Separation of charge variants of a monoclonal antibody by overloaded ion exchange chromatography.

A procedure for adjusting the content of charge variants of monoclonal antibody by ion exchange chromatography has been developed. The band splitting phenomenon was utilized to split the protein load into two parts, i.e., the flowthrough and bound fractions, which were either enriched or depleted with some of variants. The phenomenon was triggered by thermodynamic effects resulting from oversaturation of the resin binding sites at high column loadings as well as from kinetic effects arising from limited rates of mass transport. Cation exchange chromatography (CEX) and anion exchange chromatography (AEX) separations were examined, with the reverse order of the variant elution: acidic, main, basic in CEX, and basic, main, acidic in AEX, and the corresponding reverse enrichment tendency in the collected fractions. The separations were performed by pH gradient, whose course was simplified to two stages: isocratic loading and washing at mild pH to load and partly elute the protein, followed by a rapid pH change towards non-binding conditions to desorb the remains of the protein load. To improve yield of the operation, possibility of recycling of waste fractions was considered. To predict the process performance, a dynamic model was developed, which accounted for both adsorption kinetics and thermodynamics.