Aim: LC-MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices. Materials and methods: IgG1 was quantified by pellet digestion using an Acquity UPLC coupled with a Xevo TQ-S mass spectrometer. Results: Two generic LC-MS/MS methods (heavy and light chain) were developed which afforded acceptable precision and accuracy, and an lower limit of quantitation of 1 μg/ml in three preclinical matrices. A small nonsignificant bias was observed when cynomolgus serum LC-MS/MS results were compared with electrochemiluminescent immunoassay data.
Conclusion: Glu-C was successfully used as an alternative digestion enzyme for bottom-up quantitation of an IgG1 in matrices from multiple preclinical species, with good agreement with electrochemiluminescent immunoassay data.
Keywords: Bispecific antibody; ECLIA; Glu-C; IgG1; LC–MS/MS; method comparison; pellet digestion.