Thermo Fisher Scientific Richmond, Virginia, United States
Purpose: Two biotherapeutic monoclonal IgG1 antibodies (mAb X and mAb Y) are being investigated for the prophylaxis and treatment of COVID-19. To support PK assessment in the humans, a hybrid LC-MS/MS method was developed and validated for the simultaneous measurement of two biotherapeutic mAbs in human serum. Methods: An immunoaffinity approach using streptavidin magnetic beads coated with biotinylated receptor binding domain (RBD) of SARS-Cov-2 was used to enrich the two analytes from human serum. The immunocaptured analytes were subjected to proteolysis with trypsin, following standard protein denaturation, reduction, and alkylation processing steps. Two signature peptides ( X1, X2 and Y1,Y2) from each analyte monoclonal antibody were identified and monitored as quantitative and qualitative surrogate analytes. Stable isotope labeled (SIL) forms the four signature peptides were used as internal standards. Analyte separation was achieved using reversed phase chromatography on a Waters Acquity BEH C8 column. Analytes were detected with a Sciex 6500+ triple quadrupole instrument using electrospray ionization (ESI)/multiple reaction monitoring (MRM) mode. Results: Two signature peptides for each analyte were identified using in-silico digestion. The method was fully validated according to FDA guidelines. The method is specific and sensitive with a lower limit of quantitation (LLOQ) of 1 µg/mL. A quadratic calibration curve was obtained over the range of 1-100 µg/mL for both analytes. The inter-assay precision and accuracy, across three separate runs, were found to be in within 6% and ±4 %, respectively for both analytes. All other validation experiments, including dilutional linearity, selectivity, cross-analyte interference, frozen matrix stability at -25°C and -80°C, thawed matrix (on ice) stability, extract storage stability, reinjection reproducibility, recovery, matrix factor, hemolysis, lipemia, and capture capacity were demonstrated to be within acceptable criteria. Conclusion: A hybrid LC-MS/MS method was developed and validated for the simultaneous measurement of two COVID-19 biotherapeutic mAbs in human serum for use in clinical application.
