Comparison of middle- and bottom-up mass spectrometry in forced degradation studies of bevacizumab and infliximab.

Monoclonal antibodies (mAbs) used as therapeutics need comprehensive characterization for appropriate quality assurance. For analysis, cost-effective methods are of high importance, especially when it comes to biosimilar development which is based on extended physicochemical characterization. The use of forced degradation to study the occurrence of modifications for analysis is well established in drug development and may be used for the evaluation of critical quality attributes (CQAs). For mAb analysis different procedures of liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses are commonly applied. In this study the middle-up approach is compared to the more expensive bottom-up analysis in a forced oxidation biosimilar comparability study. Bevacizumab and infliximab as well as biosimilar candidates for the two mAbs were forcefully oxidized by H2 O2 for 24, 48 and 72 h. For bottom-up, the reduced and alkylated trypsin or Lys-C digested samples were analysed by LC-MS with quadrupole time-of-flight mass analyser (LC-QTOF-MS) to detect susceptible residues. By middle-up analysis several species of every subunit (Fc/2, light chain and Fd') were detected which differed in the number of oxidations. For the most abundant species, results from middle-up were in line with results from bottom-up analysis, confirming the strength of middle-up analysis. However, for less abundant species of some subunits, results differed between the two approaches. In both mAbs, the Fc was extensively oxidized. In infliximab, additional extensive oxidation was found in the Fab. Assignment to specific amino acid residues was finally possible using the results from bottom-up analyses. Interestingly, the C-terminal cysteine of the light chain was partially found triply oxidized in both mAbs. The comparison of susceptibility to oxidation showed high similarity between the reference products and their biosimilar candidates. It is suggested that the findings of middle-up experiments should be complemented by bottom-up analysis to confirm the assignments of the localization of modifications. Once the consistency of results has been established, middle-up analyses are sufficient in extended forced degradation biosimilar studies.