Quantitation of Site-Specific Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs.
During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.
School of Pharmacy, Second Military Medical University , Guohe Road, Shanghai, 200433, China.|Department of Pharmacy, Shanghai General Hospital , Haining Road, Shanghai, 200080, China.
Nan
N
Yang
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Elisha
E
Goonatilleke
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Dayoung
D
Park
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Ting
T
Song
School of Pharmacy, Second Military Medical University , Guohe Road, Shanghai, 200433, China.|Department of Pharmacy, Shanghai General Hospital , Haining Road, Shanghai, 200080, China.
Guorong
G
Fan
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Carlito B
CB
Lebrilla
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase