Selective removal of closely related clipped protein impurities using poly(ethylenimine)- grafted anion-exchange chromatography resin

Abstract Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k′) and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.