JPH03106824A - Heat-treatment of coagulation factor xiii - Google Patents

Abstract

PURPOSE:To inactivate contaminant viruses while keeping the physiological activity of coagulation factor XIII (abbreviated as factor XIII) at a sufficiently high level by heat-treating an aqueous solution containing factor XIII in the presence of an acidic amino acid or its salt as a stabilizer. CONSTITUTION:An aqueous solution containing factor XIII (e.g. extract of human placenta, blood or aqueous solution of human blood fraction containing factor XIII) is added with >=2M of an acidic amino acid (e.g. glutamic acid or aspartic acid) or its salt as a stabilizer, heat-treated at 50-70 deg.C (preferably about 60 deg.C) for 10min to 20hr (preferably 10hr) and centrifuged to remove thermally denaturated proteins. The obtained factor XIII contains viruses such as hepatitis virus or AIDS virus in essentially inactivated state and the activity of the factor XIII is maintained to a high level.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for heat treatment of an aqueous solution containing blood coagulation factor XIII.

[Conventional technology]

Human blood coagulation factor XIII (hereinafter simply referred to as factor '' exists in the blood in an inactive state, but when blood coagulation occurs due to bleeding etc. and thrombin is produced, it is activated by the action of this thrompin and calcium ions, and stabilizes fibrin. Therefore, in blood with reduced or deficient factor XIII, the coagulation time shows a normal value, but the formed fibrin clot is fragile and exhibits peculiar phenomena such as posthemorrhage.

Regarding clinical applications of factor XIII, it can be widely used to treat congenital or acquired factor XIII deficiency and deficiency, as well as to promote wound healing after general surgery.

For the production of Factor

By the way, when factor XIII is recovered from plasma, platelets, or placenta, the possibility of contaminating viruses such as hepatitis viruses cannot be denied. Therefore, some kind of virus inactivation treatment is required.

Among these, a method of processing at 60° C1σ for a time is well known as a liquid heat treatment. It has also been reported that factor XIII is subjected to liquid heat treatment for 60'C10 hours in the presence of various stabilizers. That is,
A method using monosaccharides, sugar alcohols or neutral amino acids (JP 53-59018), a method using monosaccharides, sugar alcohols or neutral amino acids in combination with carboxylic acids (JP 56-135418), Mono IJ! Method of using oligosaccharide neck or sugar alcohol in combination with 75-noic acid (Unexamined Japanese Patent Publication No. 1983-
145615), etc.

[Problem to be solved by the invention]

In view of the above circumstances, the inventors of the present invention have conducted various studies, and as a result,
By using an acidic amino acid alone as a stabilizer, the thermal stability of factor XIII during liquid heating can be improved,
Furthermore, the present invention was completed by discovering that this heat treatment facilitates high-level purification of factor XIII.

[Means for solving the problem] (i) Starting material: The starting material in the production method of the present invention is human blood coagulation factor X.
There are no particular limitations on the aqueous solution as long as it contains Factor III and impurities. Examples of such raw materials include human placenta extract (e.g., a suspension mixed with a crushed placenta and the same weight of physiological saline), blood, and an aqueous solution of a human blood fraction containing factor XIII. Ru.

It is preferable to perform pretreatment on the starting material before carrying out the purification method of the present invention.

The pretreatment is usually performed by combining known methods.

Currently known methods for producing factor
I[[Method of Purifying Factors (Brute, Volume 25, Volume 2
35 pages, 1972), a method for purifying placenta-derived crude globulin fractions by polyalkylene glycol fractionation (Japanese Patent Publication No. 60-29687), a method for purifying placenta extracts by ammonium sulfate fractionation (Japanese Patent Publication No. 60-29687), -36487
JP-A No. 58-121219), a method of fractionation by adding an alkali metal sulfate salt (JP-A-58-121219), a method of purification using an anion exchanger (JP-A-58-185525, Japanese Patent Publication No. 60-258123
JP-A-6317829), and a method of separating at low alcohol concentration at acidic pH (JP-A-6317829).

The Factor XIII-containing aqueous solution in the present invention may be a Factor Aqueous solutions are subject to heat treatment. The content of protein factor XIII is preferably 0.1 to 30% (w/v), but is not particularly limited.

Further, the pH of the aqueous solution is generally pH 6 to 8, and is preferably adjusted to about pH 7 with an appropriate buffer.

(ii) Stabilizer: The stabilizer of the present invention includes acidic amino acids or salts thereof.

Examples of acidic amino acids include glutamic acid and aspartic acid.

Examples of the salts include alkali metal salts (eg, sodium salts, potassium salts, etc.) and alkali metal salts (eg, calcium salts, etc.).

In addition, the amount added is at least 2M or more, for example,
An example is about 2 to 3M.

(iii) Heat treatment: Heat treatment may be performed at a temperature and time sufficient to inactivate contaminating viruses, specifically at 50°C to 70°C, preferably about 60°C, for 10 minutes to It is carried out for 20 hours, preferably 10 hours.

(iv) Post-treatment: After the heat treatment, it is preferable to perform centrifugation to remove heat-denatured proteins. The conditions are 3.000 ~ 2
An example is 0.00 Orpm for about 5 to 30 minutes.

The factor is sufficiently inactivated.

Further, no particular abnormality was observed in appearance or properties, and only a very small amount of contaminant protein was contained, and the physiological activity of factor XI was sufficiently retained.

In this way, the product that has undergone the method of the present invention becomes a factor XIII preparation that is medically extremely safe and highly effective.

The preparation thus treated according to the invention is in the form of a solution;
When a crude product is used, it is further processed according to a known purification method and, if necessary, subjected to dialysis and sterilization filtration before being dispensed. Moreover, the preparation may be a freeze-dried preparation if desired.

The factor XIII that has undergone this treatment is administered as it is, or after being subjected to a formulation treatment known per se and diluted or dissolved in, for example, distilled water for injection.

〔effect〕

Factor The use of factor XIII greatly reduces the possibility of infection by viruses.

[Example] Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

In addition, the activity of factor XIII in the following examples is determined by measuring the activity of factor
sis at Diathesis Haemorrh
Agica), 23, 455, 1970).

The A-D solutions used in the examples refer to solutions consisting of the following precepts.

Solution A: 0.9% Na C 42-0.2% EDTAB
Solution: 20mM phosphoric acid-5mM EDTA (pH 7.2
) Solution C: 20mM phosphoric acid-〇. 5n+M E D
TA-0.1 MN a Cl (pH 7.2) Solution D: 2On+M'J acid-5mM EDTA-1
0% (V/V) ethanol (pH 7.2) Reference example Hydrophobic chromatography (Phenyl-Se
Phrose) and 15 kg of 24 placentas purified by lectin-binding chromatography were homogenized to obtain a crushed product, and an equal weight of Solution A (15f) was added and stirred. At this time, the specific activity (U/A280) was 0.045, making the recovery rate 100%.
shall be. Obtain the supernatant from centrifugation and perform 3 as a concentration operation.
After stirring and dissolving 5% saturated ammonium sulfate (hereinafter referred to as ammonium sulfate), a precipitate was obtained by centrifugation and dissolved in 4 liters of B solution. At this time, the specific activity was 0.22 and the recovery rate was 86%.
Met. Add to this 40% of the equivalent volume (4 liters) as a desalting operation.
After adding and stirring a (W/V) polyethylene glycol (hereinafter referred to as PEG) solution, a precipitate was obtained by centrifugation and dissolved in Solution B (3 liters). The supernatant was obtained by centrifugation. The specific activity at this time was 0.40 and the recovery rate was 80%. This crude product is subjected to virus inactivation treatment at a rate of 0.3
%TNBP, 30°C in the presence of 1% Tween 80,
The cells were incubated for 6 hours.

Furthermore, this treated solution was diluted with distilled water to lower the ionic strength in order to be used in the next step of ion exchange.

Next, the anion exchanger DEA equilibrated with liquid B in advance
A sample was charged onto E-Sepharose CL-6B, washed with the same buffer solution B, and then eluted with solution C. The specific activity at this time was 4.81 and the recovery rate was 75%. This was subjected to ultrafiltration and concentration using Vericon with a molecular fraction of 1.1 million.

Next, hydrophobic chromatography using Phenyl-Sepharose CL4B was performed.

That is, a sample was charged onto a column equilibrated with Solution C in advance, washed with Solution B, and then eluted with Solution D. The specific activity at this time was 45.7 and the recovery rate was 60%. This was subjected to ultrafiltration and concentration using Vericon with a molecular weight cutoff of 100,000.

Example 1 Glutamate sodium salt was added to the factor
= 40g of Na was added) and then heat treated at 60'C for 10 hours.

Further centrifugation (10.00 Orpm for 20 minutes)
After collecting both precipitates, add 5mM EDTA to 20mM'J.
Factor XIII was extracted with acid buffer (pH 7.2). Its specific activity is 200 units/A280, recovery rate is 77
%Met.

(Purity is over 95%).

Example 2 The same procedure as in Example 1 was carried out using aspartate sodium salt.

Experimental example 1 The relationship between the type of stabilizer and the stabilizing effect was investigated.

The concentration was 2M. Experimental example 2 The relationship between the amount of stabilizer added and the stabilizing effect was investigated.

The stabilizer was glutamate sodium salt.

*Additional amount of Glu-Ha to sample iN Experimental example 3 The relationship between the concentration of factor XIII and the stabilizing effect was investigated.

The stabilizer is glutamate sodium salt, the amount added is 4
0% (corresponding to 2.IM).

Claims (1)

[Claims] Blood coagulation factor
Heat treatment method for factor II.