Abstract
Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs.
Highlights
As is generally the case for biopharmaceuticals, therapies with monoclonal antibodies are highly expensive, and access is limited worldwide
A middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS)
The posttranslational modifications (PTM) profiles of BS and reference product (RP) were compared in unstressed samples of bevacizumab and infliximab (Figures 1A and 2A)
Summary
As is generally the case for biopharmaceuticals, therapies with monoclonal antibodies (mAbs) are highly expensive, and access is limited worldwide. Kim et al applied H2O2 on infliximab By peptide mapping they showed methionine oxidations that were comparable between the reference product and the biosimilar [29]. Some studies have applied forced oxidation as well as other forced degradation treatments on bevacizumab and infliximab for analysis of aggregate formation, for binding assays and to support method validation [30,31,32,33]. As these studies were conducted on the intact mAbs, no structural information about PTMs introduced by forced degradation was provided. The results of the faster middle-up method are checked for consistency to published data obtained from the bottom-level [29]
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