Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography.

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation.

Mechanistic modeling of ligand density variations on anion exchange chromatography.

Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion-exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps. Ligand density is known to be one of the main lot-to-lot variations, affecting protein adsorption and separation performance. The use of a model-based approach can be an effective tool for comprehending the impact of parameter variations (e.g., ligand density) and their influence on the process. The objective of this work was to apply mechanistic modeling to gain a deeper understanding of the influence of ligand density variations in anion exchange chromatography. To achieve this, 13 prototype resins having the same support as the strong anion exchange resin Fractogel® EMD TMAE (M), but differing in ligand density, were analyzed. Linear salt gradient elution experiments were performed to observe the elution behavior of a monoclonal antibody and bovine serum albumin. A proposed isotherm model for ion exchange chromatography, describing the dependence of ligand density variations on protein retention, was successfully applied.