Maleimide functionalized polyethylene glycol (PEG) with varying degrees of branched chains is gaining increasing interest in drug conjugation. Characterization of these molecules in solution is highly challenging due to the multiplicity of the hydrolysis products and the subtle differences between them, especially for high molecular weight PEG-maleimide. A highly selective, sensitive and stability indicating HPLC method employing charged surface hybrid (CSH) stationary phase was developed for 8-arm PEG-maleimide characterization. The method has been validated per ICH Q2 guideline for specificity, sensitivity, linearity, accuracy and precision for quality and process control. Hydrolysis of 8-arm PEG-maleimide in different buffer media, pH and temperature was studied. The kinetics revealed the hydrolysis is a first-order reaction. Hydrolysis rate constant and half-life were obtained to guide protein conjugation process development.
Keywords: Conjugation; Maleimide; Maleimide hydrolysis; Method development; Multi-arm polymer; Polyethylene glycol (PEG).
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