Hydrochloric Acid → Glycine

Participants

Reaction Steps

60×1.6 cm siliconised column was filled with dextran sulphate Sepharose 4B matrix prepared as in Example 1. The resin was equilibrated at 4° C. with 14 mM trisodium citrate, 2.1 mM calcium chloride in 1.0 M glycine pH 7.3. Two bags of Blood Transfusion Service cryoprecipitate were thawed at 37° C., combined and diluted to 100 ml with equilibrating buffer and adjusted to pH 7.3 with 0.05 M hydrochloric acid. After centrifugation at room temperature to remove any particulate material, 20 ml of the supernatant was diluted to 50 ml and applied to the chromatography column and developed at 4° C. at 42 ml per hour. Wben all the unbound material was judged to have eluted the bound proteins were developed with a simple two-chamber gradient comprised of 450 ml of the initial equilibrating buffer and 450 ml of 1.0 M glycine, 1.0 M sodium chloride, 2.1 mM calcium chloride pH 5.5, resulting in an elution profile that was linear with respect to sodium chloride concentration, but convex with respect to lowering pH value. As with Example 4 there was good resolution of Factor VIII from most plasma proteins. Fibrinogen eluted at 0.15 M sodium chloride pH 7.0, fibronectin at 0.28 M sodium chloride pH 6.7 and Factor VIII at 0.38 M sodium chloride pH 6.5 with overall yields of 100% Factor VIIIR:Ag, 100% Factor VIIIR:CoF, 65% Factor VIIIC:Ag and 35 % Factor VIII:C.

Provenance & Evidence