Conformational difference in human IgG2 disulfide isoforms revealed by hydrogen/deuterium exchange mass spectrometry.
Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies. By comparing the protection factors between amino acid residues in isoforms B and A1 (the classical form), we successfully identified several local regions in which the IgG2-B isoform showed more solvent protection than the IgG2-A1 isoform. On the basis of three-dimensional structural models of IgG2, these identified regions were located on the Fab domains, close to the hinge, centered on the side where the two Fab arms faced each other in spatial proximity. We speculated that in the more solvent-protected B isoform, the two Fab arms were brought into contact by the nonclassical disulfide bonds, resulting in a more compact global structure. Loss of Fab domain flexibility in IgG2-B could limit its ability to access cell-surface epitopes, leading to reduced antigen binding potency. The A2 isoform was previously found to have disulfide linkages similar to those of the classical A1 isoform, but with different biophysical behaviors. Our data indicated that, compared to IgG2-A1, IgG2-A2 had less solvent protection in some heavy-chain Fab regions close the hinge, suggesting that the A2 isoform had more flexible Fab domains.
Process Development, Amgen, Inc., Thousand Oaks, California 91320, United States.
Aming
A
Zhang
Process Development, Amgen, Inc., Thousand Oaks, California 91320, United States.
Jing
J
Fang
Process Development, Amgen, Inc., Thousand Oaks, California 91320, United States.
Robert Y-T
RY
Chou
Process Development, Amgen, Inc., Thousand Oaks, California 91320, United States.
Pavel V
PV
Bondarenko
Process Development, Amgen, Inc., Thousand Oaks, California 91320, United States.
Zhongqi
Z
Zhang
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D020836:Protein Structure, Quaternary
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G