Protein and virus-like particle adsorption on perfusion chromatography media

• Architecture and pore size distribution of POROS HS 50 are determined.
• Perfusion effects on the adsorption of biomolecules are visualized.
• Perfusion is negligible for proteins up to 150 kDa.
• Perfusion becomes significant for a 700 kDa protein at 1000 cm/h.
• 100 nm VLPs are largely excluded and bind only at the particle surface.

The structural and protein adsorption characteristics of the perfusion chromatography matrix POROS® HS 50 are determined. Transmission electron microscopy shows a broad distribution of pore sizes with 100–500 nm through-pores transecting a network of much smaller pores formed by aggregates of microgranules about 100 nm in size. Dextran standards, proteins, and virus-like particles (VLPs) show size-exclusion behavior consistent with such a bimodal distribution of pore sizes. For non-binding conditions, the trends in height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity and molecular size are consistent with perfusion suggesting that a fraction of the mobile phase between 0.0005 and 0.0008 flows through the particles. This small fraction provides little or no enhancement of intraparticle mass transfer for relatively small proteins (lysozyme and IgG) even at 1000 cm/h, but can contribute substantially to transport for large proteins (thyroglobulin) and VLPs. Intraparticle concentration profiles during transient adsorption are determined by confocal microscopy in batch and flow systems. The profiles are spherically symmetrical indicating a dominance of diffusion for smaller proteins in both batch and flow systems but become highly asymmetrical and skewed in the direction of flow for thyroglobulin at 1000 cm/h. Estimates of the convective enhancement of intraparticle transport for these conditions based on the confocal measurements are consistent with estimates of the intraparticle Peclet number and previously published models. Adsorption of VLPs, however, was found to be confined to a thin layer on the outer surface of the particles indicting that bound VLPs block access to the underlying pore network and suggesting that pores larger than those present on the resin studies are needed to take advantage of the effects of perfusion for the adsorption of large VLPs.