title: Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.
abstract: Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.
authors.affiliation: Genor Biopharma Co., Ltd., Shanghai, China.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Xu
authors.forename: Jason
authors.initials: J
authors.lastname: Li
authors.forename: Joe X
authors.initials: JX
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D016466:CHO Cells; D002411:Cation Exchange Resins / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration
chemical_list: D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins; D002412:Cations