title: IgG2 disulfide isoform conversion kinetics.
abstract: Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.
authors.affiliation: Department of Process and Product Development, Amgen Inc., Thousand Oaks, CA 91320, USA.
authors.forename: Yaoqing Diana
authors.initials: YD
authors.lastname: Liu
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
authors.forename: Robert
authors.initials: R
authors.lastname: Chou
authors.forename: Louise
authors.initials: L
authors.lastname: Chen
authors.forename: Gunasekaran
authors.initials: G
authors.lastname: Kannan
authors.forename: Riki
authors.initials: R
authors.lastname: Stevenson
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Goetze
authors.forename: Xinzhao Grace
authors.initials: XG
authors.lastname: Jiang
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Dillon
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D000818:Animals; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D020033:Protein Isoforms