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

ABSTRACT

Non-enzymatic hinge fragmentation of monoclonal antibodies (mAb) is considered a critical quality attribute since it changes the primary sequence of the proteins, thereby leading to structural changes which can affect stability, function, and efficacy. While peptide bonds are exceptionally stable under physiological conditions, reactive side chains of a few residues, the flexibility of the backbone, and physicochemical parameters such as pH, temperature, and the reaction of radicals and metal ions can promote the cleavage of peptide bonds. In this study, the relative extent and rate of fragmentation are compared with respect to the presence of several different factors (including hydrogen peroxide, metal ion, and temperature) as measured by size exclusion chromatography. A kinetic model of monomer degradation as a function of time (mAb only) is created. In the presence of either H2O2 or Cu2+, or both, the reaction kinetics follow different orders depending on the reaction conditions. The half-life for peptide bond cleavage of the mAb hinge region was 385 days at 40 °C and decreases to 250, 48, and 45 days in the presence of H2O2, Cu2+, and a combination of H2O2 and Cu2+, respectively. A temperature dependence of peptide bond cleavage at 35 °C, 40 °C, 45 °C, and 50 °C showed Arrhenius behavior with an apparent activation energy of 76.9 ± 16.4 kJ/mol. The reaction rates obtained from the Arrhenius equation were then extrapolated to predict fragmentation rates under real storage conditions (e.g., at 2-8 °C). We demonstrate that trace levels of impurities including peroxide left after surface sterilization or degradation of non-ionic surfactants or metal ions from the buffer components can significantly affect the stability of a mAb.