Author: Till Briskot
  • Model development
  • |
  • IEX
  • |
  • Antibody
  • Other protein

Modeling of ion exchange chromatography: From mechanistic to empirical and back

Adsorption of colloidal proteins in IEX under consideration of charge regulation

Protein adsorption onto ion-exchange (IEX) resins is a complex process that is not only affected by the properties of the protein itself, but also by resin properties, buffer pH, and ionic strength. Commonly, protein adsorption is described by stoichiometric displacement models, taking into account a stoichiometric exchange of counter-ions bound to the adsorber surface with charged proteins in the mobile phase, and vice versa. Despite the importance of these models in model-based development, the stoichiometric representation of the adsorption process is rather abstract and not accurate for long-range electrostatic interactions in IEX chromatography. Complex dependencies, such as the effect of the buffer pH on protein adsorption, can therefore often only be described in an empirical way, limiting model application to a rather small pH range.

Monoclonal antibody in blue setting

Colloidal adsorption model

In this case study, we introduced a mechanistic model that is based on a more fundamental description of the electrostatic interactions taking place in IEX chromatography. Using a colloidal representation of proteins, the proposed model allows a description of the pH-dependent retention behavior of proteins based on their primary sequence. The model was used to describe the retention behavior of multiple antibodies with known primary sequence as a function of ionic strength and pH. A model validation beyond observed pH conditions demonstrated that the model is able to describe protein retention over several pH units.

Separation of antibody charge variants

Antibody isoforms often pose a difficult separation problem due to the similar physicochemical properties to the main component. Depending on the post-translational modification (PTM), they differ only slightly in the number of amino acids. Since the theory is based on the primary sequence of the protein, differences in the adsorption behavior due to PTMs can be predicted by the proposed mechanistic model. Without the need of additional experiments, the model could accurately describe the elution behavior of multiple antibody charge variants solely based on differences in their primary structure.

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