Development of a Heat-Treated Factor VIII/ von Willebrand Factor Concentrate Prepared from Heparinized Plasma

Abstract

SummaryA high yield, intermediate purity factor VIII concentrate derived from heparinized plasma has been developed which can be heat-treated at 60 °C, 68 °C or 80 °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 °C. After centrifugation to remove the precipitated fibrinogen and fibronectin, a factor VIII-rich supernatant can be recovered which contains > 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 °C/72 h without a significant loss of VIII :C activity. However, at 68 °C or 80 °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 °C or 80 °C/72 h were achieved if the 8 °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 > 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 °C, 68 °C or 80 °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 > 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 °C. In addition, the von Willebrand factor multimers are similar in size and triplet pattern to those observed in routine cryoprecipitate preparations.

Rapid serum tube technology overcomes problems associated with use of anticoagulants

“…Plasma samples produced from heparinised blood collection tubes have been used as an alternative in the analysis of many common chemical analytes ( 26 ). Plasma tubes are thought to have some advantages over blood serum tubes as they can be centrifuged immediately to produce plasma with higher yield ( 27 ). When repeated studies were carried out, measuring several key analytes of sera stored at room temperature or 4°C for 0, 8, 24 and 36 hours, it is expected that there would be significant differences in stability and reproducibility of these four clinically relevant analytes between plasma/heparin and serum/PtPA samples, even between serum/GBO-SST and serum/PtPA samples.…”

Experience with Haemate P in von Willebrand’s Disease in Adults

“…Reports of successful use of Hae mate P in vWD were also published by Scharrer and Vigh [5], Takahashi et al [6], Czapek et al [7], Fukui et al [1], Vigh et al [8], Berntorp and Nilsson [2], Ieko et al [9], Rose et al [10], Logan [11], Mannucci et al [12], Rodeghiero et al [13], Yoshioka et al [14], and Foster [15], Several other new FVIII concentrates look promising for treatment of vWD. Successful use of SD-FVIII concentrates and other FVIII preparations were reported by Furlan et al [16], Gazengel et al [ 17], Mazurier et al [18], Logan and Higgins [ 19], Pasi et al [20], Palmer et al [21], Cumming et al [22], Lawrie et al [23], Oates et al [24], Retzios et al [25], and Hanna et al [26]. The different concentrates are similarly effective in attaining high levels of FVIII.…”

Characterization of Factors Affecting the Stability of Frozen Heparinized Plasma

“…though SP formation has not been observed in hepari nized plasmas initially frozen at -80°C and then stored at -80°C [1][2][3][4][5]10], it does occur at warmer freezing and stor age temperatures, even after relatively brief periods of storage. We wished to determine the mechanism of SP formation in frozen heparinized plasma and further to delineate the effects of such variables as initial freezing and subsequent storage temperatures, period of storage, method of plasma preparation and the effect of citrate concentration on SP recovery.…”

“…Blood was collected into triple blood packs containing 63 ml of either CPDA-1 (Fenwal MR 7733; Fenwal Laboratories, Deerfield, 111., USA) or 2,250 units USP of heparin anticoagulant (Travenol Canada Inc., Fenwal Division, Mississauga, Ontario, Canada) [10]. Plasmas were separated from whole blood within 2-4 h of collection by centrifugation (7,000 g for 7 min) at 4 °C and then expressed into a satellite pack [Method I (MI)].…”

“…Further, we have recently developed a factor VIII/von Willebrand factor concentrate that can be heat treated at 80°C for 72 h to inactivate potential viral contaminants [10]. The fractionation results obtained by our group were from heparinized plasma initially frozen and stored at -80°C for a period not in excess of 3 weeks [1,4] prior to routine cryoprecipitation at 4°C or were obtained by others [5,6,9] from plasma snap frozen at -70 °C in an alcohol-dry ice bath and then thawed at 4°C in a water bath for 75-90 min.…”