JPH0530812B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for the preparation of plasma preparations substantially free of active virus or concentrates of blood coagulation factors, coagulation factors, and blood coagulation factors substantially free of such viruses by warming in the presence of stabilizers and prepared thereby. The present invention relates to formulations of these factors. Such formulations may be used in the treatment of blood coagulation disorders. Blood coagulation is a complex, stepwise process triggered by a variety of physiological and pathological causes, and its course is dependent on about 20 promotive or inhibitory factors. A decrease or increase in these blood coagulation factors causes blood coagulation disorders,
Some of them manifest as diseases. Concentrations of factors 1, 2, 3 and 3 are suitable for the treatment of various congenital or acquired disorders of synthesis of these factors. Treatment of patients with concentrates of these factors has hitherto been associated with the risk of viral infections and, in particular, hepatitis infections. Albumin poses no risk of hepatitis if it is heated to 60°C in an aqueous solution and in the presence of stabilizers (Gellis, SS et al. J. Clin. Invest. No. 27
(see Vol. 239, 1948). It is therefore assumed that concentrates of factor 1, 2 and factor 1 heated in the presence of suitable stabilizers are likewise free of hepatitis risk. DE 2916711 describes a method for stabilizing other coagulation factors in aqueous solutions against heat by adding amino acids and mono- or oligosaccharides or sugar alcohols. However, this method cannot prevent most of the first and second factors from being inactivated upon heating. The observation that factor 1 and factor 1 are protected from thermal inactivation in aqueous solution by chelating agents (DE3043857A1) and by calcium (DE3045153A1) was therefore an advance. However, both methods do not allow preparation of all four factors in one operational step, as desired. This is because in case of vitamin K deficiency or under treatment with oral anticoagulants, all four factors are reduced and therefore have to be replaced simultaneously. Therefore, the challenge was to find a way to stabilize aqueous solutions containing factors against thermal inactivation. It has now surprisingly been found that aqueous solutions containing all four primary and secondary factors can be protected against the adverse consequences of heat treatment by the addition of calcium ions and chelating agents. Antithrombin (AT) and heparin may optionally be added to prevent activation of factor A and factor A. Therefore, the prerequisites were provided to prepare these four factors in high purity and high yield in one operational step without active virus. The chelating agent is added in addition to the citrate already present. Even if, according to DE 3043857, chelating agents are advantageous for the protection of factor 1 and factor, whereas calcium ions are used for the same purpose for factor 1 and factor (DE 30451653), and chelating agents can also be used for calcium ions. It is surprising that a mixture of calcium ions and chelating agents, even when forming a complex, exerts the desired protective effect on all factors against loss of activity due to heating. Therefore, depending on whether chelating agents or calcium ions were present in excess, only two corresponding factors each, but not all four, should be protected against thermal degradation. The present invention therefore provides preparations of blood coagulation factors, which are virtually virus-free and without the risk of hepatitis, by heating an aqueous solution, optionally in the presence of amino acids and/or sugars or sugar alcohols. A process for the preparation of a chelating agent, characterized in that it is heated in the presence of calcium ions and a chelating agent and optionally antithrombin and/or heparin. Such solutions can be solutions containing these factors or concentrates containing them as well as plasma or plasma fractions or placental fractions. The optimum concentration of calcium ions is 1-50 mmol/preferably 25-50 mmol/. Suitable calcium ion-producing salts are, for example, the chlorides, acetates or nitrates, as well as all water-soluble calcium salts of sugar acids, such as gluconic acid or lactonic acid. Preference is given to using chloride and acetic acid solutions. Suitable chelating agents are, for example, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis-(2-aminoethyl ether)-tetraacetic acid (EGTA), diaminocyclohexane-tetraacetic acid (CDTA), diaminopropane-tetraacetic acid or nitrilotriacetic acid. Acetic acid and especially their soluble alkali salts. Aliphatic aza-tri- or -tetra-carboxylic acids having 6 to 20 carbon atoms and 1 or 2 nitrogen atoms and their soluble alkali salts and especially ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis( Preferably, the sodium salt of 2-aminoethyl ether)-tetraacetic acid (EGTA) is used. The optimum concentration of chelating agent is 1-20 mmol/and preferably 5 mmol/. 25 mmol/calcium and 5 mmol/
A mixture of EDTA has been found to be particularly suitable. Antithrombin is used at a concentration of 0.05 to 2 U/ml based on the activity (1 unit (U)) of 1 ml of citrated plasma and together with heparin at a concentration of 0.5 to 20 USP-U/ml, preferably 2 USP- 0.2 U of antithrombin with U of heparin is used. An aqueous solution of coagulation factors in the presence of calcium ions and a chelating agent and optionally an antithrombin-heparin complex can be heated until infection with viruses and certain hepatitis pathogens is virtually eliminated according to the present state of knowledge. This is of value when pasteurization is combined with adsorption and precipitation methods, in which case the active substance remains in the supernatant and the hepatitis virus can be separated out together with the insoluble precipitate. Preparations kept in aqueous solution at about 60°C for at least 10 hours are considered virtually free of hepatitis risk, especially when starting from human tissue fluids, in which hepatitis viruses are not detected according to third-generation tests. I don't get it. A particularly preferred embodiment of the invention is a solution containing all four factors, preferably 0.2 to 2 U/ml in plasma or plasma fractions or placental fractions.
