A PURE FACTOR I PROTEIN AND A PROCESS FOR PRODUCING SAID PROTEIN

Abstract: A pure Factor I protein essentially free of infectious virus, Factor B and C3. The protein is derived from plasma and pasteurized by heating to a temperature of 50 to 65 DEG C for 0.5 to 100 hours in the presence of one or more stabilizers for Factor I. Preparations containing the protein are useful in the treatment of Factor I deficiency and autoimmune diseases.

Description: A pure Factor I protein and a process for producing said protein. The invention relates to a pure Factor I protein, essentially free of infectious virus, Factor B and C3. The invention also 5 relates to an injectable preparation containing the pure Factor I protein, and derived from plasma. Such a preparation may be used in the treatment of Factor I deficiency and in the treatment of autoimmune diseases. The invention also relates to a process for producing the pure factor I protein, Θ recovered from plasma. The complement system is an enzyme cascade system consisting of more than 2Θ plasma proteins. The system is important in the defence against microbial infections. It is activated partly by antigen antibody complexes via the classical pathway, partly by e.g. bacterial membranes and soluble substances via the alternative pathway. In both situations, the activation leads to formation of some active enzyme complexes, C3 covertases, having the central component C3 as a substrate. The C3 protein is cleaved in a small fragment, C3a, and in a larger fragment, C3b. C3b forms part of the C3 convertase of the alternative pathway. Thus, an amplification principle of the alternative pathway of the complement system is incorporated. The C3 convertase of the alternative pathway also includes a Factor Bb, a degradation product from the complement factor, Factor B. The system is regulated by several proteins which inhibit the activated enzyme complexes. Factor I (C3b inactivator or KAF) is such a regulator protein since it enzymatically degrades the C3b fragment to an intermediate fragment iC3b and further to C3c and C3d. Factor I deficinecy entails that C3b cannot be degraded to smaller cleavage products. This results in a continuous and uncontrolled formation of the alternative C3 convertase, with a consequent reduction or depletion of proteins from the alternative pathway. This involves less resistance to certain types of infections. Thus, increased frequency of e.g. cerebrospinal menigitis and pneumonia has been reported in Factor I deficient patients, cf. S.C. Ross et al., Medicine (1984) 63(5), 243. Treatment with intravenous infusion of Factor I preparation, purified from plasma, has been described, cf. J.B. Ziegler et al., J. Clin. Invest. (1975) 55, 668. This treatment has been found to be effective in providing near normalization of most plasma protein concentrations in a Factor I deficient patient. However, the preparation was not virus inactivated. Treatment with plasma infusions has likewise been found effective, cf. D.J. Barret et al., J. Pediatri. (1984) 104(1), 76, V. ahn et al., Allergol. Immunopathol. (198Θ) 8(4), 422 as well as V. Wahn et al, J. Clin. Immunol. (1981) 1(4), 228. However, this treatment has some serious draw¬ backs: Factor I deficient patients have large amounts of 03 con¬ vertase in the blood. 03, administered via the plasma, is therefore immediately cleaved to C3a and C3b, as described above. 03a, which is an anaphylotoxin, can cause anaphylactic reactions in connection with plasma infusion, cf. V. Wahn et al., J. Pediatr. (1984) 105(4), 673. Further, the resulting C3b, in combination with Factor B, likewise administered with the plasma, has a stimulating effect on the alternative pathway. The presence of the complement components 03 and Factor B in the administered plasma thus weakens the efficacy of Factor I regulation of the alternative pathway. Finally, it is not possible to eliminate the risk of viral transmis¬ sion by plasma infusions. Temporary decreased levels of Factor I and Factor H are observed in patient suffering from systemic lupus erythemato- sus (SLE). Kaneko (EP ø 222 611) showed that administration of Factor H and/or Factor I to mice suffering from autoimmune diseases resembling those of human SLE, rheumatoid arthritis and glomerulonephritis, were effective in improving or preventing the proteinuria, that represents the pathological conditions of the autoimmune diseases. Kaneko, however, dit not submit the Factor I