The process of blood clotting begins with an injury to a blood vessel. The damaged vessel wall initiates hemostasis by causing adherence and accumulation of platelets at the point of vascular injury and by activating the plasma proteins which initiate the coagulation process. Sequential activation, via specific proteolytic cleavages and conformational changes, of a series of proteins (including Factor VIII) initiate a coagulation cascade that eventually leads to deposition of insoluble fibrin. The fibrin, together with aggregated platelets, curtails the escape of blood through the point of injury in the damaged vessel wall.
One of these coagulant proteins, Factor VIII, is a plasma protein that has the ability to correct the clotting defect in plasma from patients with Hemophilia A. The activity of Factor VIII is also measured by its ability to induce clotting in plasma obtained from these patients. One unit of Factor VIII is defined as the amount of Factor VIII present in one milliliter of normal, adult male plasma. This standard is a World Health Organization standard and is available from the National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NY3 6RD, England.
During normal blood coagulation, Factor VIII is activated by thrombin, a protease, which affects a proteolytic modification (cleavage) of Factor VIII. (Weiss et al., Science, 182: 1149-1151, 1973; Hoyer, Chapter 4, Hemostasis and Thrombosis, 2d Ed., Colman, Ed., J. B. Lippincott & Co., Philadelphia, Pa., 1987; Fulcher et al., J. Clin. Invest., 6: 117-124, 1985; and Eaton et al., Biochemistry, 25: 505-512, 1986). Following an initial increase in activity after exposure of Factor VIII to thrombin, there is a decay in activity to below base line levels. This decay in activity is attributed to further proteolytic cleavage of activated Factor VIII by thrombin, instability of the partially cleaved molecules Hultin, M. B. et al., Blood, 57: 476-482, 1981, and during pathological states, to inactivation by thrombin-activated protein C (Eaton, D. et al., Bioch. 25: 505, 1986). The activation and inactivation of Factor VIII by thrombin and protein C allows a fine measure of control over coagulant activity in vivo. Unfortunately, it may also cause premature degradation and instability of Factor VIII, and particularly of the isolated, purified Factor VIII or purified plasma fractions containing Factor VIII, prior to its administration to mammalian hosts in need of such treatment.
The primary therapeutic use of Factor VIII has been its intravenous administration to patients with hemophilia A to stop or to prevent bleeding. Early administration was by the infusion of whole blood or fresh-frozen plasma. These methods required the infusion of very large volumes of proteins and long infusion times, often causing hypervolemia.
The use of plasma cryoprecipitate ("cryo") eliminated the need for whole blood or fresh frozen plasma infusion but the moderate volumes and high protein concentrations still required long infusion times. The cryo was not completely soluble, the cryo solution used for infusion required filtration, and the cryo's AHF content was low and highly variable. Moreover, cryo required storage at -20.degree. C. in large plastic blood bags, and therefore, required large refrigeration facilities.
The primary source of native Factor VIII used today to treat those in need of clotting factor is still plasma, and the use of lyophilized, higher purity Factor VIII concentrates has solved many of the above problems. However, the yield obtained by conventional methods of purification is relatively low, making the cost of the factor excessive. Furthermore, some of the low purity concentrates contain high amounts of contaminating proteins including specific blood-type antibodies that cause hemolysis, proteins that cause immunological abnormalities including a temporary inversion of the T-cell ratio (helper/suppressor) which resembles AIDS (Carr, R. et al., Lancet 1: 1431, 1984), fibrinogen, fibronectin, Von Willebrand factor, and other proteins.
When a clinical Factor VIII concentrate shows evidence of prior activation, several in vitro and in vivo problems may occur. If the Factor VIII assay was performed by a one stage procedure, it may be falsely high (Niemitz and Nossel, Brit. J. of Hematol. 16: 337, 1969); this is not the case, if the assay is performed by a two stage procedure, but very few laboratories are equipped to use the method. Additionally, the activated material is unstable and tends to lose activity during short term storage in the liquid state and during long term storage in the lyophilized state, resulting in poor results when infused in vivo. Use of these Factor VIII concentrates could lead to inadequate treatment for the patient and excessive bleeding.
