title: 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 VIII (F VIII) directly from plasma. Previous reports have focused on the use of DEAE-Fractogel 650 (M) (Dimethylaminoethyl) ion exchange medium to capture F VIII from cryoprecipitate and plasma. Our main objectives were (I) to standardise the purification of FVIII from human plasma by column chromatographic technique. (II) to study the recovery of FVIII activity in purified fraction at 18-20°C process condition. (III) 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 FVIII/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.
authors.affiliation: National Plasma Fractionation Centre, K.E.M. Hospital, Parel, Mumbai.
authors.forename: V M
authors.initials: VM
authors.lastname: Baikar
authors.forename: M V
authors.initials: MV
authors.lastname: Kamath
authors.forename: C
authors.initials: C
authors.lastname: Vishwanathan
authors.forename: A M
authors.initials: AM
authors.lastname: Varadkar
authors.forename: S E
authors.initials: SE
authors.lastname: Bharmal
keywords: Coagulation; Factor VIII; Haemophilia A; Plasma; Separation