JPS60155120A - Manufacture of blood plasma medicine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the preparation of plasma preparations substantially free of active virus or blood coagulation products substantially free of such viruses by warming in the presence of stabilizers. The present invention relates to a method for preparing concentrates of factor X and to formulations of these factors prepared thereby. 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. Due to a decrease or increase in these blood clotting factors9
Blood clotting disorders occur, some of which manifest as diseases.

Concentrates of Factors II, II, II and X 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 aqueous solution and in the presence of stabilizers (G
ellis, S. S. J. et al. Clin.

Invest. See Vol. 27, p. 239 + 1948.

Therefore, No. 1, No. 2, No.
and concentrates of factor X are also assumed to be free of hepatitis risk.

DE 29 16 711 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 factors ①, ②, ② and X from being inactivated upon heating. Therefore, factor ■ and factor
D (DE 30 45 857 A1) and factors II and
The observation that 153 AI) is protected from thermal inactivation in aqueous solution was an advance. However, both methods do not allow preparation of all four factors in one operational step, as desired. This is because in the case of vitamin deficiencies or under treatment with oral anticoagulants, all four factors are reduced and therefore have to be replaced simultaneously.

Therefore, the problem was to find a way to stabilize aqueous solutions containing factors ①, ②, ② and X against thermal inactivation.

It has now surprisingly been found that aqueous solutions containing all four factors 1, 2, 2 and X can be protected against the adverse effects of heat treatment by the addition of calcium ions and chelating agents. . Optionally 9-antithrombin (AT) to prevent activation of factor II and factor II.
) III and heparin can be added. Therefore, the prerequisites were given to prepare these four factors with high purity and high yield in one operational step containing active virus.

The chelating agent is added in addition to the citrate already present.

For example, according to Dg 3043857, chelating agents are advantageous for the protection of factors and factor
and calcium ions are used for the same purpose for factor X (DE 3045153 ”).

(No.), and even though the chelate-forming agent also forms a complex with calcium ions, the mixture of calcium ions and the chelate-forming agent is effective against loss of activity due to heating.
It is surprising that factors 1, 2 and X all fail to exert the desired protective effect. Therefore, depending on whether chelating agents or calcium ions are present in excess, only two corresponding factors each should be protected against thermal degradation, but not all four.

Therefore, the present invention provides an aqueous solution that optionally contains amino acids and/or amino acids.
or calcium ions in the preparation of preparations of blood coagulation factors Ⅰ, Ⅲ, ③ and and a method characterized by heating in the presence of a chelating agent and, optionally, antithrombin (2) 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 immersion rate of calcium ions is 1 to 50 mmol/l, preferably 25 to 50 mmol/l, per day.

Suitable calcium ion-producing salts are, for example, all water-soluble calcium salts of sugar acids, such as the chlorides, acetates or nitrates, as well as gluconic or lactonic acids. Preference is given to using chlorides and acetates. Suitable chelating agents are, for example, ethylenediaminetetraacetic acid (BDTA), ethylene glycol, -bi.

Sue(2-a is noether ether)-tetraacetic acid, ::.

(E()TA), diaminocyclohexane-4-(C
DTA), diaminopropane-tetraacetic acid or nitrilotriacetic acid and especially their soluble alkali salts.

Aliphatic azatri- or monotetracarboxylic acids having 6 to 20 carbon atoms and 1 or 2 nitrogen atoms and their possible, soluble alkali salts and especially ethynediaminetetraacetic acid @ ( shoulder, q or ethylene green
Ru, bis-(2-azeta-no-ethybeether)-tetraacetic acid (EGTA), thorium, salt are used.
I like it. , sharp,,-to.

Form, formation, agent, optimum concentration a1. ~20 mmol,
/, tsoshi -teko, mashi, gumak・5mi1rimo,ru/1
It is. ,,.

25 mmolpht,, Calciu,, mu and tomiri dirt 1 l/l Q ID'I! A mixture of A was found to be particularly suitable. , : ・,,.

, ,An, Tito Town, Nbin 11I′i, 1. - 1 liter of acid storage mixture.

