Separation of closely related monoclonal antibody charge variant impurities using poly(ethylenimine)-grafted cation-exchange chromatography resin

The removal of protein charge variants due to complex chemical and enzymatic modifications like glycosylation, fragmentation and deamidation presents a significant challenge in the purification of monoclonal antibodies (mAb) and complicates downstream processing. These protein modifications occur either in vivo or during fermentation and downstream processing. The presence of charge variants can lead to diminished biological activity, differences in pharmacokinetics, pharmacodynamics, stability and efficacy. Therefore, these different product variants should be appropriately controlled for the consistency of product quality and to ensure patient safety. This investigation focuses on the development of a chromatography step for the removal of the charge variants from a recombinant single-chain variable antibody fragment (scFv−Fc−Ab). Poly(ethyleneimine)-grafted cation-exchange resins (Poly CSX and Poly ABX) were evaluated and compared to traditional macroporous cation-exchange and tentacle cation-exchange resins. Linear salt gradient experiments were conducted to study the separation efficiency of scFv–Fc–Ab variants using different resins. A classical thermodynamic model was used to develop a mechanistic understanding of the differences in charge variant retention behaviour of different resins. High selectivity in separation of scFv−Fc−Ab charge variants is obtained in the Poly CSX resin.