Determination of Fab−Hinge Disulfide Connectivity in Structural Isoforms of a Recombinant Human Immunoglobulin G2 Antibody

• Published in: Analytical chemistry (Washington) Vol. 82; no. 3; pp. 1090 - 1099
• Main Authors: Zhang, Bing, Harder, Adam G, Connelly, Heather M, Maheu, Lorna L, Cockrill, Steven L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.2010
• Subjects: Amino Acid Sequence; Analytical chemistry; Antibodies, immunoglobulins; Biological and medical sciences; Chemical compounds; Disulfides - analysis; Disulfides - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin G - chemistry; Immunoglobulin G - genetics; Immunoglobulins; Molecular immunology; Molecular Sequence Data; Molecular structure; Peptide Mapping - methods; Peptides; Protein Isoforms - chemistry; Protein Isoforms - genetics; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Human; Cysteine; IgG2; Peptides; Antibody; Disulfide bond
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/ac902466z

The detection and characterization of unexpected disulfide-mediated structural variants of human immunoglobulin G2 (IgG2) antibodies was recently the subject of two copublications. , In this paper, we present data to confirm the previously reported structures and elucidate the complete disulfide connectivity of each variant through the application of a novel analytical methodology. In this manner, the data illustrate the presence of at least five structural variants, including the classical structure with independent Fab domains and a hinge region. Multiple subvariants of the IgG2-A/B and IgG2-B structures are identified; these subvariants of each structure differ through the order of attachment of Fab peptides to the sequential hinge cysteines. Furthermore, the connectivity of a novel subvariant of IgG2-B containing an intrachain disulfide linkage in the lower hinge region is elucidated. The results presented in this paper reveal that the population of IgG2 disulfide structural variants is yet more complex than recently reported.