Development of a Size-Exclusion Chromatography Method to Characterize a Multimeric PEG-Protein Conjugate

Conjugation of poly(ethylene glycol) (PEG) to therapeutic molecules is a widely used technique to increase in vivo half-life of therapeutics. A multimeric PEG protein conjugate, which contains eight antigen-binding fragments (Fabs) conjugated to an 8-arm PEG core, was developed as a therapeutic candidate for age-related macular degeneration (AMD). The increased molecular size of the conjugate, compared to the Fab alone, produces a significant longer half-life, and requires less frequent intravitreal (ITV) injections, which can greatly benefit the patient. Due to the major impact that molecular size has on in vitreous half-life, it is crucial to have an analytical method to monitor the size attribute of the conjugate. Here we report a simple and robust size-exclusion chromatography (SEC) method that was developed for the conjugate. A wide range of size variants of the conjugate, ranging from 50 kDa to >1000 kDa, can be resolved and quantitated. The method was evaluated for precision, accuracy, linearity, and robustness, and was deemed suitable for routine use in product quality control.