Size-exclusion chromatography of plasma proteins with high molecular masses

Two different hydrophilic materials with large pores, Superose 6 and Fractogel EMD BioSec (S), which are designed for size-exclusion chromatography (SEC) of plasma proteins with high molecular masses, are tested for their performance on a preparative scale. The model mixtures are preparations of the clotting factors VIII (FVIII) and IX (FIX). A combination of a Fractogel EMD BioSec (S) column and a Superose 6 column has proved to be particularly effective for separations in a wide molecular size range, from several millions down to about 20,000. Superose 6 showed good results on a small scale as well as on a large scale, even in the molecular mass range over 1,000,000. However, recovery of FVIII clotting activity was less than 70% with this material and therefore not satisfactory. Fractogel did not perform well in terms of separation on a small scale. However, in the case of biopolymers with high molecular masses, separation was improved by using larger columns. With Fractogel, recovery of activity of the two clotting factors FVIII and FIX was satisfactory, above 80%. On a large scale, the active fraction in the clotting factor concentrate was successfully separated from the non-active fraction with either size exclusion (SE) material. In the preparation under investigation, the clotting factor VIII is found in a complex with the von Willebrand factor (vWF). The FVIII-vWF complex has a molecular mass of several millions. It dissociates in the presence of high concentrations of Ca2+ ions. Under such conditions FVIII and vWF were successfully separated with both SEC columns.