JPS6210019A - Heat-treatment of drug preparation containing human blood coagulation factor ix - Google Patents

Abstract

PURPOSE:To inactivate virus in a drug preparation containing virus-contaminated human blood coagulation factor IX, by heat-treating the preparation in the presence of a specific stabilizing agent in dried state. CONSTITUTION:A drug preparation containing virus-contaminated human blood coagulation factor IX (e.g. human blood coagulation factor II, factor VII, factor X, etc.) is freeze-dried, and heated under a condition having a humidity of 0.05-3%. A stabilizing agent consisting of sodium chloride and sodium citrate is added to the system preferably prior to the freeze-drying treatment of the plasma protein. The heat-treatment is carried out at 30-100 deg.C, preferably at 60 deg.C for 10min-200hr, preferably for 10-100hr to inactivate the contaminant virus and to obtain a drug preparation containing human blood coagulation factor IV having improved stability and water-solubility of the factor IV. The amount of the stabilizer is 100-200mg of sodium chloride and 50-150mg of sodium citrate based on the amount of factor IV which is >=300 times that of the factor IX in 1ml of normal human plasma.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a heat treatment method for virus inactivation of a preparation containing human blood coagulation factor (I) (hereinafter referred to as factor X).

[Conventional technology]

Traditionally, heat treatment in an aqueous solution (hereinafter referred to as liquid heating method) has been the most reliable method for inactivating viruses that may be contaminated with plasma proteins such as albumin. Hurray) et al. [The New York Academy of Op Medicine (The Ne
w York Academy of Medicine
e).

31 (51, 341-35B (1955)), and has been widely used for many years until today, and the virus inactivation effect of the liquid heating method has been epidemiologically proven.

However, only a limited number of plasma proteins, such as albumin, can withstand liquid heating, and plasma proteins that have physiological or biological activity are particularly sensitive to heat and easily undergo thermal denaturation. It is easy to cause a decrease in activity or disappearance.

On the other hand, separate from the liquid heating method, if plasma proteins are heated in a dry state that does not contain or contains little water (hereinafter referred to as dry heat treatment), their activity decreases and decreases compared to the liquid heating method. The blood coagulation tube is markedly inhibited.■
This was revealed through an experiment using factors as a model. However, in general, even in dry heat treatment, unless a stabilizer is added, the activity of plasma proteins cannot be avoided, and the solubility and solubility in water deteriorates.

By the way, the mechanism of action of virus inactivation by heating is that liquid heating is mainly based on the denaturation of the protein components of the virus, whereas dry heat treatment is mainly based on the oxidation of the lipid components of the virus, which damages the virus and makes it pathogenic. It is said that the virus inactivation mechanisms of both types overlap with each other, but it has been suggested that they are fundamentally different [Learn, Physical Methods of Sterilization of Macroorganisms and Bacteriology Rev. Rahn, Physical
Methods of 5terilization
f Macroorganisms Bact, Re
v.) Lord, 1-47 (1945)).

[Problem that the invention seeks to solve]

An object of the present invention is to provide a heat treatment method for inactivating contaminant viruses without inactivating a factor X-containing preparation.

c. Means for Solving Problems] The present inventors have demonstrated that they can remove viruses without losing the activity of factor X by dry heat treating a factor The present invention was completed by discovering that factor X can be inactivated, that factor X is significantly stabilized, and that factor

That is, the present invention involves heating a virus-contaminated blood coagulation factor-containing preparation in a substantially dry state in the presence of sodium chloride and sodium citrate as stabilizers until the virus is inactivated. Characteristics of human blood coagulation
This invention relates to a heat treatment method for factor-containing preparations, which inactivates contaminating viruses and improves the stability and water solubility of factor X.

The factor

Examples of such factor The composition ratio is, for example, 400 to 50 each of factor X, factor II, factor II, and factor X per vial.
0 units, 100-500 units, 5-150 units, 100
~500 units.

The present invention is usually carried out by freeze-drying a factor By adding it, the stability of factor X is promoted and the solubility and liquid state of factor X are improved.

The stabilizers used in the present invention are sodium chloride and sodium citrate.

The amount of the stabilizer used is, for example, as follows.

That is, the amount of factor X contained in normal human plasma 1-300
more than twice, preferably more than 400 times (i.e., activity value 300 times more)
Sodium chloride as a stabilizer is used as a stabilizer for a factor
fIg, preferably 140 to 160 mg, and sodium citrate 50 to 150 mg, preferably 90 to 1)0
It is added in the proportion of mg. At this level of addition,
It has the best balance between stabilizing effect, water solubility, solubility and formulation.

Factor

Furthermore, although the stabilizer may be removed after the drying treatment of the present invention, it is preferable to include it as it is in the factor X-containing preparation.

The heating temperature in the heat treatment is usually 30 to 100°C, preferably about 60°C, and the heating time is usually 10 minutes to 100°C.
It is about 200 hours, preferably about 10 to 100 hours.

Viruses to be inactivated by the heat treatment of the present invention are viruses that are likely to contaminate human direct proteins, particularly hepatitis viruses.

Further, by performing the heat treatment of the present invention in an inert gas atmosphere, stability during heating can be further improved.

