Interchain disulfide bonding in human IgG2 antibodies probed by site-directed mutagenesis.
Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.
Allen
Martin J
MJ
Process and Product Development, Amgen, Inc., Seattle, Washington 98119, USA. mart.j.allen@gmail.com
Guo
Amy
A
Martinez
Theresa
T
Han
Mei
M
Flynn
Gregory C
GC
Wypych
Jette
J
Liu
Yaoqing Diana
YD
Shen
Wenyan D
WD
Dillon
Thomas M
TM
Vezina
Christopher
C
Balland
Alain
A
D019943:Amino Acid Substitution / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D003545:Cysteine