Investigation of pore diffusion hindrance of monoclonal antibody in hydrophobic interaction chromatography using confocal laser scanning microscopy

In this article, hindrance of intraparticle mass transfer during the adsorption of a monoclonal antibody (mAb) on Butyl Sepharose 4FF was investigated under different conditions. In addition to common fluid phase measurements, confocal laser scanning microscopy (CLSM) was applied to evaluate the respective intraparticle concentration profiles. In order to ensure that the observed intraparticle protein distributions are not disturbed by artefacts of CLSM, microscopic data are carefully analysed considering signal attenuation and competitive adsorption between labelled and native species. Using this setup, lower protein concentration in the inner particle region was observed even after long equilibration times in protein solution. Since the observed phenomenon showed a dependency on the amount of adsorbed protein, we assumed that the intraparticle diffusion was hindered by the adsorbed protein molecules. We propose hypotheses on the diffusion hindrance, and compare the experimental results with model-based simulations of single particles that include novel terms for the description of hindered diffusion.