Evidence for trisulfide bonds in a recombinant variant of a human IgG2 monoclonal antibody.
The hinge region of human IgG2 contains four cysteine residues involved in disulfide linkages between the heavy chains, as well as the heavy and light chains. These linkages provide the fundamental framework of three distinct IgG2 disulfide isoforms recently described. Here, we detail another, disulfide-related post-translational modification in a recombinant variant of human IgG2. Heterogeneity associated with this antibody was separated into several fractions by anion-exchange chromatography (AEX), which is an important initial step that highlights the resolving power of surface charge-based HPLC techniques. Mass spectrometry of the intact antibody revealed weakly resolved discrete covalent additions of 25-35 Da in one of the two main AEX fractions. Digestion by endoproteinase Lys-C performed under nonreducing conditions, as well as tandem MS experiments, narrowed the modification to the peptide-containing disulfide-bridged hinge structure. High mass resolution and accuracy measurements of the peptide strongly suggested an addition of one or two S atoms. The modification could be eliminated by a mild reducing treatment of the intact antibody. Overall, these findings are consistent with the replacement of up to two disulfide bridges (S-S) with a like number of trisulfides (S-S-S) in the antibody hinge. The trisulfide modification is rather uncommon for proteins and its possible origins in the IgG2 variant are discussed.
Pristatsky
Pavlo
P
Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Cohen
Steven L
SL
Krantz
Debra
D
Acevedo
Jillian
J
Ionescu
Roxana
R
Vlasak
Josef
J
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013440:Sulfides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013440:Sulfides; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase