title: Development of a heat-treated factor VIII/von Willebrand factor concentrate prepared from heparinized plasma.
abstract: A high yield, intermediate purity factor VIII concentrate derived from heparinized plasma has been developed which can be heat-treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h to permit inactivation of viral contaminants which may be present. After cold reprecipitation of the heparinized cryoprecipitate (CRC), the resolubilized CRC precipitate was adjusted to 25-30 mg/ml protein and pH 6.35 +/- 0.1 and incubated for 1 h at 8 degrees C. After centrifugation to remove the precipitated fibrinogen and fibronectin, a factor VIII-rich supernatant can be recovered which contains greater than 500 units of VIII:C per liter of starting plasma (Method I product) at a purity of 1.5 U/mg protein. Adjusted to 50 mM glycine and pH 6.8, the product can be lyophilized and heat-treated at 60 degrees C/72 h without a significant loss of VIII:C activity. However, at 68 degrees C or 80 degrees C/72 h, temperatures now reported to be more effective in viral inactivation, the recoveries were reduced to 68 and 33% respectively. Significantly improved recoveries after heat-treatment (HT) at 68 degrees C or 80 degrees C/72 h were achieved if the 8 degrees C supernatant product was prepared by a modified procedure (Method II). This further reduces the fibrinogen content of the product while maintaining VIII:C yields greater than 500 U/l at a purity of 1.9 U/mg. When adjusted to 50 mM glycine and 1-2% (w/v) sucrose (pH 6.8), lyophilized and heat treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h, the VIII:C recoveries of Method II product were 88-100%, 79-84% and 80-83% of pre-HT levels respectively. The yield of VIII:C was greater than 400 U/l at a purity of 1.6-1.4 U/mg at 1-2% (w/v) sucrose even after the severe heat-treatment at 80 degrees C. In addition, the von Willebrand factor multimers are similar in size and triplet pattern to those observed in routine cryoprecipitate preparations.
authors.affiliation: Ottawa Centre, Canadian Red Cross, Blood Transfusion Service, Ontario.
authors.forename: D S
authors.initials: DS
authors.lastname: Palmer
authors.forename: P R
authors.initials: PR
authors.lastname: Ganz
authors.forename: H
authors.initials: H
authors.lastname: Perkins
authors.forename: D
authors.initials: D
authors.lastname: Rosborough
authors.forename: G
authors.initials: G
authors.lastname: Rock
mesh_terms: D011232:Chemical Precipitation; D003080:Cold Temperature; D005169:Factor VIII / Q000302:isolation & purification*; D005612:Freeze Drying; D006493:Heparin; D006358:Hot Temperature; D006801:Humans; D046911:Macromolecular Substances; D010949:Plasma / Q000032:analysis / Q000187:drug effects; D012995:Solubility; D014867:Water / Q000032:analysis; D014841:von Willebrand Factor / Q000302:isolation & purification*
chemical_list: D046911:Macromolecular Substances; D014841:von Willebrand Factor; D014867:Water; D005169:Factor VIII; D006493:Heparin