Examples of the inert gas include nitrogen gas, argon, and helium.

Furthermore, there is a poor correlation between the degree of purification and heat resistance of a factor X-containing preparation, and no matter what degree of purification a factor Therefore, the heat treatment of the present invention may be performed at any stage of the factor X-containing preparation purification process.

The dry state in the drying process of the present invention is substantially anhydrous, preferably with as little moisture as possible. The water content is usually 3% or less, preferably 1% or less, and usually about 0.05 to 3%.

The present invention is useful as an industrial method for producing plasma protein preparations, since it is possible to inactivate viruses that may be mixed into the preparation without significantly losing the activity of factor X, which is a valuable blood product.

In the present invention, the titer of each factor was measured by the following method.

■ Blood coagulation measurement method using the factor ■ two-step method [Lavaboat,
Niss, I, et al., New England, Medical (R
apaport, S, 1. + et al,, N
ew Engl.

Med, ), 267125-130 (1962))
■ Factor ■: Prothrombin alone measurement method [Osin.

P., N. et al., Scandinavian Journal of
Clinical Laboratory Investigation (O
wren+ P, A,, et al,, 5cand
.

J, Cl1n, Lab, Invest,), 3
201-208 (1951)) ■ Part ■ Prisoner 2 One-Step Measurement Method [Shirifuji. M, Varnish 3. Laboratory Evaluation Ophematology, Philadelphia:
Lee & Huebiger (Sirridge, M.S.
,, Laboratory Evaluation
of Haematology+ Ph1ladel
phia: Lee and Febiger), 1
974. P, 145-148) ■ MXX factor knee side measurement method syrifuge. M, varnish 0. Laboratory Evaluation of Hematology, Philadelphia: Lee & Huebiger (Sirridge, M.
S., Laboratory Evalu-atio
n of llaematology, Ph1lad
elphia: Lee and Febiger),
1974. P, 145-148) [Examples] The present invention will be explained below using experimental examples and examples, but the present invention is not limited by these in any way.

Example 1 From normal human plasma, DEAE cellulose column chromatography method [Dike, G., Double.

Earl et al., Pritish Journal of Hematology (Dike, G.W.R., et al., B
r1tishJournal of Haematol
ogy), 22469 (1972))
Factor-containing powder product (factor activity 15 per mg protein)
unit) was obtained. 6.5 g of this factor X-containing preparation
After dissolving with 1 part of water, 49 g of sodium chloride and 33 g of sodium citrate were added.

After adjusting the pl+ of this factor The factor X activity after freeze-drying was 440 u/V, and the humidity content was 0.8%. This freeze-dried factor As a result of testing for the following items, no significant changes were observed even after heat treatment, and it was found that the factor X-containing preparation was stable under these heating conditions.

Experimental Example 1 The amount of stabilizer listed in Tables 1 and 2 was added to the factor
Factor ■ activity 400 units/bottle) and pH
was adjusted to 6.4 to 7.4, and then freeze-dried. After heat-treating the factor

The results are shown in Tables 1 and 2.

As a result, due to the use of stabilizers, when no additives are used (Chapter
It was found that the stability of Factor, Factor Ⅰ, Factor X, and Factor Furthermore, the effect of the stabilizer was more enhanced when used in combination than when used alone.

(Leaving space below) Table 1: Thermal stabilization effect of sodium citrate on heating of dried human blood coagulation factor (400 volumes/bottle) (
60℃, 72hr, sodium chloride 150IIIg
/v) (blank space below) Table 2: Dried human blood coagulation No.
°C, 72hr, sodium citrate: 1OQmg/V
) Experimental Example 2 To the factor ■ preparation prepared in Example 1 (400 units/bottle), 100 mg/bottle of sodium citrate and 150 B/bottle of sodium chloride were added, and to this was added O.05M phosphoric acid overlay (pH 7). , 1) was added, mixed uniformly, and then freeze-dried in a glass bottle. After the freeze-drying was completed, the pressure was returned to normal using nitrogen gas, the tube was sealed tightly, and the tube was immersed in a hot bath at 60.degree. C. and heated. Note that since it takes 10 to 15 minutes for the temperature inside the bottle to reach 60°C, the heating time was actually extended for 30 minutes.

The infectivity of each virus is determined by black forming (pla
It was measured by the que forming method.

The results are shown in Table 3.

(Blank below) Procedural amendment (October 28, 1985 Patent Application No. 148964 2, title of invention 3, heat treatment method for human blood coagulation factor-containing preparations 3, person making amendment case) Relationship Patent applicant name Midori Juji Co., Ltd. 4, agent 541 Address New Life Hirano-cho 406, 4-53-3 Hirano-cho, Higashi-ku, Osaka (06) 227-1) 56

Claims (2)

[Claims] (1) A virus-contaminated blood coagulation factor IX-containing preparation is heated in a substantially dry state in the presence of sodium chloride and sodium citrate as stabilizers until the virus is inactivated. Person Blood Coagulation No. I
Method for heat treatment of factor-containing preparations. (2) The method according to claim (1), which comprises heating under conditions where the humidity content is 3% or less.