PEGylation kinetics of recombinant hirudin and its application for the production of PEGylated HV2 species

A critical challenge of PEGylation is the production of the desired PEGylated protein form at a high yield. In this study, a kinetic model was constructed successfully to describe the PEGylation reaction of recombinant hirudin variant-2 (HV2) with monomethoxy-PEG-succinimidyl carbonate (mPEG-SC) by fitting the experimental data. Moreover, PEGylation reaction conditions were investigated using the established model and the corresponding experiments to determine the optimal condition to achieve the mono-PEG-HV2 at the desired yield. The model predictions agreed well with the experimental data. Several important process parameters (maximum theoretical yield of mono-PEG-HV2 (y_max), critical PEG/HV2 molar ratio (m_crit) and reaction time to achieve y_max (t_max)) and their mathematical equations were obtained to determine the optimum reaction conditions. Among reaction conditions affecting the PEGylation rates, pH and temperature displayed little effect on y_max, but y_max increased as PEG size increased. Optimal reaction condition to produce mono-PEG-HV2 was as follows: pH and temperature could vary in a certain range; whereas PEG/HV2 molar ratio should be slightly greater than m_crit and the reaction should be stopped at t_max. The results of this study indicate that the proposed reaction kinetic model can provide a possible mechanism interpretation for real PEGylation reactions and optimize efficiently the PEGylation step.