Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.
The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.
Protein Analytical Chemistry, Genentech, California 94080, USA. yonghuaz@gene.com
Taylor
T
Zhang
Jennifer
J
Zhang
Daniel
D
Hewitt
Ben
B
Tran
Xiaoying
X
Gao
Zhihua Julia
ZJ
Qiu
Max
M
Tejada
Helene
H
Gazzano-Santoro
Yung-Hsiang
YH
Kao
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D018951:Antigens, CD20 / Q000276:immunology; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D013058:Mass Spectrometry; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine