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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.

The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps.