Comparison of diffusion and diffusion–convection matrices for use in ion-exchange separations of proteins

Abstract

A comprehensive study has been undertaken to characterise a range of chromatographic properties for a series of modified polystyrene–divinylbenzene (PS–DVB) chromatography matrices. The matrices studied included diffusion matrices and matrices that allowed convective mass transfer of liquid into the particles at high flow-rates, so-called “perfusion” matrices. The matrices tested included the following: CG1000sd 20–50 μm (TosoHaas), PLRP4000s 15–25 μm, 50–70 μm (Polymer Labs.), Source 15RPC and 30RPC, 15S, 30S, (Pharmacia Biotech), POROS 20SP type 1 matrix and OH activated POROS 20 type 2 matrix (PerSeptive Biosystems) and SP Sepharose Fast Flow (Pharmacia Biotech). A Van Deemter equation was used to determine bead tortuosities and split ratios. Frontal analysis, resolution studies, ionic capacities and isotherms were measured. It was found that diffusion–convection chromatographic particles had smaller plate heights to comparable diffusion particles. The smallest diffusion bead, Source 15, had the lowest plate heights at low superficial velocities, but the small particle size resulted in a high back pressure at high flow-rates. The equilibrium binding capacities for lysozyme and IgG on the diffusion–convection matrices were substantially lower than the equilibrium binding capacities on the diffusion matrices. The dynamic capacities for these proteins were also lower on the diffusion–convection particles, compared to the diffusion particles, over the tested flow-rates. At high protein loading, resolution between proteins was higher on diffusion particles than on diffusion–convection particles. Diffusion–convection particles showed low or no resolution at high protein loading. At analytical level loadings, the diffusion–convection particles achieved a high resolution over the whole flow-rate range tested and were more suitable for this application than diffusion particles.