of antithrombin, 2-20 USP-U/ml of heparin, 25-50 mmol of calcium ion, 1-20 mmol of EDTA, glycine,
α- or β-alanine, lysine, leucine, valine, asparagine, serine, hydroxyproline, proline, glutamine, α-, β- or γ
- at least one of aminobutyric acid, preferably glycine
Types of amino acids 1-3 mol/, and solution 100
Add 20-60 g of monosaccharide or oligosaccharide or sugar alcohol per g, preferably 1-3 mol/g of glycine and 20-60 g of sucrose per 100 g of solution,
~100℃ preferably heated to 60-100℃ and 1
It is characterized by a holding at this temperature for between minutes and 48 hours, preferably between 8 and 12 hours, the shortest time being combined with the highest temperature and vice versa. PH is kept between 6 and 8. This method results in a virtually virus-free preparation of the first and second factors. Depending on the solubility of the calcium salt, amino acid or carbohydrate, the corresponding concentrations of 0.3 and 3.0 mol/and 60 g/100 g will be higher if the calcium salt, amino acid or carbohydrate has a correspondingly higher solubility at the desired temperature. can be expanded to The temperature treatment can also be carried out in several sequential steps. Using the preferred combination of antithrombin, heparin, calcium chloride and EDTA with glycine and sucrose, a preparation without hepatitis risk is obtained by heating. In order to obtain a virus-free plasma containing the factors in their natural and active form, the plasma is preferably treated with a mixture of chelating agents.
A mixture of EDTA and calcium ions present at an EDTA concentration of 5 mmol/ and a calcium chloride concentration of 25 mmol/ is added and sucrose (60 mmol/
g/100 g)-glycine (2 mol/) in a mixture
Warm to 60°C and hold at this temperature for 10 hours. In order to obtain natural and active concentrates of the factors, a mixture of chelating agents, preferably 5 mmol/ Add a mixture of EDTA and calcium ions in a concentration ratio of 25 mmol EDTA and 25 mmol calcium chloride. Preferably 0.2 U/ml of antithrombin and preferably 2 USP-U/ml of heparin are added before heating to 60° C. in a sucrose (60 g/100 g)-glycine (2 mol/) mixture and at this temperature for 10 hours. Hold. Such a flux is e.g. Soulier et al.
Thrombosis Diath.Haemorrh.Suppl.Vol. 35, No. 61
(1969). For this, plasma obtained from blood anticoagulated with 0.01 mol/EDTA is adsorbed onto calcium phosphate and centrifuged. The factor is then quantitatively bound to the adsorbent and can be isolated by elution several times with 0.2 mol/sodium citrate. The combined eluates are further purified by a combination of alcohol and acetic acid precipitation at -8°C to +4°C. In that case the factors are concentrated at the same time. This concentrate is mixed with a suitable buffer, preferably 0.06 and 0.02 mol/salt, respectively, having a pH of 7.5.
