How do metal ions, hydrogen peroxide, and temperature increase monoclonal antibody fragmentation?

Monoclonal antibodies (mAbs) are protein-based medicines used to treat various life-threatening diseases, including cancers. However, the proteinaceous nature of these medicines makes them susceptible to degradation through multiple pathways, including aggregation, fragmentation, oxidation, isomerization, deamidation, and so on. These degradation products reduce the medicine’s effectiveness and induce side effects in the patients. Fragmentation is one of the degradation pathways and is defined as the disruption of chemical bonds, including peptide bonds in a protein, via enzymatic or non-enzymatic reactions. Usually, peptide bonds of these protein-based medicines are extremely stable to non-enzymatic hydrolysis with a half-life of hundreds of years. However, various factors make them prone to hydrolysis, including pH, temperature, the flexibility of protein backbone, the side chain of amino acids, presence of metal ions and free radicals. The peptide bond of the hinge region of the mAbs is flexible and more susceptible to degradation. In this study, we have demonstrated how trace metal ion and hydrogen peroxide levels promote the degradation of the mAb hinge region and their synergistic effect on the degradation. This study shows that with an increase in the level of these impurities, the rate of fragmentation increases. Thus, it is important to avoid adding or reducing these impurities throughout the manufacturing of the product. This study also shows that lowering storage temperature reduces degradation.