preparation to neither a virus inac- tivation step nor a specific Factor B depletion step. As essential problem in the therapeutic use of preparations or fractions produced from human blood or blood plasma is that this blood or blood plasma may contain infectious viruses. Examples of such infectious viruses are hepatitis B virus, non-A, non-B hepatitis virus and human immunodeficien- cy virus. Transmission of infectious viruses is known in general from the use of preparations produced from human blood or blood plasma. Examples of such preparations are coagulation Factor VIII, coagulation Factor IX, coagulation Factor XIII, immunoglobulin and antithrombin III. Various processes are known for removal of infectious viruses from therapeutic preparations or solutions containing these produced from human blood or blood plasma. Such a known process comprises heating for 10 hours at 60°C of aqueous solutions containing therapeutic protein, thereby providing safety against transfer of infectious viruses when using preparations containing these plasma proteins. To avoid denaturation or other damage to the therapeutically active protein in the aqueous solution, it is necessary, however, to add stabilizers during the heat treatment. Thus, salts of fatty acids or amino acids are added as stabilizers in a heat treatment for 10 hours at 60°c of solutions containing human albumin or protein concentrate, cf. Gellis et al., J. Clin. Invest. (1948), 27, 239. Use of this process for treatment of human albumin or protein concentrate has entailed that since the process was introdu¬ ced in 1948, no infectious viruses have been transmitted when using these preparations. This process involved no or only insignificant losses of the therapeutic plasma protein albumin. Solutions containing plasminogen may be stabilized with the amino acid lysine during a heat treatment af 60°C for 10 hours, cf. Bau garten et al., US Patent 3 227 626, (1966). This process destroys the presence of hepatitis virus according to the patent. Solutions containing the coagulation Factor XIII may be heated at 60°C for 10 hours in the presence of an amino acid, a monosaccharide or a sugar alcohol, cf. Fukuhima, Patent Sho 51-134878, (1976). A loss of Factor XIII activity of about 50% has been reported with this process. Furthermore, solutions containing the proteinase inhibitor anti-thrombin III may be stabilized with salts of citric acid prior to heating for 10 hours at 60°C, cf. Holleman et al., Throm. & haemo. (1977), 38, 201. The loss of the therapeutic protein antithrombin III amounts to about 30% in this process. Finally, the DE O fenlegungschrift 2916711 (1980) discloses a process for heat treatment of Factor VIII for 10 hours at 60°C while reducing a contaminating fibrinogen content. The stabilizer used is a saccharide or a sugar alcohol in combination with a high concentration of amino acid. The amino acid present causes the fibrinogen of the Factor VIII solution to be precipitated. In this process, the Factor VIII solution is admixed with e.g. 50% w/w saccharose and 2 M glycine during the heat treatment. No methods of producing a virus inactivated Factor I prepara- tion has been described in the literature. However, the present invention provides a process for producing a solution of Factor I essentalily free of infectious viruses and Factor B, which is safe against transfer of infectious viruses, the method comprising subjecting a solution containing Factor I, optionally recovered from plasma, to pasteurization by heating to 55 to 65°oC for 0.5 to løø hours, preferably at 6ø ° oC for 4 to 24 hours, in the presence of one or more stabilizers for Factor I. The heat treatment is more pre- ferably performed at about 60 ° C for 5 to 15 hours. The invention enables production of a Factor I preparation which is practically protected against transmission of active viruses and the complement factor, Factor B, in one step. If the invention is combined with a fractionation step in which 03 activity is removed, e.g. by PEG precipitation as described in example 1, a preparation wil be obtained which has two very important advantages over plasma in the treat¬ ment of Factor I deficiency since it is, in practice, free of viruses and is in practice without C3 and Factor B activity, which, as described above, may cause anaphylactic reactions. The product is also superior to the Factor I preparations described by Kaneko (EP 0 222 611) for treatment of auto¬ immune