General CommentaryAn Automated and Qualified Platform Method for Site-Specific Succinimide (cas 123-56-8) and Deamidation Quantitation Using Low-pH Peptide Mapping
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Add time:09/08/2019 Source:sciencedirect.com
ABSTRACTMonoclonal antibodies undergo several post-translational modifications, including the formation of Succinimide (cas 123-56-8) from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of monoclonal antibodies, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here we report the development of a platform low pH peptide mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient monoclonal antibody digestion equivalent to conventional tryptic peptide mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a Good Manufacturing Practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness.
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