Trisulfide is a post-translational modification (PTM) commonly found in recombinant antibodies. It has been demonstrated that trisulfide had no impact on the bioactivity of mono-specific antibodies (MsAbs). However, the impact of trisulfide on multi-specific antibodies has not been evaluated. In this study, two mass spectrometric methods were developed for comprehensive trisulfide characterization. The non-reduced peptide mapping method combined with the unique electron activated dissociation (EAD) provided signature fragments for confident trisulfide identification as well as trisulfide quantitation at individual sites. A higher throughput method using Fab mass analysis was also developed and qualified to support routine monitoring of trisulfide during process development. Fab mass analysis features simpler sample preparation and shorter analysis time but provides comparable results to the non-reduced peptide mapping method. In this study, a bi-specific (BsAb) and a tri-specific antibody (TsAb) were compared side-by-side with a MsAb to evaluate the impact of trisulfide on the structure and function of multi-specific antibodies. Results indicated that trisulfide dominantly formed at similar locations across different antibody constructs and had no impact on the size heterogeneity, charge heterogeneity, or bioactivities of any assessed antibodies. Together with the in vitro stability under heat stress (25 °C and 40 °C for up to four weeks) and rapid conversion from trisulfide to disulfide during in vivo circulation, trisulfide could be categorized as a non-critical quality attribute (non-CQA) for antibody products.
Keywords: Antibody; Biopharmaceutical characterization; HPLC (high-performance/pressure liquid chromatography); Mass Spectrometry (MS); Protein structure.
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