Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 2. Investigation of...

The application of a model-based approach for industrial chromatography development requires the capability of the model to describe protein elution under high loading and overloading conditions. In a previous work, an extensive dataset was created to model the elution behavior of a bispecific antibody (bsAb) on the strong cation exchange resin POROS™ XS. Thereby, the pH-dependence of the model parameters in the Steric Mass Action (SMA) model could be examined and described over a pH range of 4.5 to 8.9. However, discrepancies between simulated and experimental data were observed under high loading and overloading conditions, particularly in the lower pH range (pH 4.5 to 5.3) and in the higher pH range (pH 6.0 to 9.0). In this work, these discrepancies are studied by performing new experiments which show that these differences were primarily not caused by limitations of the SMA model. At lower pH values, overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak shoulders occurred. The application of various experiments performed with different Na⁺ concentrations and different loading times during sample loading revealed that intraparticle diffusion effects and conformational changes of the bsAb are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate and should be extended to consider these effects. At higher pH, the assumption of describing the bsAb's elution behavior with only one simulated species was insufficient to predict complex peak shapes that arise because of multi-component elution of the bsAb's charge variants. The extension of the model to a simple multi-component system consisting of two variants allowed the prediction of a majority of the complex elution profiles.