Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody.
Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase-high-performance liquid chromatography mass spectrometry (RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.
Zhang
Wei
W
Wyeth BioPharma, One Burtt Road, Andover, MA 01810, USA. wei.zhang@immunogen.com
Marzilli
Lisa A
LA
Rouse
Jason C
JC
Czupryn
Marta J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D019791:Guanidine; D003545:Cysteine; D005033:Ethylmaleimide