Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion.
Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Yi
Y
Wang
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Xiaojuan
X
Li
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Yan-Hui
YH
Liu
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Daisy
D
Richardson
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Huijuan
H
Li
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Mohammed
M
Shameem
a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , NJ , USA.
Xiaoyu
X
Yang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014357:Trypsin
High throughput; monoclonal antibody; multi-attribute liquid chromatography-mass spectrometry method; peptide mapping; product quality attributes