Non-Cryoprecipitation Separation of Coagulation FVIII and Prothrombin Complex Proteins by Pseudoaffinity Calcium Elution Chromatography Using Anion Exchange Resin

Abstract

Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

Intensified capture of recombinant blood coagulation factors by continuous, integrated downstream processing

“…32 More recent literature using ion-exchange chromatography reports yields of 40-80% for FVIII. [33][34][35][36][37] The ion-exchange chromatography was also converted into a continuous operation by applying countercurrent loading. The best result obtained from chromatographic separation was a recovery of FVIII co-purified with VWF of >86%, where the specific activity was at least 140 IU mg −1 .…”

“…In an early study with affinity chromatography using lectins an overall yield of 40–57% for FVIII and vWF has been reported 32 . More recent literature using ion‐exchange chromatography reports yields of 40–80% for FVIII 33‐37 . The ion‐exchange chromatography was also converted into a continuous operation by applying countercurrent loading.…”