diseases because these products were not safe with regard to the risk of viral transmission. Thus, the invention makes it possible to introduce a consi¬ derable improvement in the treatment of Factor I deficient patients over the common treatment with plasma infusions. The process with heat treatment for 1/2 to 100 hours at 55 to 65°oC of a solution containing Factor I with a saccharide and/or a sugar alcohol and/or an amino acid as a stabilizer may be performed on any fraction or preparation containing Factor I. Solutions containing Factor I are admixed with a saccharide and/or a sugar alcohol and/or an amino acid. The stabiliza¬ tion of Factor I increases with increased addition of saccharide, sugar alcohol or amino acid; thus, addition may be effected until saturation of one or more of these types of stabilizers. Stabilization of Factor B likewise increases with increased addition of saccharide and/or sugar alcohol, but there will be a range for the concentration of these stabilizers where no Factor B is present in the heat treated solutions, and a great yield of Factor I is obtained after heat treatment for 10 hours at 60°C. It is remarkable e.g. that stabilization with 55% (w/w) saccharose results in an occurrence of Factor B of 21% after heat treatment at 60°C for 10 hours, while the use of 30% (w/w) saccharose gives total denaturation of Factor B already within 4 hours at 60°C. The same distinct effect is not seen for Factor I, cf. example 1, part B and example 3. The use of amino acids as a stabilizer shows no stabilization of Factor B, while a great yield of Factor I can be obtained after heat treatment for 10 hours at 60°C. This fact renders it possible to produce a Factor I preparation which is without Factor B activity. Removal of saccharide, sugar alcohol or amino acid from the solution containing Factor I after the heat treatment can be effected by ion exchange or dialysis. After removal of the stabilizer, the solution may be concentrated to the desired concentration, e.g. by means of ultrafiltration, following which the preparation may optinally be freeze-dried in order to improve the stability until use when it is reconstituted. Factor I may be determined by rocket immuno electrophoresis with rabbit anti-human-antibody monospecific for Factor I (produced by the Institute of Medical Microbiology at the University of Odense, Denmark). The sample size is 10 μl. The standard is freeze-dried human plasma (KABI) . Factor B may be determined by radial immunodiffusion with rabbit antihuman Factor B antibody (Behring, anti 03 ac¬ tivator). The sample size is 10 μl. The standard is likewise freeze-dried human plasma (KABI). Since antibody against C3 is not commercially available, presence of 03 can be excluded by the following indirect method, cf. I. Brandslund et al., Scand. J. Clin. Lab. Invest. (1984) 44 suppl. 168, 57: antibodies against the 03 degradation product C3c react with C3 as well as C3c, but not with C3d. Correspondingly, antibodies against C3d react with both 03 and C3d, but not with C3c. The total 03 and C3c activity may be determined by rocket immuno electrophoresis with rabbit anti-human-antibody aganist C3c (Dakopatts). The sample size is 15 μl. The total 03 and C3d concentration can be determined correspondingly by rocket immuno electrophore- sis with rabbit anti-human-antibody against C3d (Dakopatts). In both assays, the standard is freeze-dried human plasma (KABI). If just one of these electrophoreses gives a negatige response (no rocket), this means that there is no 03 in the sample. The concentration of saccharose may be determined by io- dometric titration according to a method, modified with respect to the one described in Ph. Nord. 63 Vol. II, page 558. The process of the invention is illustrated by the following examples. 8 EXAMPLE 1 Part A Fresh frozen plasma is thawed and PEG (18.5%) is added. The precipitate is centrifuged off. Then pH is adjusted to 4.8, so that most of the proteins are precipitated. The precipita¬ te is centrifuged off and redissolved in water until 86% of the original plasma volume. This solution contains: Factor I: 0.6 U/ml Factor B: 0.3 U/ml 03: 0 U/ml Further, 0.3 U C3d/ml is present, but no C3c since all 03 and C3c activity is precipitated in the first precipitate. Part B The starting material is an aqueous solution containing 0.4 U of Factor I/ml and 0.16 U Factor B/ml, produced according to the method described in part A. In the solution, pH is adjusted to 7.0, and 5 g of solution are weighed in each of 8 vials. The vials are placed in water bath at 37°C for 6 minutes. 