Separation of antihemophilic factor VII from human plasma by column chromatography |
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| Authors: | V M Baikar M V Kamath C Vishwanathan A M Varadkar S E Bharmal |
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| Institution: | (1) Dept. of Biochemistry, Seth G.S. Medical College, Parel, Mumbai;(2) National Plasma Fractionation Centre, K.E.M. Hospital, Parel, Mumbai;(3) 182/3, “Swapna Siddhi” Co. Op. Hsg. Society, MHB Colony, Jai Maharashtra Nagar, Magthane, Borivali (East), 400 066 Mumbai, Maharashtra |
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| 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. |
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| Keywords: | Haemophilia A Coagulation Plasma Factor VIII Separation |
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