JPH0635391B2 - Heat treatment method for human blood coagulation factor IX-containing preparation - Google Patents

Description

TECHNICAL FIELD The present invention relates to a heat treatment method for virus inactivation of a human blood coagulation factor IX (hereinafter, factor IX) -containing preparation.

[Conventional technology]

Conventionally, regarding plasma proteins such as albumin, a heat treatment method in an aqueous solution state (hereinafter, referred to as a liquid heating method) has been known as the most reliable method for inactivating a virus that may be mixed in with plasma proteins. Murray) et al. (The New York A Academy of Medicine
cademy of Medicine), 31 (5), 341-358 (1955)], and it has been widely used for many years since then, and the virus inactivation effect of the liquid heating method is epidemiologically effective. Proven.

However, the ones that can withstand liquid heating such as albumin are extremely limited among plasma proteins.In particular, plasma proteins that have physiological or biological activity are extremely sensitive to heat and are prone to thermal denaturation and Is likely to decrease or disappear.

On the other hand, when the plasma protein is heat-treated (hereinafter referred to as dry heat treatment) in a dry state containing no or almost no water, in addition to the liquid heating method, its activity is lower than that in the liquid heating method. Blood coagulation VIII significantly suppressed
It became clear in the experiment using the factor as a model. However, in general, even in the dry heat treatment, the activity of the plasma protein cannot be reduced unless a stabilizer is added, and the solubility and solubility in water deteriorate.

By the way, the action mechanism of virus inactivation by heating is based on the denaturation of the protein component of the virus in liquid heating, whereas it is impaired by dry heat treatment due to the oxidation of the lipid component of the virus and the loss of pathogenicity. It has been suggested that both virus inactivation mechanisms, although overlapping with each other, are fundamentally different [Learn, Physical Methods, Obstellization of Macroorganism Bacteriology Rev. (Rahn, Physical Methods of Sterili
zation of Macroorganisms Bact. Rev.) 9 , 1-47 (194
Five)〕.

[Problems to be solved by the invention]

An object of the present invention is to provide a heat treatment method for inactivating a contaminating virus without inactivating a Factor IX-containing preparation.

[Means for solving problems]

The present inventors can inactivate a virus without losing the activity of Factor IX by subjecting a Factor IX-containing preparation to dry heat treatment in the presence of a specific stabilizer described below, and further
The present inventors have completed the present invention by discovering that Factor IX-containing preparations in which Factor IX is remarkably stabilized and which has undergone dry heat treatment under such conditions have good solubility and solubility in water.

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

The factor IX-containing preparation to be heat-treated in the present invention is one having biological activity or physiological activity as factor IX, for example, obtained by fractionating plasma protein.

Such a Factor IX-containing preparation also contains, for example, human blood coagulation Factor II, Factor VII, and Factor X,
Those that conform to the standards defined by the standards for biological products are listed. The composition ratio is, for example, IXth IX per vial.
Factor 400, factor II, factor VII, and factor X are each 400-500
The units are 100 to 500 units, 5 to 150 units, and 100 to 500 units.

The present invention is usually carried out by freeze-drying a Factor IX-containing preparation solution and then usually heating it under the condition of a moisture content of 0.05 to 3%, in which case the above-mentioned specific stabilizer is added. By preserving, the stability of Factor IX is promoted and the solubility and liquid state of Factor IX 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, it is stable against the amount of factor IX contained in 1 ml of normal human plasma, which is 300 times or more, preferably 400 times or more (that is, an activity value of 300 to 500 units) (about 1 g of protein). Sodium chloride as an agent is 100-200 mg, preferably 140-160 m
g, sodium citrate 50-150 mg, preferably 90-1
It is added at a rate of 10 mg. With this amount of addition,
It has the best balance of stabilizing effect, water solubility, and formulation of soluble form.

The factor IX-containing preparation is usually used as a lyophilized product, but the stabilizer is preferably added before the freeze-drying treatment of plasma protein.

Further, the stabilizer may be removed after the drying treatment of the present invention, but it is preferable to add it as it is to the Factor IX-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 200 hours, preferably about 10 to 100 hours.

The virus to be inactivated by the heat treatment of the present invention is a virus in which human plasma protein may be contaminated,
Especially hepatitis virus.

Further, by performing the heat treatment of the present invention in an inert gas atmosphere, the stability during heating can be further enhanced.
Examples of the inert gas include nitrogen gas, argon and helium.

