Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2

Abstract

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.

Keywords: Antibody structure; DTT; Disulfide formation; Disulfide isoforms; EDTA; GFB; HC; HL; L-1-tosylamido-2-phenylethyl chloromethyl ketone; LC; LC/MS; N-ethylmaleimide; NEM; Oxidation–reduction; Protein degradation; SDS-PAGE; SEC; TFA; TPCK; V(o); dithiothreitol; ethylene diamine tetraacetic acid; glufibrinopeptide B; half molecule; heavy chain; light chain; liquid chromatography/mass spectrometry; size-exclusion chromatography; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; trifluoroacetic acid; void volume.