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

Autor: Dyck YFK; Department of Pharmaceutical & Medicinal Chemistry, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany; Department of Life Sciences & Technology, Berlin University of Applied Science, Seestraße 64, 13347 Berlin, Germany., Rehm D; Department of Pharmaceutical & Medicinal Chemistry, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany; ProBioGen AG, Herbert-Bayer-Straße 8, 13086 Berlin, Germany., Winkler K; ProBioGen AG, Herbert-Bayer-Straße 8, 13086 Berlin, Germany., Sandig V; ProBioGen AG, Herbert-Bayer-Straße 8, 13086 Berlin, Germany., Jabs W; Department of Life Sciences & Technology, Berlin University of Applied Science, Seestraße 64, 13347 Berlin, Germany., Parr MK; Department of Pharmaceutical & Medicinal Chemistry, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany. Electronic address: maria.parr@fu-berlin.de.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2023 Oct 25; Vol. 235, pp. 115596. Date of Electronic Publication: 2023 Jul 22.
DOI: 10.1016/j.jpba.2023.115596
Abstrakt: 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 H 2 O 2 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.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests, Karsten Winkler reports a relationship with ProBioGen AG that includes: employment and equity or stocks. Volker Sandig reports a relationship with ProBioGen AG that includes: employment and equity or stocks. Yan Dyck reports equipment, drugs, or supplies were provided by ProBioGen AG. Daniel Rehm reports financial support and equipment, drugs, or supplies were provided by ProBioGen AG. Daniel Rehm reports a relationship with ProBioGen AG that includes: employment.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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