Characterization of Cysteinylation and Trisulfide Bonds in a Recombinant Monoclonal Antibody.
A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab')2 fragment generated from IdeS digestion. LC-MS analysis of nonreduced tryptic peptides indicated trisulfide bonds are associated with the interchain disulfide bonds of both A isoform and A/B isoform and cysteinylation is associated only with the A isoform. A low percentage of trisulfide bonds was detected in between the light chain and heavy chain disulfide bond of the A and A/B forms. While the majority of trisulfide bonds and cysteinylation is associated with the hinge region peptide that involves the four closely spaced cysteine residues of the heavy chain. The locations of trisulfide bond and cysteinylation were determined using a combination of Edman sequencing and LC-MS. In the A isoform, the major site of the trisulfide bond and cysteinylation is between the first disulfide bond in the hinge region. In the A/B isoform, the trisulfide was also located in between the disulfide bond that is formed by the second pair of cysteine residues.
Product Characterization, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
Adriana
A
Kita
Product Characterization, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
Gomathinayagam
G
Ponniah
Product Characterization, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
Christine
C
Nowak
Product Characterization, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
Hongcheng
H
Liu
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003545:Cysteine / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013440:Sulfides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013440:Sulfides; D003545:Cysteine