Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography.
Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG(1) monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG(1) mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG(1) mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG(1) molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture.
Aono
Hiromasa
H
Biogen Idec Inc., Department of Bioprocess Development, 14 Cambridge Center, Cambridge, MA 02142, USA.
Wen
Dingy
D
Zang
Li
L
Houde
Damian
D
Pepinsky
R Blake
RB
Evans
David R H
DR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D013440:Sulfides; D003545:Cysteine