Take in sodium citrate. Recovery and purification of coagulation factors from heated solutions can be accomplished by precipitation with ammonium sulfate at 30-45% saturation, adsorption and elution of the supernatant on 0.4-1 g/100 ml potassium phosphate. The monitoring of substances and measures for concentrating and purifying the factors is well known to the expert with knowledge of how to measure the substances in question. Using these monitoring methods operating conditions can be controlled with a view to satisfactory yield and purity of product. The first factor is, for example, Koller, F. et al.'s Dtsch.
It can be measured by the method described in med.Wschr., Vol. 81, p. 516 (1956). For this, one part of factor-deficient plasma, for example 0.1 ml, and one part of diluted normal plasma are mixed. This mixture is held at 37°C for 30 seconds.
Two parts of calcium-containing thromboplastin, for example prepared as described in German Pat. No. 2,356,493, are then added and the time elapsed until a clot appears is determined. For quantitative measurement, the clotting time obtained from a solution containing factor is read based on a calibration curve obtained using a normal plasma dilution series. One unit of factor corresponds to the factor activity of 1 ml of normal plasma. The first factor is, for example, Acta of Koller, F. et al.
haemat. Vol. 6, p. 1 (1951). For this, one part of factor-deficient plasma, for example 0.1 ml, and one part of diluted normal plasma are mixed. This mixture is held at 37°C for 30 seconds. Two parts of calcium-containing thromboplastin, such as that prepared as described in German Patent No. 2,356,493, are then added and the time elapsed until the appearance of a clot is determined. For quantitative measurement, the clotting time obtained from a solution containing factor is read based on a calibration curve obtained using a normal plasma dilution series. One unit of factor corresponds to the factor activity of 1 ml of normal plasma. Measurement of the first factor is carried out, for example, according to the following method. 1 part of partial thromboplastin, e.g. 0.1 ml, prepared e.g. as described in DE-AS2316430
is mixed with 1 part factor-deficient plasma and 1 part diluted normal plasma. This mixture is held at 37°C for 6 minutes. After adding 1 part of a 0.025 molar calcium chloride solution previously warmed to 37° C., the time elapsed from the addition of the calcium chloride solution to the appearance of a clot is measured. For quantitative measurement, the clotting time obtained from a solution containing factor is read based on a calibration curve obtained using a normal plasma dilution series. Factor 1 international unit (=1) is 1 ml of normal plasma
This corresponds to the factor activity of The first factor is, for example, Blood of Duckert et al.
Coagulation, Hemorrhage and Thrombosis,
It can be measured by the method described in Tocantins, LM and Kazal, LA (1964). For this, one part of factor-deficient plasma, for example 0.1 ml, and one part of diluted normal plasma are mixed. This mixture was heated at 37°C.
Hold for 30 seconds. Two parts of calcium-containing thromboplastin, for example prepared as described in German Patent No. 2,356,493, are then added and the time elapsed until the appearance of a clot is determined. For quantitative measurement, the clotting time obtained from a solution containing factor is read based on a calibration curve obtained using a normal plasma dilution series. One unit of factor corresponds to the factor activity of 1 ml of normal plasma. To kill the hepatitis virus, calcium ions, EDTA, glycine and sucrose and optionally antithrombin and heparin are added to the solution and heated. Human plasma was used after pasteurization (60°C, 10 hours) with the addition of sucrose (60 g/100 g) and glycine (2 mol/) and in the presence of 25 mmol/calcium chloride and 5 mmol/EDTA. The following activities were found based on plasma. namely factor 90%, factor 85%, factor 83% and factor 80%. The influence of various stabilizers on obtaining the activity of the respective prothrombin factor in the plasma or placental fraction upon heating is shown in the table. Pasteurization using sucrose and glycine alone yields poor factor yields. However, this is very disadvantageous. This is because prothrombin concentrate is mainly used for the treatment of hemophilia type B. The additional use of calcium ions at a concentration of 6.25 mmol/min without chelating agent results in activation of factor and factor and also reduces the yield of factor and factor. Such formulations are not suitable for human use due to the potential risk of thrombosis. 5 mmol/along with sucrose and glycine without using calcium ions
When using EDTA at a concentration of Antithrombin, heparin, sucrose and glycine alone cannot stabilize prothrombin factor. Best results are obtained by combining calcium ions and EDTA using sucrose and glycine and adding antithrombin and heparin. Under these conditions the yield for all four factors is in the 80% range and the formulation is also free of activated factor and thrombin. The concentration of the coagulation factor in the aqueous solution in which the method described above may be carried out lies in particular within the following ranges: Factor (F) 0.3-50U/ml Factor (F) 0.3-30U/ml Factor (F) 0.5-60U/ml Factor (F) 0.4-60U/ml Particularly preferred ranges are 0.5~25, 0.5~15, 0.7~ for the first factor, respectively.
