| The manufacture of biological products has increased rapidly recently, leading to a greater focus on rational process design and the removal of process bottlenecks, which are commonly found in purification steps. The association of protein molecules can complicate purification when multiple associated forms are present in processing environments. Denaturants, such as urea and guanidine hydrochloride, are frequently used to dissolve inclusion bodies and to prevent association during processing. However, few studies have been conducted to explain how these associative properties or the use of denaturants impact chromatographic purification steps.;In this work, we probe the self-association and ion exchange behavior of apolipoprotein A-IMilano (apo A-IM), using four anion exchange resins, Q-Sepharose-HP, Q-Sepharose-FF, Source-30Q, and Macro-Prep-HQ, and urea as a modifier of protein-protein interactions and protein structure. Macroscopic measurements, such as equilibrium isotherms, batch uptake, and column experiments are used to determine binding capacities, mass transfer rates, and elution behavior under different processing conditions, while microscopic methods, such as refractive index-based microscopy and confocal laser scanning microscopy, are used to provide insight into the underlying mass transfer mechanisms. In addition, protein cross-linking and dynamic light scattering experiments are used to characterize the protein association and diffusivity in solution under various conditions.;Column experiments performed with the strong anion exchangers reveal complex chromatographic behavior for apo A-IM over the range of processing conditions tested. High equilibrium binding capacities are found in the absence of urea for all resins. However, these high binding capacities are accompanied by slow mass transfer and complex elution behavior due to protein self-association. Conversely, ideal elution behavior and rapid mass transfer are observed at high urea concentrations where self-association is minimal, but at the expense of greatly reduced binding capacities. RIBM and CLSM experiments reveal sharp protein fronts in the absence of urea and smooth adsorption fronts in high urea concentrations. Overall, the observed changes in capacity and uptake kinetics as a function of urea are attributed primarily to a transition in transport mechanism from pore to surface diffusion as the urea concentration is increased, due to the formation of a more flexible protein structure. |
