Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore\nrepresent critical quality attributes (CQA) that require evaluation. To complement classical CQA,\nbevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to\nprobe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing\nliquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2\nsubunit was completely oxidized. Additional oxidations were found in the light chain (LC) and\nin the Fdâ?? subunit of infliximab, but not in bevacizumab. By direct comparison of methionine\npositions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fdâ??. The forced\noxidation approach was further exploited for comparison of respective biosimilar products. Both for\nbevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high\nsimilarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab,\ncomparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP.\nIt may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment,\nas well as for quality control of protein drugs.
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