30 and 0.6-30. Concentrates usually refer to solutions containing active substances, ie coagulation factors in the present case, in a higher concentration than the natural starting material.

[Table] For further purification, the heated solution can optionally be centrifuged. Impurities may be removed by precipitation with ammonium sulfate at 30-45% saturation. The supernatant can then be adsorbed to 0.04-1.0 g of calcium phosphate per 100 ml of solution, the loaded adsorbent washed, eluted with citrate buffer and the eluate dialysed. The product may be sterilized for human use. The present invention particularly relates to active virus-free first, second, third and factor formulations obtainable by the method. In order to increase the storage stability, it is advantageous to add protein stabilizing substances, such as proteins, amino acids or carbohydrates to the formulation. Finally, the preparations subjected to this treatment can be provided in lyophilized form, the addition of anticoagulants, such as eg heparin, may be preferred. The product according to the invention, especially in the form of a concentrate, is a medicament for the treatment of coagulopathy in a solution suitable for pharmaceutical administration and intravenously, preferably as an infusion for the treatment and prevention of bleeding caused by factor deficiency. can be used for The pasteurized product in the form of freeze-dried, coagulation-activated plasma can be used, for example, for blood volume filling,
It covers the indications for fresh frozen plasma in the maintenance of colloid osmotic pressure, the treatment of various forms of blood loss after major surgery and disasters, as well as in the emergency care of multiple traumas. The invention is illustrated by the following examples. Example 1 1000 ml of human citrate plasma was stirred for 25-30 min.
℃ and contains 1.86 g of EDTA and
20 ml of a solution adjusted to pH 7.8 with 2N NaOH was added. 1000 sucrose in the solution heated to 30℃ after 10 minutes.
I slowly stirred in g. After the sucrose was dissolved, crystalline glycine was slowly added with stirring.
After this has completely dissolved, add 25 ml of 1M CaCl ₂ solution.
and PH the solution using 2N NaOH.
Adjusted to 7.2. The solution, made up to 1.7 using stabilizers, was heated to 60° C. on a water bath for 10 hours and remained clear even after heating. Removal of stabilizer: After cooling the pasteurized solution, convert it to 3.0 and
It was clarified by passing through a 1.2μ Sartorius membrane filter.
This solution was then mixed with citrate-NaCl-buffer (0.01 mol/trisodium citrate, PH
7.5, 0.06 mol/NaCl) to 5 and dialyzed three times against buffer 5 of the same composition with simultaneous concentration in an ultrafilter and 1000
Concentrated to ml. After the plasma starting volume reaches 1000ml, remove the clear solution.
Sterilized and clarified in a Sartorius membrane filter,
Filled to ml volumes and lyophilized. 50ml
The following activities were found based on standard human plasma in the dried product reconstituted with distilled water:
i.e. factor 87%, factor 81%, factor
83% and factor 80%. Example 2 1. Isolation of prothrombin factor by adsorption and elution on DEAE-Sephadex.
DEAE-Sephadex A50 containing mmol/EDTA and equilibrated to PH6 (0.6 g/
) was added. This suspension was stirred at 15° C. for 60 minutes to allow adsorption. After the adsorbent has settled, the supernatant liquid is siphoned off and DEAE-Sephadex containing the adsorbed factors is added at 5 mmol/
Washed with 50 ml of physiological saline solution containing EDTA until free of contaminating proteins. Next, the adsorbent was added to the PH containing 5 mmol/EDTA.
