this novel form. The novel form, before full denaturation, migrated at the position of unheated, intact antibody (137 kDa), while full denaturation in SDS converted the novel form to a species migrating at about 190 kDa. The novel form, before full denaturation, was identified as HHL ***L, where the asterisks indicate noncovalent binding of one light chain to HHL. This noncovalent complex remains intact during SDS-PAGE. The novel form, after full denaturation, was identified as HHL. This study, with ea...Continue Reading

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Citations

Jul 6, 2012·Protein Engineering, Design & Selection : PEDS·Jonas V Schaefer, Andreas Plückthun
Sep 11, 2007·Journal of Pharmaceutical Sciences·Hongcheng LiuJoanne Sun
Jul 23, 2013·Homeopathy : the Journal of the Faculty of Homeopathy·Jeremy Swayne
Apr 20, 2021·Advanced Healthcare Materials·Qian-Ni YeJun Wang
Aug 1, 2019·Journal of the American Society for Mass Spectrometry·Christopher J BrownDavid E Clemmer

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