Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains.
A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.
Tous
Guillermo I
GI
Department of Analytical Biochemistry, MedImmune, Inc., One MedImmune Way, Gaithersburg, Maryland 20878, USA. tousg@medimmune.com
Wei
Ziping
Z
Feng
Jinhua
J
Bilbulian
Susanna
S
Bowen
Stephen
S
Smith
Jaime
J
Strouse
Robert
R
McGeehan
Patrick
P
Casas-Finet
Jose
J
Schenerman
Mark A
MA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D013440:Sulfides / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D013440:Sulfides