Separation of antihemophilic factor VII from human plasma by column chromatography

Abstract

TMAE-Fractogel 650 (M) (Trimethylaminoethyl) is an ion exchange medium can be used to capture factor Vlll (F VIII) directly from plasma. Previous reports have focused on the use of DEAE-Fractoge1650 (M) (Dimethylaminoethyl) ion exchange medium to capture F Vlll from cryoprecipitate and plasma. Our main objectives were (I) to standardise the purification of FVIII from human plasma by column chromatographic technique. (11) to study the recovery of FVIII activity in purified fraction at 18 -20~ process condition. (Ill) to study the effect of virucidal step on recovery of FVIII activity and (IV) to study the effect of lyophilisation on FVIII activity. In this report, Citrate Phosphate Dextrose (CPD) plasma was batch stirred with dry DEAE-Sephadex A50, filtered, diluted, loaded on to a column packed with TMAE-Fractogel and chromatographed. Most of the unwanted proteins flowed through the gel unadsorbed. Bound F VIII was eluted by increasing the ionic strength of the buffer. This purification step gave an overall 80% recovery from the plasma with a specific activity of 0.97 IU FVlll/mg protein. The purified F VIII fraction was made virus safe by employing the virucidal technique developed by New York Blood Centre (NYBC). There was 48.43% loss of FVIII activity in Virus inactivation treatment and the loss of FVIII activity in lyophilisation was 8.45% which is acceptable. This method of purification gave a higher yield of FVIII than cryoprecipitation, and is a promising alternative method to cryoprecipitation of F VIII.

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

“…Compared with the traditional Cohn method, in the proposed purification method it is possible to obtain an enriched FVIII fraction without the need of cryoprecipitation, thus avoiding refrigerated thawing and centrifugation at low temperatures. Attempting to avoid cryoprecipitation is not new [ 24 , 25 , 26 , 27 ]; however it was necessary to use 2 ion exchange columns to separate FVIII from PCC, one of them being used in batch mode, which makes it more difficult to scale up the process. Finally, the method we describe in this work can be easily integrated into any fractionation process.…”

Application of dye-ligand chromatography for purification of coagulation factor VIII

“…Тривале застосування афінних лігандів "еволюціонувало" від ензиматичних субстратів, коензимів, гормонів, лектинів, кофакторів, антитіл, нуклеїнових кислот, ефекторів та інгібіторів до різних пептидів, поліпептидів, а також інших синтетич-них структур [1].…”

Implementation of Plasma Fractionation in Biological Medicines Production

“…The quality of plasma has a great impact on the product ( 45 ). The first step of plasma fractionation produces Cohn Fraction I, which the cryoprecipitate from this fraction is a suitable source as starting material for factor VIII production ( 46 ).…”