A universal strategy for development of a method for absolute quantification of therapeutic monoclonal antibodies in biological matrices using differential dimethyl labeling coupled with ultra performance liquid chromatography-tandem mass spectrometry.

Although the strategic use of enzymatic digestion combined with isotope dilution mass spectrometry has been increasingly developed and used for the absolute quantification of therapeutic and endogenous proteins in the biopharmaceutical industry over the past several years, the lack of an appropriate internal standard has become the rate-limiting step in the development of a standardized analytical approach to provide bioanalytical support for both preclinical and clinical studies. In this study, we present a universal strategy for fast development and validation (within 1-2 weeks) of a method for absolute quantification of a therapeutic monoclonal antibody in biological matrices using differential dimethyl labeling coupled with UPLC-MS/MS. Differential dimethyl labeling of tryptic peptides generated from the purified therapeutic monoclonal antibody and those derived from proteins in cynomolgus monkey serum with either d(2)- or d(0)-formaldehyde provided a fast, cost-effective, and standardized approach to generate internal standards for any surrogate peptides that are used to quantify the therapeutic monoclonal antibody in biological matrices. This labeling reaction employs inexpensive and commercially available reagents, d(0)- or d(2)-formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys in a peptide via reductive amination. Moreover, the process is simple, relatively fast (<2 h reaction time), specific, and quantitative under mild reaction conditions. The chromatographic run time is 6 min per sample. The linearity of the assay for the selected monoclonal antibody was established from 1.00 to 1000 mug/mL with accuracy and precision within 15% at all concentrations. The intraday and interday assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QCs (2.00, 4.00, 20.0, 100, 750 mug/mL) prepared in three different serum pools from male and female cynomolgus monkeys.