Substantial activation of antihemophilic factor in plasma and cryoprecipitate is usually prevented by the addition of citrate as the blood is drawn. However, the addition of too much citrate at the time of drawing or later in the purification procedure can cause inactivation of Factor VIII and a corresponding decrease in yield (Rock, et al., Thromb. Res. 29: 521-535, 1983, Foster, P. R. et al., Scan. J. Haematol. Supp. 40, 33: 103-110, 1984). Consequently, heparin and calcium are often added to the cryoprecipitate or the partially purified Factor VIII during fractionation. In addition, most of the contaminating procoagulant clotting factors or proproteases which are present, such as Factor II, VII, IX and X, and protein C, are adsorbed with aluminum hydroxide. However, it is difficult, if not impossible, to adsorb all of the contaminating Factors II, VII, IX, and X and protein C with aluminum hydroxide without also adsorbing Factor VIII. For example, when Factors II, VII, IX and X are adsorbed from cryoprecipitate or plasma with 5 to 20% aluminum hydroxide and the residual cryoprecipitate or plasma is brought to approximately 3 mM Ca.sup.+ , coagulation still occurs in a relatively short period. It was believed that this was due to residual Factors II, VII, IX, and X in the preparation.
Additionally, minute amounts of thrombin tend to form during blood collection and during large scale cryoprecipitation. When Factor VIII is purified and concentrated, some thrombin is purified and concentrated in addition. The effects of various proteolytic enzymes, including thrombin on purified Factor VIII, were shown by Eaton et al. Bioch. 25: 505 (1986), and the activation of Factor VIII by thrombin with redistribution of polypeptide chains in Factor VIII was demonstrated by polyacrylamide gel electrophoresis by Fulcher et. al., Blood 61: 807-811, (1983), Vehar et al., Nature, 312, 337-342 (1984), and Rotblatt et al., Biochem. 24: 4294-4300 (1985). Similar biochemical changes were found in commercial Factor VIII concentrates purified by large scale methods for clinical use when analyzed by polyacrylamide gradient autoradiograms of Factor VIII-Factor VIII antibody complexes (Barrowcliffe, Lancet, 1: 1448-1449 (1986).
Chromatographic techniques, including ionic, hydrophobic, and immunoaffinity chromatography, have been used to purify Factor VIII, but the yield is relatively low. Several groups of investigators have purified Factor VIII with specific activities as high as 3,000 units per milligram of protein (Fulcher et al., Proc. Natl. Acad. Sci. USA, 79: 1648-1652, (1982); Tuddenham, et al., NIH Symposium on Factor VIII/von Willebrand Factor, pg. 1., Scripps Clinic and Research Foundation, La Jolla, Calif. (1982); Johnson et al., NIH Symposium on Factor VIII/von Willebrand Factor, pg. 2., Scripps Clinic and Research Foundation, La Jolla, Calif. (1982).
Much effort has been made to increase the purity of Factor VIII concentrates. This included the use of immunoaffinity chromatography and monoclonal antibodies (Fulcher et al., Proc. Natl. Acad. Sci. USA. 79: 1648-1652 (1982) and Zimmerman et al., U.S. Pat. No. 4,361,509. Overall recovery was estimated to be less than 15% without a heating step.
Further attempts were described by J. J. Morgenthaler, Thromb. Haemostas, 47(2): 124 (1982) wherein the use of acetate-glycine-lysine buffer on modified sepharose columns to purify Factor VIII:C from polyethylene glycol precipitated AHF was disclosed.
The addition of 0.05M dextrose in the eluting buffer of bovine Factor VIII during ion exchange column chromatography (on DEAE-cellulose) slightly improved the purity of the product and increased the yield as reported by Lundblad et al. Thrombosis Research, 1: 197, Pergamon Press, Inc. (1972). However, resolution was sightly reduced.
Purification of Factor VIII by column chromatography in the presence of additives, including sugars and polyhydric alcohols which serve to increase the electrostatic forces on the surface of the proteins while decreasing the hydrophobicity of the proteins, was reported to result in high recovery of preparations having high purity and resolution (Mathews et al., U.S. Pat. Nos. 4,743,680, 4,847,362, and 4,952,675).
Fulton et al., U.S. Pat. No. 4,970,300, disclose conjugates of proteins having antihemophilic factor activity linked to non-antigenic ligands. These conjugates were stated to have a longer half-life than unconjugated proteins while maintaining substantial Factor VIII activity. Fulton et al. also used short columns of ionic resins to remove residual amounts of Factors II, VII, IX and X.
Finally recombinant Factor VIII has become available through the cloning of the Factor VIII gene. (Gitschier, et al. Nature 312: 326-330 (1984); Wood et al., Nature 312: 330-337, 1984; Vehar et al., Nature, 312: 337-342, (1984); and Toole, et al., Nature, 312: 342, (1984).
A novel method for stabilizing partially purified Factor VIII and Factor VIII from biological fluids or mixtures of cellular products derived from recombinant materials, often containing calf serum and/or endopeptidases, has now been discovered. This method prevents activation or degradation of moderately, or highly purified Factor VIII during viral inactivation, purification, recovery, lyophilization and storage and produces a higher yield from the starting material in vitro and a higher recovery in vivo. This results, in turn, in a safer, more effective therapeutic agent at lower cost.