Multi-state steric mass action model and case study on complex high loading behavior of mAb on ion exchange tentacle resin

Abstract

Tentacle resins for IEX are increasingly applied in preparative chromatography for their higher selectivity and higher capacities in comparison to IEX resins without tentacles. However, tentacle resins are often observed to cause unusual elution behavior of monoclonal antibodies under high loading conditions. Understanding this elution behavior is important for a quality by design approach, as it is now mandated by regulatory agencies. A model-based analysis of load, wash and gradient elution is performed for a monoclonal antibody (mAb) on Fractogel SO3−. Four experiments with increasing loaded mass show complex peak shapes and formation of a shoulder under overloaded conditions. We hypothesize that the observed peak shapes are caused by mAbs binding in multiple states on the tentacle ion-exchange resin. A new multi-state SMA binding model is used for testing this hypothesis. A two-state binding model is found to quantitatively reproduce all four experiments. An in-depth analysis reveals that the shoulder formation under overloaded conditions can be explained by multi-state binding that particularly manifests in rapid but weak re-adsorption of eluting molecules near the column end. The introduced multi-state SMA model combines features of the so-called spreading model (multiple bound states) and of the standard SMA model (salt dependency). It is by no means limited to ion-exchange chromatography on tentacle resins, but the same concept can be applied for studying systems that are based on other physical mechanisms. The new model can potentially improve mechanistic understanding and facilitate quantitative simulation of various phenomena, such as caused by reorientation, reconformation or unfolding of bound species. Similar concepts can be applied for studying surface-induced aggregation and denaturation.