Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry.

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.