Human Factor VIII (FVIII) is a 265 kDa glycoprotein which circulates in plasma (0.1 μg/mL) bound to von Willebrand factor (vWf). The glycoprotein is highly sensitive to proteolytic processing which brings about both its activation and destruction, therefore regulating its role as a co-factor in the coagulation cascade (blood clotting). Activated FVIII (FVIIIa) is a co-factor in the activation of Factor X to Factor Xa. A deficiency in FVIII may lead to the bleeding disorder Haemophilia A.
The average industrial yield of FVIII from plasma is 140 to 270 international units (IU) per litre of plasma (1 IU is the average amount of FVIII activity found in 1 mL of pooled plasma=0.2 μg). The first step of FVIII conventional purification is typically cryoprecipitation (traditional Cohn fractionation) which yields 40–50% of FVIII. The cryoprecipitate containing the FVIII is then treated using chromatography, typically immunoaffinity and ion exchange. Minimizing the loss of FVIII at each of the processing steps is desired to improve yield since current supply of suitable FVIII is inadequate. Hemasure Denmark A/S has developed technology in an attempt to combat current problems with traditional Cohn fractionation of plasma. Hemasure uses a high capacity gel filtration step to replace the initial cryoprecipitation step reported to provide a step yield of 60–70% FVIII with a total process yield of 200 IU of FVIII/litre plasma. J. Dam, Downstream, vol. 31, p. 65 (Dec. 1999). Although this method results in an improved yield, there are still significant losses of FVIII in the process. Recombinant production is another source of FVIII, but this source has not replaced FVIII obtained from natural sources. Current purification schemes are time consuming, result in a significant loss of FVIII, and do not adequately remove pathogens, particularly viruses, without adversely affecting FVIII activity or yield.
It has been reported that several thousand different proteins coexist in plasma. Obtaining a given protein from such a complex mixture can be difficult, especially if the given protein must retain its biological activity in its isolated state. Currently, it is very difficult to purify or separate FVIII in reasonable quantities with good yields from plasma. As an example, for plasma having an average protein concentration of 70 mg/mL, FVIII (˜0.1 μg/mL) constitutes approximately only 0.00014% of total plasma protein. The presence of FVIII in such low concentrations in plasma or recombinant sources usually requires large amounts of plasma or other sources to obtain reasonable commercial yields. Hence, production costs are increased and typically require process step reduction.
FVIII is a relatively unstable protein in plasma. As a result, standard purification technology applied to FVIII separation has difficulty obtaining a method for obtaining large amounts of biologically active FVIII. Current processes involve stabilizing the FVIII preparation at the end of the purification scheme with stabilizing agents, the most common being human albumin. However, addition at the end of the purification process may be too late to protect the activity of the separated FVIII. “Kogenate-F”, a recombinant FVIII (rFVIII) therapeutic product formulated with sucrose, may eliminate the need to add human albumin to the preparation.
Viral contamination of FVIII preparations is also a potential problem. Typically, solvent detergent (SD), pasteurization, Methylene Blue (MB) and UV treatment or a combination thereof are used to inactivate viruses in FVIII preparations. However, traditional viral removal steps often result in loss or inactivation of FVIII.