Protein and VLP Adsorption on Perfusion Chromatography Media and Monoliths

This work studies the adsorptive and chromatographic behavior of proteins and human papillomavirus (HPV) virus-like particles (VLPs) on perfusion chromatography media and tube monolith column. The perfusion resins have a bimodal distribution of pore sizes including large through-pores and smaller diffusive pores. The effect of perfusion under non-binding conditions was obtained from HETP measurements for various proteins and VLPs and the results show that the dominant mechanism of intraparticle mass transfer gradually shifts from diffusion to perfusion as the reduced velocity increases. For strong binding conditions, confocal laser scanning microscopy (CLSM) images show that the intraparticle concentration profiles at higher reduced velocity become skewed in the direction of flow, deviating from the symmetrical profiles that are characteristic of diffusional transport. The perfusive enhancement intraparticle mass transfer can be predicted based on the structural properties of the resin particles using a perfusion model. In the case of VLPs, however, the advantage of perfusion under strong binding conditions vanishes as, in this case, the adsorption is restricted to a thin layer on the adsorbent particle surface with little penetration, which is due to the blockage of the pores by bound VLPs.
The effects of particle size on the separation performance of perfusion media is examined by comparing the adsorption behavior of proteins and VLPs on differently sized particles with similar internal structure. For a smaller particle, the fraction of intraparticle flow rate increases and the shift of diffusion to perfusion occurs at lower reduced velocity.
Monoliths are also studied as another type of convective stationary phase. The internal structure of the monoliths studied comprises large flow channels where convective transport takes place. For both non-binding and strong binding conditions, the effects of flow rate on performance are negligible. However, due to the smaller binding surface area when compared with perfusion particles, the monoliths studied showed lower binding capacities. The load-wash-elute of VLPs experiments on monoliths column show low VLP recovery in the elution step, with a strong dependence on the flow directions indicating that physical trapping of VLPs may have occurred on a dense “skin” layer observed by scanning electron microscopy on the side wall of the monolith.