Quantification and characterization of antibody deamidation by peptide mapping with mass spectrometry

Capturing a complete assessment of deamidation in monoclonal antibodies is challenging due to the structural complexity of multiple potential deamidation sites and deamidation pathways. In this study, a peptide mapping approach has been developed to quantify the extent of deamidation of a therapeutic recombinant monoclonal antibody. To obtain an accurate measurement, a rapid sample preparation procedure was developed to minimize formation of deamidation during sample preparation and analysis. Ammonium formate mobile phase was used in the reversed phase separation to completely separate deamidated peptides from their native peptides. To improve detection sensitivity and prevent interference from chemical background noise and coeluting peptides, mass spectrometry (MS) was utilized to quantify the low levels of deamidation in the product. The method was subsequently qualified as a characterization test for comparability studies, forced degradation studies and for characterizing reference standards. The method demonstrated suitable linearity, precision and accuracy. The limit of detection (LOD) and the limit of quantification (LOQ) of the method for specific deamidation sites were estimated to be as low as 0.1% and 0.3%, respectively. The deamidation sites and the deamidation products were identified using several orthogonal methods including tandem MS, N-terminal sequencing and protein isoaspartate methyl transferase (PIMT) enzymatic reactions.

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► A rapid peptide mapping method with MS was developed for deamidation quantification.
► The method was developed and qualified for a recombinant monoclonal antibody.
► The method is specific for multiple deamidation sites and deamidation products.