The adsorbent was removed by centrifugation and discarded. 2. Pasteurization 0.2 U/ml of antithrombin and 2 U/ml of heparin were added to the supernatant. Then add 1050 g of solid glycine and after these have dissolved, correspond to a final concentration of 25 mmol/
25.7 g of _{CaCl2.2H2O was} added. constant PH value
After reaching 7.2, the solution was heated to 30-37°C and 10.5Kg of sucrose was added (final concentration solution
Approximately 60g per 100g). After adjusting the pH value of this highly viscous solution to 7.2 again, this solution was placed in a water bath.
Heated to 60°C for 10 hours. 3. Removal of undesired proteins by precipitation with ammonium sulfate Cool the pasteurized solution to room temperature and correspond to a final concentration of 50 mmol/
Distilled water 31.5 containing 334 g CaCl ₂ 2H ₂ O
Added inside. Into this solution of prothrombin factor 24.5 ml of saturated ammonium sulfate solution at PH 7.5 was introduced, the precipitate was centrifuged and discarded. 4 High-level purification of prothrombin factor by adsorption to calcium phosphate.
7.6 Add 700 g of solid Ca ₃ (PO ₄ ) ₂ and at room temperature
Stir for 20-30 minutes. Calcium phosphate was obtained by centrifugation and had a pH of 7.6 at 0.5 mol/
of NaCl and 1g/100ml of glycine
I washed it at 20. Prothrombin factor was extracted from the adsorbent with 0.2 mol/sodium citrate and 0.15 mol/sodium citrate.
It was eluted with 750 ml of buffer (PH approximately 7.5) containing NaCl, 1 g/100 ml glycine, 0.6 U/ml antithrombin and 28 U/ml heparin and by stirring (20 minutes) at room temperature. Coarse particles were removed from the eluate by centrifugation at 3000 g. 5. Formulation 0.15 g/100 ml of Aerosil was added to the supernatant and solids were removed by ultracentrifugation at 30000 g. Approximately 0.6 g of human albumin per 100 ml of liquid was added to the clear supernatant liquid, the pH was adjusted to 6.8, and physiological saline solution was added at 50 g.
Dialysis was performed for 3 hours. Add this solution to the factor approx.
60 U of factor, 50 U of factor and 25 U of factor were adjusted, sterilized, filled in 5 ml aliquots, and lyophilized.

Claims (1)

[Scope of Claims] 1. An aqueous liquid containing blood coagulation factors, blood coagulation factors, and optionally amino acids and/or
or in the preparation of virtually virus-free and hepatitis-free preparations of these factors by heating in the presence of sugars or sugar alcohols;
A method characterized by heating in the presence of calcium ions and a chelating agent and optionally antithrombin and/or heparin. 2 The concentration of calcium ions is 1 to 50 mmol/
, preferably from 25 to 50 mmol/. 3. The chelating agent is an aliphatic aza-tri- or -tetra-carboxylic acid having 6 to 20 carbon atoms and 1 or 2 nitrogen atoms or a soluble alkali salt thereof. The method described in Scope 1. 4 The concentration of the chelating agent is between 1 and 20, preferably 5.
2. A method according to claim 1, characterized in that the amount of mol/mmol/mmol. 5 0.2-2U/ml antithrombin, 2-
20USP-U/ml heparin, 25-50 mmol/
of calcium ions, 1-20 mmol/of
EDTA, glycine, α- or β-alanine,
1 to 3 mol/at least one amino acid of lysine, leucine, valine, asparagine, serine, hydroxyproline, proline, glutamine, α-, β- or γ-aminobutyric acid, preferably glycine, and 20 to 3 mol per 100 g of solution. 60g monosaccharides or oligosaccharides or sugar alcohols, preferably 1
~3 mol/g of glycine and 20 per 100 g of solution
30-100℃ preferably 60℃ in the presence of ~60g sucrose
~100℃ temperature for 1 minute ~48 hours preferably 8~12
The method according to claim 1, characterized in that the heating is carried out for a period of time. 6. Process according to claim 1, characterized in that the aqueous liquid is citrate plasma or a fraction of plasma or placenta. 7. A method according to claim 1, characterized in that the aqueous liquid is a concentrate of factors 1, 2, and 3.