Based on plasma activity (1 unit (b)), 0.0 empty ~
At a concentration of 2U/d, it's been 1 year! Te 0,5~20
Heparin at a concentration of U8P7 U/-, preferably 2. USP-U-15. Rin, do, -Ono・α2. Longbin, the anti-city of U.

Ling is used.

Calcium, cum ions, and chelating agents, and in some cases, Aichito, C! Nbin ■ - hebarin complex,
In the presence of water soluble coagulation factors, and infection with certain hepatitis pathogens, it can be heated until it is virtually eliminated according to today's standards of wisdom.

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 third-generation tests have shown that the Ibaraki hepatitis virus Cannot be detected.

A particularly preferred embodiment of the invention is a solution containing all four factors, preferably 0.2 to 2 U/m/m of antithrombin, 2 to 20 UEIP, in the plasma or plasma fraction or placental horn. heparin, 25
~50 mmol/l of calcium ions, 1-20 mmol/l of EiDTA, glycine, α- and Lβ-
Alanine, lysine, leucine, valine, asparagine,
serine, hydroxyproline, proline, glutamine,
1 to 3 mol/l of at least one amino acid, α-1β- or γ-aminobutyric acid, preferably glycine, and 920 to 6011 monosaccharides or oligosaccharides or sugar alcohols, preferably 1 to 3 mol per 1ooy of solution. /l glycine and 20-609 parts sucrose per 10011 solution, heated to 30-100°C, preferably 60-1°C and 0°C and kept at this temperature for 1 minute to 48 hours, preferably 8-12 hours. , where the shortest time and highest temperature are combined and vice versa.

The pH is maintained at 6-8. In this way virtually fils-free formulations of Factors I, II, II and X are obtained.

Depending on the solubility of the calcium salt, amino acid or carbohydrate, the corresponding 0, 3 and 3.0 mol/l and 61+/100 & if the calcium salt, amino acid or carbohydrate has a correspondingly higher solubility at the desired temperature.
The concentration can be scaled up to higher concentrations. The temperature treatment can also be carried out in several sequential steps.

By heating with antithrombin, heparin, calcium chloride and the preferably used combination of EDTA with glycine and sucrose, a preparation is obtained without the risk of hepatitis.

In order to obtain a virus-free plasma containing Factors 1, 2, 3 and
5 mmol/l and calcium chloride concentration 25 mmol/! A mixture of EDTA and calcium ions present at 100° C. is added and warmed to 60° C. in a sucrose (60II/100.9)-glycine (2 mol/1) mixture and kept at this temperature for 10 hours.

In order to obtain natural and active concentrates of factors 1, 2, 2 and Add a mixture of EDTA and calcium ions in a concentration ratio of 5 mmol/l TMA and 25 mmol/l calcium chloride. Preferably 0.20/- of antithrombin ■ and preferably 2 USP-U/d of hepari/, followed by sucrose (60 g/100.9)-
Warm to 60° C. in a glycine (2 mol/1) mixture and hold at this temperature for 10 hours.

Such fractions are described, for example, by 8oulier et al.
rom'bosis Death, Haemorrh
, 8upp1. Obtained by the method described in Vol. 35, p. 61 (1969).

For this, α01 mol/l (D) Plasma obtained from blood anticoagulated with EDTk is adsorbed on calcium phosphate and centrifuged. The factor is quantitatively bound to the adsorbent and 0.2 mol/l The combined eluates are further purified by a combination of alcohol and acetic acid precipitation at -8°C to +4°C, in which case the factor is simultaneously concentrated. be done.

This concentrate is added to a suitable buffer, preferably p) 0.06 and 0.02 mol/l NaCl/L with p) 17.5, respectively.
Take in sodium citrate.

Recovery and purification of coagulation factors from heated solutions
Precipitation with ammonium sulfate at ~45% saturation, 0.
This can be achieved by adsorption and elution of the supernatant on catasium phosphate of 4 to 1.9/100.

The monitoring of measures for reducing and elucidating factors ①, ②, ② and Using these monitoring methods, operating conditions can be controlled with a view to satisfactory yield and purity of product.

Factor Ⅰ is, for example, the Dtsch method of Koller, F. et al.
.

med, Wschr, Vol. 81, p. 516 (1956
year) can be measured by the method described. This includes 1 part of factor II-deficient plasma, e.g. 1 part of diluted normal plasma.
Mix parts. 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. 2,356,495, 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 (I) is read based on a calibration curve obtained using a normal plasma dilution series.

Factor 1 corresponds to the activity of 1 unit of factor 1 in normal plasma.

Factor Ⅰ is, for example, Actah of Koller, F. et al.
aemat, Vol. 6, p. 1 (1951). To this was diluted 1 part of Ko factor I, deficient plasma, for example 0.1-. Mix 1 part of normal plasma. This mixture is held at 67°C for 30 seconds. Next, two parts of a calcium-containing Trompoplar, lXtin, prepared as described in West German Patent No. 2,556,493, are added, and the mixture is allowed to stand until the appearance of clots.
Measure time. For constant quantitative measurement, the clotting time obtained from a solution containing factor (I) is read based on a calibration curve obtained using a normal plasma dilution series.

One unit of factor Ⅰ corresponds to the factor Ⅰ activity of the normal level*1-.

Measurement of factor X is carried out, for example, according to the following method.

For example, the part prepared as described in DB-A 苧2316450, thoron giblastin 1. 1 part, for example, 0.1 d'i factor 1 K-deficient plasma and 1 part diluted normal plasma. Add this mixture to 3
Hold at 7°C for 6 minutes and 1 minute. After adding 1 part of 0.025 molar calcium chloride solution pre-warmed to 37°C, the time elapsed from the addition of the calcium chloride solution to the appearance of clots. Measure. For quantitative measurement, factor X can be obtained from a sipping solution containing
The clotting and incubation times are based on the assay line 1 obtained using a normal plasma dilution series.

Number ■, factor 51 country missing unit 4 (lock 1! ■) is normal plasma 11
1!7! The second factor is equivalent to the activity of the factor.

- Factor X is described, for example, by Duclcert et al., BL
oo, dCoagulation, )Iemorrh
age and Thrombosi, 4゜'Toca
ntipql L, ,M, and ,ICaza14
, L.A., ed., (1,964). This includes 1 part of factor X-deficient plasma, e.g. 0.1
- and 1 part diluted normal plasma are mixed. This mixture is held at 67°C for 30 seconds. Two parts of calcium-containing thromboplastin, for example prepared as described in DE 23 56 493, are then added and the time elapsed until the clot arms appear is determined. For quantitative determination, the clotting time obtained from a solution containing factor X is read based on a calibration curve obtained using a normal plasma dilution series. One unit of factor X corresponds to one unit of factor X activity in normal plasma.

Calcium ions, ED in solution to kill hepatitis virus
Add TA, glycine and sucrose and optionally lianthithrombin and heparin and heat.

Sucrose (60#/100ji) and glycine (2mol/
1) and pasteurization (60°C, 10 hours) of human plasma in the presence of 25 mmol/l calcium chloride and 5 mmol/l EDTA, the following activity was found based on the plasma used: It was done. namely factor X 80%, factor X 80%, factor X 86chi and factor X 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 Table 1. Pasteurization using sucrose and glycine alone yields poor factor X yields. However, this is very disadvantageous. This is because prothrombin concentrate is mainly used for the treatment of hemophilia type B. Concomitant use of calcium ions at a concentration of 6.25 mmol/l without chelating agents resulted in activation of factors II and X and also decreased the yield of factors II and X. let Such formulations are not suitable for human use because of the risk of thrombosis.

When using 'BDTA at a concentration of 5 mmol/l along with sucrose and glycine without using calcium ions, the yields of Factors II and II are good and the yields of Factors II and X are poor. antithrombin■, heparin,
Sucrose and Glycun alone cannot stabilize prothrombin factor.

The best results are obtained using sucrose and glycine, adding antithrombin and heparin, and combining calcium ions and ED.
It can be obtained by combining with TA. Under these conditions the yield for all four factors is about 80% units 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'l[) 0.3~50U/-Factor ■(
F■) 0.5-50U/ml Factor ■ (FK) 0.
5~+50U/-Factor X (FX) 0.4~60U/
d Particularly preferred ranges are 0.5 to 25.0.5 to 15.0.7 for factors
~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.

For further purification, the heated solution can optionally be centrifuged. Impurities may be removed by precipitation with ammonium sulfate at 30-45% saturation.

Next, the supernatant liquid was adsorbed on calcium phosphate of solution 100-10,04~tO,V, and the loaded adsorbent was washed.
Elution may be performed with citrate buffer and the eluate dialysed.

The product may be sterile filtered for human use.

The present invention particularly relates to active virus-free factor .

In order to increase storage stability, it is advantageous to add protein stabilizing substances such as proteins, amino acids or carbohydrates to the formulation. Finally, the R agent which has been subjected to this treatment can be provided in lyophilized form, the addition of an anticoagulant such as, for example, heparin, being advantageous.

A medicament for the treatment of coagulopathy in a solution suitable for pharmacological administration of the product according to the invention, especially in the form of a concentrate.
) and can be used intravenously, preferably as an infusion, for the treatment and prevention of bleeding caused by factor deficiency.

Freeze-dried, coagulation-activated plasma or pasteurized products in the form can be used, for example, for filling blood volumes, maintaining colloid osmotic pressure, treating blood loss after major surgeries and disasters, and various forms of shock and multiple traumas. It covers the indications for fresh frozen plasma in emergency cases.

The invention is illustrated by the following examples.

□ Example 1 Human citrate blood W1000sdl 25-6 under stirring
Warm to 0°C and add 20- of a solution containing KDTA186N and adjusted to pH 7.8 with 2N NaOH. After 10 minutes, 1 sucrose was added to the solution heated to 30°C.
I slowly stirred in 00ON.

After the sucrose had dissolved, crystalline glycine was slowly added with stirring. After this is completely dissolved, 1 mop CaCl2
Solution 25- was added and the solution was adjusted to pm (7,2) using 2N NlOH. The solution, which had been made up to t7t using stabilizers, was heated to 60 °C on a water bath for 10 h and remained clear even after heating.・□ Removal of stabilizer: ・ After cooling the pasteurized solution, it was filtered through a 5 artorius membrane filter with &, 0 and t2 μ to clarify it.・
This solution was then mixed with citrate-NaC1-buffer (0,.
0.1! -L/l of citric acid, linato-lium,
5 using NaCj (pH 7.5, α06 momo 7t)
Diluted to t and dialyzed 6 times against 5 volumes of buffer of the same composition with simultaneous concentration in an ultrafilter and 100
Concentrated to 0-.

After the plasma starting volume reaches 1000 m, a clear solution f5
It was sterilized and clarified using an Artorius MtE filter, filled to a volume of 50 d and lyophilized.

507! The following activities were found based on standard human plasma in the dried product reconstituted with distilled water: namely factor X 80chi, factor X 80chi, factor X 83chi and factor X 80chi.

Example 2 Isolation of prothrombin factor by adsorption and elution on DgAE-8ephadex' Citrated plasma 550 μm DEkH containing 5 mmol/l EDTA in saline solution and equilibrated to pa 6 -8ephadex■person 5'O(0,611
/l) was added. This suspension was stirred at 15° C. for 60 minutes to cause adsorption. After the adsorbent has settled, the supernatant liquid is siphoned and the adsorbed factor is D]1
iAE-8ephadex” with 5 mmol/l EDT
The plate was washed with 50 t of physiological saline solution containing A until all contaminating proteins were removed. Next, the adsorbent was added to 5 mmol/l of E
After stirring with 1 mol/l NaC17t pH 8 containing DTA, the adsorbent was removed by centrifugation and discarded.

2. 0.2 U/- of antithrombin and 2 U/- of heparin were added to the pasteurized supernatant. Next, solid glycine 1050
11 and, after these have dissolved, 25.7 & CaCl2, corresponding to a final concentration of 25 mmol/l.
o' added to 2H20' After reaching a constant pI-J@7.2, heat the solution to 30-57°C and 10.
Added 5 Kf of sucrose (approx. 6 kg per 100 g of final concentration solution)
01i). After the pH value of this highly viscous solution was adjusted once again to Z2, the solution was heated to 60° C. in a water bath for 10 hours.

3. Removal of unwanted proteins by precipitation with ammonium sulfate. Cool the pasteurized solution 142 to room temperature and 5.
634I CaC corresponding to a final concentration of 0 mmol/l
The solution was added to 31.5 liters of distilled water containing 12.2H20.

A saturated ammonium sulfate solution at pH 75 was introduced into this solution of prothrombin factor, and the precipitate was centrifuged and discarded.

4. Highly Purified Prothrombin Factor by Adsorption on Calcium Phosphate Solid Ca5 (PO4) 27001 was added to the supernatant containing prothrombin factor at pH 76 and stirred at room temperature for 20-30 minutes. Calcium phosphate was extracted by centrifugation and had a pH of 7.6, 0.5 mol/
l o Washed with 20t of a solution of Na, Cl and IJi'/1oo-o glycine.

Prothrombin factor was extracted from the adsorbent with 0.2 mol/l trisodium citrate, 0.15 mol/l NaCl,
Elution was carried out using 750 tons of buffer (pH approximately 7.5) containing 11/100-glycine, 0,6 U/- antithrombin and 28 U/- heparin and by stirring (20 min) at room temperature. It was done. 3
Coarse particles were removed from the eluate by centrifugation at 000g.

5. Add α1511/100-Aerozil (Aer
osil) and then! +0000. Solids were removed by ultracentrifugation at p.p. Approximately 0.69 human albumin per 100 mm of liquid was added to the clear supernatant liquid, and p
The solution was adjusted to H6.8 and dialyzed against 50 tons of physiological saline for 6 hours. Add this solution to 80% of factor X and 50U of factor
and factor ■, adjusted to 25U, sterilized and filtered,
Filled in 5-volume portions and lyophilized.

Continuation of Gesellschaft page 1 @) Int, CI,' Identification symbol Internal serial number 0
Written by: Hans Martin, Labreis, Federal Republic of Germany 4

Claims (1)

[Scope of Claims] 1) Water/liquid containing blood'coagulation factor ■, factor ■, factor 1■, and factor To prepare preparations of these factors that are practically virus-free and without the risk of hepatitis, it is possible to do so by heating in the presence of □'1 alcohol).
9. Calcium ions and chelating agents and their deterioration? 2) A method that detects skin symptoms by heating the soda and /l sauce to a temperature of □ in the presence of ``balin''. 2) The degree of crushing of calcium ions is □1~'50□'
□ #lmol/l, preferably 2'5 to 50 mmol/l: ゛ □ 6゛ □ 6》 A method in which the chelate forming agent has 6□ to 20 molecules and The above-mentioned □ patent claim is characterized in that the first encounter is an alkali salt of a carbon aliphatic 7r- or -tetracarboxylic acid having two nitrogen atoms. Scope 1 Current method described □. 4) The above-mentioned [! F.Claim 1: The method according to claim 1. 5》α2～□□2 Koji antithrombin III, 2～
20 UI9P-Rue heparin, 25-50 lmol/l calcium ions, 1-20 mmol/l R
DTA, glycine, α-t or β-alanine, □lysine, leucine, valine, asparagine, □sericine, drochyreproline, proline, glutamic acid, α-, □β7
F-1 is r'-aminobutyric acid, preferably glycine, at least one amino acid 1 to 1 mole/1, and 20 to 601 monosaccharides or oligosaccharides or sugar alcohols, preferably glycine, per 100 g of solution. In the presence of 1 to 3 mol/l of glycine and 920 to 6011 parts of sucrose per 100 g of solution, heating to a temperature of 30 to 100°C, preferably 60 to 100°C, for 1 minute to 48 hours, preferably 8 to 12 hours. 6) The method according to claim 1, characterized in that the aqueous liquid is citrated plasma or a fraction of plasma or placenta. 7) 2. A method according to claim 1, characterized in that the aqueous liquid is a concentrate of Factors I, II, II and X.