Recently, different authors have reported a two-peak elution behavior for a number of monoclonal antibodies (mAbs) when purified on strong cation exchange resins. The two-peak elution behavior is observed when a prolonged hold time after injection is applied, while the antibody elutes as a single peak during salt gradient elution without a hold time. In this work, the influence of ligand density variations on the elution behavior of an antibody and its purified charge variants is investigated. Ligand density variation is known to influence protein separation and affect process robustness. Three strong cation exchange prototypes with different ionic capacities (196, 256 and 334 mmol/Lpore ) were used. Additionally, the influence of several operating parameters (pH, column loading, flow rate/residence time and Na+ concentration during the bind/elute process) on the extent of the double peak pattern was tested. Analytical cation exchange chromatography (CEX) and size exclusion chromatography (SEC) was used to assess the change in the distribution profile of the charge variants and aggregate formation, respectively. The thermal stability of proteins was measured by screening experiments using the nanoDSF technology. The results show that a higher ligand density leads to a more pronounced two-peak elution behavior. The extent of this increase depends on the type of mAb charge variant (acid, neutral or basic species). Results from charge variants unfolding experiments do not show a clear trend between the thermodynamic stability of the protein and the two-peak pattern. Moreover, results from pH and Na+ variations indicate that the local environment in the pore volume the protein is exposed to may influence the elution behavior. To understand these effects, it is important to consider the complex ionic equilibrium between the mobile and the stationary phase. The Donnan equilibrium describes the intraparticle conditions and helps to elucidate the correlation observed between the charge variants and the ligand density of the resin.

Influence of ligand density on two-peak elution behavior of mAb charge variants in cation exchange chromatography

2019 Spring National Meeting

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