# US 4,395,396 A · Blood-coagulation-promoting preparation based on human proteins and a method of producing the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,395,396 A - Document ID: US4395396A - Status: Granted - Kind: A - Application number: 06/283,143 - Filed: 1981-07-14 - Granted: 1983-07-26 - Earliest claimed: 1980-07-22 - Assignees: Immuno Aktiengesellschaft fur chemisch-medizinische Produkte - Inventors: Johann Eibl; Otto Schwarz; Fritz Elsinger; Anton Philapitsch - Web page: https://patentgrep.com/patent/US4395396A ## CPC - C07K 14/745: Blood coagulation or fibrinolysis factors - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract In a method of producing a blood-coagulation-promoting preparation based on human proteins and having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), human plasma is treated with sulphated high-polymer carbohydrates and/or with basic ion exchangers, the protein mixture with generated FEIB-activity is adsorbed on the ion exchanger, and the preparation is gained by elution and concentration. ## Claims ### Claim 1 A blood-coagulation-promoting preparation based on human proteins, having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-by-passing-activity (FEIBA), which preparation is characterized in that it is free of thrombogenic activity up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, it is free of kallikrein activity and free of prekallikrein activator activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, it is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, said eluate containing said protein with factor IX activity and said eluate containing said protein with FEIB-activity contain .alpha.- and .beta.-globulins when electrophoretically separated thus obtaining a separation curve having a main peak in the .alpha.-globulin region corresponding to a content of 60 to 80% of the total protein, a shoulder of 10 to 20% of the total protein following thereupon, and a slightly pronounced peak in the .beta.-globulin region corresponding to a content of 10 to 20% of the total protein following upon the shoulder-like course of said separation curve. ### Claim 2 (depends on 1) A preparation as set forth in claim 1, wherein said protein with factor IX activity elutes at an NaCl concentration of 0.1 to 0.5 molar and said protein with FEIB-activity elutes at an NaCl concentration of 0.3 to 0.5 molar, the maximum factor IX activity eluting at 0.3 molar and the maximum FEIB-activity eluting at 0.4 molar. ### Claim 3 (depends on 1) A preparation as set forth in claim 1, wherein said FEIB-activity, after a one-hour incubation in factor VIII inhibitor plasma, is preserved by at least 50%. ### Claim 4 (depends on 1) A preparation as set forth in claim 1, wherein said factor IX activity, after a one-hour incubation in factor IX deficient plasma, is preserved by at least 50%. ### Claim 5 (depends on 1) A preparation as set forth in claim 1, further comprising a content of inter-alpha-trypsin inhibitor (ITI) of 0.05 to 5 mg per FEIBA unit. ### Claim 6 (depends on 1) A method of producing a blood-coagulation-promoting preparation as set forth in claim 1, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration. ### Claim 7 (depends on 6) A method as set forth in claim 6, wherein said human plasma at first is briefly treated with sulphated high-polymer carbohydrates, said protein mixture with generated FEIB-activity is then adsorbed on an ion exchanger on dextran basis and is then immediately eluted and concentrated. ### Claim 8 (depends on 6) A method as set forth in claim 6, wherein said human plasma is treated with an ion exchanger on dextran basis, and after at least two hours of exposure said protein mixture with generated FEIB-activity, adsorbed on said ion exchanger, is eluted and concentrated. ### Claim 9 (depends on 6) A method for promoting the coagulation of blood containing an inhibitor to factor VIII which comprises administering to a patient having blood which contains an inhibitor to factor VIII, an amount, effective to provide factor-VIII-inhibitor-by-passing-activity, of a blood coagulation-promoting preparation, which is produced according to the method described in claim 6. ### Claim 10 A blood-coagulation-promoting preparation based on human proteins, having a content of coagulation factors II, VII, IX and X and a factor-VIII-inhibitor-by-passing-activity (FEIBA), which preparation is characterized in that it is free of thrombogenic activity up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler; it is free of kallikrein activity and free of prekallikrein activator activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml; and it is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity. ### Claim 11 (depends on 10) A preparation as set forth in claim 10, wherein said FEIB-activity, after a one-hour incubation in factor VIII inhibitor plasma, is preserved by at least 50%. ### Claim 12 (depends on 10) A preparation as set forth in claim 10,further comprising a content of inter-alpha-trypsin inhibitor (ITI) OF 0.05 to 5 mg per FEIBA unit. ### Claim 13 A method for promoting the coagulation of blood containing an inhibitor to Factor VIII, which comprises administering to a patient having blood which contains an inhibitor to factor VIII, an amount, effective to provide factor-VIII-inhibitor-bypassing activity, of a blood coagulation-promoting preparation based on human proteins, having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), which preparation is characterized in that it is free of thrombogenic activity up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to WEssler; it is free of kallikrein activity and free of prekallikrein activator activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml; and it is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an elute containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, said eluate containing said protein with factor IX activity and said eluate containing said protein with FEIB-activity contain .alpha.- and .beta.-globulins when electrophoretically separated thus obtaining a separation curve having a main peak in the .alpha.-globulin region corresponding to a content of 60 to 80% of the total protein, a shoulder of 10 to 20% of the total protein following thereupon, and a slightly pronounced peak in the .beta.-globulin region corresponding to a content of 10 to 20% of the total protein following upon the shoulderlike course of said separation curve. ### Claim 14 A method for promoting the coagulation of blood containing an inhibitor to factor VIII, which comprises administering to a patient having blood which contains an inhibitor to factor VIII, an amount, effective to provide factor-VIII-inhibitor-bypassing-activity, of a blood coagulation-promoting preparation based on human proteins, having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-by-passing-activity (FEIBA), which preparation is characterized in that it is free of thrombogenic activity up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler; it is free of kallikrein activity and free of prekallikrein activator activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml; and it is affinity-chromatographically separable on dextran sulphate agarose by means of a NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an elute containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity.