SIZE-EXCLUSION CHROMATOGRAPHY OF PLASMA-PROTEINS WITH HIGH-MOLECULAR MASSES

Two different hydrophilic materials with large pores, Superose 6 and F ractogel EMD BioSec (S), which are designed for size-exclusion chromat ography (SEC) of plasma proteins with high molecular masses, are teste d for their performance on a preparative scale. The model mixtures are preparations of the clotting factors VIII (FVII) and IX (FIX). A comb ination of a Fractogel EMD BioSec (S) column and a Superose 6 column h as proved to be particularly effective for separations in a wide molec ular 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, ev en in the molecular mass range over 1 000 000. However, recovery of FV III clotting activity was less than 70% with this material and therefo re not satisfactory. Fractogel did not perform well in terms of separa tion on a small scale, However, in the case of biopolymers with high m olecular 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 fractio n in the clotting factor concentrate was successfully separated from t he non-active fraction with either size exclusion (SE) material. In th e preparation under investigation, the clotting factor VIII is found i n a complex with the von Willebrand factor (vWF). The FVIII-VWF comple x has a molecular mass of several millions. It dissociates in the pres ence of high concentrations of Ca2+ ions. Under such conditions FVIII and VWF were successfully separated with both SEC columns. (C) 1998 El sevier Science B.V.