Non-enzymatic hinge fragmentation of monoclonal antibodies (mAb) through hydrolysis is considered a critical quality attribute since it changes the primary sequence leading to structural changes and thus, affecting the stability, function, and efficacy of the drug. Peptide bonds are exceptionally stable to non-enzymatic hydrolysis with a half-life of hundreds of years. However, reactive side chains of a few residues, the flexibility of the backbone, and physicochemical conditions such as pH, temperature, radicals, and metal ions affect the cleavage of peptide bonds. In this work, the relative amount and rate of fragmentation are compared in the presence of different factors (including hydrogen peroxide, metal ion, and temperature) as measured by size exclusion chromatography. Kinetic model of monomer degradation with time in control (mAb only), mAb in the presence of either H2O2 or Cu2+ or both are developed and follows first-order kinetic. The peptide bond cleavage half-life of hundreds of years declined to days. Degradation of monomer at 35 °C, 40 °C, 45 °C, and 50 °C is studied and follows Arrhenius law. This work shows that trace levels of impurities including, peroxide after surface sterilization or degradation of non-ionic surfactants; or metal ions from the buffer components, can affect the stability of mAb to a greater extent. Hence, this study suggests monitoring the trace level of metal ion and peroxide impurities throughout the process and in formulated products and control fragmentation by either using a chelating agent or superfine grades of buffer components.

Impact of various factors on the kinetics of the non-enzymatic fragmentation of monoclonal antibody

ACS Spring 2022

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