0-55% (w/w) saccharose is added to each vial, cf. the table below. After dissolution of the saccharose, pH is again adjusted to 7.0. Then the vials are sealed and heat treated for 10 hours at 60°C. Factor I and Factor B are determined by rocket immuno electrophoresis and radial immuno electrophoresis, respectively, both in heat-treated and non- heat-treated, formulated samples. % (w/w) saccharose in the formulated % recovery of % recovery of solution Factor I activity Factor B activity Part C The aqueous heat treated solution formulated with 30% (w/w) saccharose, as described in part B, is the starting material. pH is adjusted to 7.8. The solution is applied to a column packed with DEAE Sepharose FF (Pharmacia) ion exchange gel, equilibrated in phosphate buffer having a conductivity of 2.0 mS and pH 7.8. After application of the sample to the gel, it is flushed with 1.5 column volumes of equilibration buffer, followed by elution with a phosphate buffer having a conduc¬ tivity of 4.0 mS and pH 7.8. The eluate is collected. Both Factor I activity (rocket immune electrophoresis) and saccharose concentration (ionometric titration) are determi¬ ned in the starting sample for ion exchange as well as eluate. The recoveries of Factor I and saccharose are 64% and 1.1%, respectively. The eluate is concentrated by means of ultrafiltration and is freeze-dried. 10 EXAMPLE 2 The starting material is an aqueous solution containing 0.6 U Factor I/ml and 0.3 U Factor B/ml, cf. example 1, part A. In the solution, pH is adjusted to 7.0, and 5 g of solution are weighed in each of 13 vials. 0-50% (w/w) sorbitol or 0.5- 1.0 M lysine or 0.5-2.0 M glycine are added to the vials, cf. the following table. After dissolution of the stabilizer, pH is adjusted again to 7.0. Then the vials are sealed and heat treated for 10 hours at 60°C. Factor I and Factor B are determined by rocket immuno electrophoresis and radial immunodi fusion, respectively, both in heat-treated and non- heat-treated sa les. Amount of stabilizer in the formulated % recovery of % recovery of solution Factor I activity Factor B activity ø ø 5% (w/w) sorbitol 21 ø 10% (w/w) sorbitol 59 ø 15% (w/w) sorbitol 75 ø 20% (w/w) sorbitol 85 ø 30% (w/w) sorbitol 93 approx. 3 40% (w/w) sorbitol 96 5 50% (w/w) sorbitol 100 8 0.5 M lysine løø ø 1.0 M lysine løø ø 0.5 M glycine ø 0 1.0 M glycine 9 0 2.0 M glycine 59 0 11 EXAMPLE 3 The starting material is an aqueous solution containing 0.63 U Factor I/ml and 0.30 U Factor B/ml, produced as described in example l, part A. In the solution, pH is adjusted to 7.0, and 30% (w/w) saccharose is added. The formulated solution is distributed in vials so that 1/2 ml is added in each. The vials are sealed and placed at 60 ° C. At different times, cf. the following table, vials are moved from storage at 60°C to -20°c. Solutions are analysed for Factor I activity and Factor B activity by rocket immuno electrophoresis and radial im unodiffusion, respectively. Number of minutes % recovery of % recovery of at 60°c Factor I activity Factor B activity ø 100 løø 30 100 6 60 100 4 120 100 3 240 92 0 420 84 0 600 78 0 4446 46 0

Claims: 12 PATENT CLAIMS: 1 A pure Factor I protein, essentially free of infectious virus, Factor B and 03. 2. A pure Factor I protein according to claim 1, derived from plasma. 3. An injectable preparation containing pure Factor I as defined in claim 1 og claim 2. 4. A process for producing a solution of Factor I essentially free of infectious viruses and Factor B, characterized by subjecting a solution containing Factor I to pasteuriza¬ tion by heating to 55 to 65°C for Θ.5 to 100 hours in the presence of one or more stabilizers for Factor I. 5. A process according to claim 4, characterized in that the solution containing Factor I is recovered from plasma. 6. A process according to claim 4, characterized by perfor¬ ming the heat treatment at about 60°C for 4 to 24 hours, preferably 5 to 15 hours. 7. A process according to any of claims 4 to 6, characterized in that the stabilizer is a saccharide, a sugar alcohol or an amino acid or a combination thereof.

Inventors: ALSOEE KARINA OESTERGAARD, KIHL JESPER, ALSOEE, KARINA, OESTERGAARD, KIHL, JESPER
Applicants: NOVONORDISK AS, NOVO-NORDISK A/S
Application year: 1989
Main classification: C07K15
IPC codes: A61K35, A61K38, A61P37, C07K1, C07K14, C12N7