Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

Abstract

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale.

Adsorption equilibrium and kinetics of monomer–dimer monoclonal antibody mixtures on a cation exchange resin

“…In conclusion, it should be noted that multimodal chromatography (MMC), where electrostatic and hydrophobic interactions are combined, has also recently emerged as a viable alternative for monomer-aggregate resolution [52]. While, obviously, our results are not directly applicable to MMC, we think that many of the experimental approaches used here could be used readily to obtain insights about the fundamental underpinnings of MMC.…”

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…Chen et al () was able to not only achieve aggregate separation but also isolated charge variant impurities into separate peaks. However, MMC applications to charge variant separation have been limited, and most of the applications have focused on resolving aggregation (Chmielowski et al, ; Gao, Wang, Lin, & Yao, ; Gospodarek, Hiser, O'Connell, & Fernandez, ; Wang, Zhou, Gowtham, Harcum, & Husson, ). As most of the MMC research has focused on flow‐through mode, more research is needed for understanding bind‐and‐elute mode operation to design more economically viable resins (Liu, Wickramasinghe, & Qian, ).…”

High‐capacity multimodal anion‐exchange membranes for polishing of therapeutic proteins

“…These results align with a study by Weaver et al 32 Increased hydrophobicity and charged patches of aggregates lead to better clearance by ligands with electrostatic and hydrophobic moieties. 33 Purexa™-MQ is well suited for clearance of HMW species at higher conductivities or in the presence of phosphate, and could provide an orthogonal step capable of clearing HCPs with a different profile than typical AEX technologies, which show similar profiles for cleared HCPs. 34 This is a noted advancement in technologies that can improve manufacturing efficiency of antibody processes while maintaining or improving product quality.…”