Further, the degree of purification of the factor IX-containing preparation and the heat resistance are poorly correlated, and the stabilizing effect of the stabilizer does not change regardless of the degree of purification of the factor IX-containing preparation. Therefore, the heat treatment of the present invention may be performed at any stage of the Factor IX-containing preparation purification process.

The dried state in the drying treatment of the present invention is a substantially anhydrous state, and it is preferable that the water content is as low 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 production method of a plasma protein preparation, because it can inactivate a virus that may be contaminated in the preparation without significantly losing the activity of factor IX, which is a valuable blood preparation.

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

Factor IX: One-step method for measuring blood coagulation [Rapaport,
S. Eye. Et al, New England, Medical (Rap
aport, SI, et al., New Engl.Med.), 267 125-130 (196
2)] Factor II: Prothrombin single assay [Ohren,
Pee. A. Et Scandinavian Journal of
Clinical Laboratory Investigation (O
wren, PA, et al., Scand.J.Clin.Lab.Invest.), 3 201
~ 208 (1951)] Factor VII: One-step measurement method [Siliridge, M .; S. , Laboratory Evolution of Hematology, Philadelphia: Lee and Feviger (S
irridge, MS, Laboratory Evaluation of Haematology,
Philadelphia: Lee and Febiger), 1974, P.145-148] Factor X: One-step measurement method [Siliridge, M .; S. ，
Laboratory Evolution of Hematology, Philadelphia: Lee and Feviger (Sirri
dge, MS, Laboratory Evaluation of Haematology, Phil
adelphia: Lee and Febiger), 1974, P. 145-148] [Examples] The present invention will be described below with reference to experimental examples and examples, but the present invention is not limited thereto.

Example 1 From normal human plasma, DEAE cellulose column chromatography method [Dyk, G. double. Earl et al., British Journal of Hematology (Dike, G.
WR, et al., British Journal of Haematology), 22 46
9 (1972)] to obtain a powder containing Factor IX (15 units of Factor IX activity per 1 mg protein). After dissolving 100 g of this Factor IX-containing preparation in 6.5 water, sodium chloride 4
9 g and 33 g of sodium citrate were added. This IX
After adjusting the pH of the factor-containing preparation solution to 6.4 to 7.4, 50
20 ml was dispensed into 20 ml vials and freeze-dried. After freeze-drying, the factor IX activity was 440 u / V and the moisture content was 0.8%. This freeze-dried Factor IX-containing preparation was heat-treated at 60 ° C. for 72 hours, and compared with the pre-heat-treatment Factor IX-containing preparation, solubility, Factor IX activity, cellulose acetate membrane electrophoresis, gel filtration As a result of testing about the item, no remarkable change was observed even after the heat treatment,
It was found that the factor IX-containing preparation was stable under this heating condition.

Example 1 In a Factor IX-containing preparation solution prepared according to Example 1, the amount of stabilizer shown in Tables 1 and 2 was added to each Factor IX-containing preparation (Factor IX activity 400 units / bottle). After adding and adjusting pH to 6.4-7.4, it was freeze-dried. The Factor IX-containing formulation was heat treated at 60 ° C. for 72 hours and then tested for each factor activity. The results are shown in Tables 1 and 2.

As a result, in the case of no addition due to the use of stabilizer (No. II
It was found that the stability of the factor, the factor VII, the factor IX, and the factor X is extremely improved as compared with the residual solubility of 38, 30, 31, and 30%, respectively. Further, the effect of the stabilizer was more enhanced when used in combination than when used alone.

Example 2 The Factor IX formulation (400 units / bottle) prepared in Example 1
Sodium citrate 100mg / bottle, sodium chloride 1
50 mg / bottle was added, and a virus suspension of 0.05 M phosphate buffer (pH 7.1) was added thereto, and the mixture was mixed uniformly and then freeze-dried in a glass bottle. After the freeze-drying was completed, the pressure was returned to normal pressure with nitrogen gas, and the vessel was sealed, immersed in a warm bath at 60 ° C. and heated.
Since it takes 10 to 15 minutes until the temperature in the bottle reaches 60 ° C., the heating time was actually extended for 30 minutes.

The infectivity of each virus is plaque forming (plaque
forming) method.

The results are shown in Table 3.

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Claims (2)

[Claims] 1. A method comprising heating a virus-contaminated blood coagulation factor IX-containing preparation in a substantially dry state in the presence of sodium chloride and sodium citrate as stabilizers until the virus is inactivated. Method for heat treatment of human blood coagulation factor IX-containing preparation. 2. The method according to claim 1, which comprises heating under conditions of a moisture content of 3% or less.