Monoclonal antibodies (mAbs) are inherently heterogeneous molecules. Protein post-translational modifications (PTMs) can arise at different stages of antibody manufacturing or during drug product storage. Even though forced degradation studies are performed at relatively harsh conditions within a short time period, the information gathered can provide highly relevant data of the mAbs to predict effects of real time environmental conditions. The most commonly observed pathways during forced degradation studies on monoclonal antibodies are aggregation, fragmentation, deamidation and oxidation.
Liquid chromatography coupled with high resolution mass spectrometry (LC–HRMS) analysis has been widely utilized for protein therapeutic development and characterization. It is one of the most sensitive technologies providing site-specific identification and quantitation of PTMs in biotherapeutics such as mAbs.
In this case study, we investigate the susceptibility of oxidation and deamidation by subjecting the IgG antibody to either oxidative stress by exposing the mAb to 1% hydrogen peroxide and to basic hydrolysis by incubation with 0.1M NaOH. Samples were assessed at the intact protein, subunit and peptide level by using HR-MS. Four methionine residues, prone for oxidation after oxidative stress, could be localized, while peptide mapping analysis of samples exposed to basic hydrolysis showed clearly higher levels of deamidated peptides. These results indicate that LC-HRMS is a powerful tool in the development and quality control of (bio) therapeutics.
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