JP2965069B2 - Method for producing protein-containing composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a substantially protein-containing pharmaceutical composition virus is inactivated processed and protein-containing reagents from protein-containing composition is the possibility of viral contamination
It relates to a method for producing the composition .

[0002]

2. Description of the Related Art Virus-containing proteins such as hepatitis virus and AIDS virus may be mixed into protein-containing compositions derived from human blood.

[0003] In order to prevent the spread of these viruses, a method of heating a protein-containing liquid composition is known (JP-A-55-145615, JP-A-56-139422,
JP-A-56-106594).

[0004] A method of heating a protein-containing dry composition is also known (Japanese Patent Application Laid-Open No. 58-55048;
JP-A-58-213721).

Further, a method is known in which a virus is removed by bringing a protein-containing composition into contact with a trialkyl phosphate (JP-A-60-51116).

However, there has been a problem that the activity of the protein is reduced during the treatment with the trialkyl phosphate.

[0007]

[0008] The present invention is trialkyl phosphate Fe - DOO process efficiently viruses without losing most of the active protein to inactivation treatment in step, safe protein-containing pharmaceutical composition or protein-containing reagent composition It is intended to provide a method for manufacturing an article .

[0008]

SUMMARY OF THE INVENTION In order to solve these problems, the present invention comprises contacting a liquid composition containing a protein with a possibility of virus contamination with a trialkyl phosphate in the presence of a protease inhibitor. The present invention provides a method for producing a protein-containing pharmaceutical composition or a protein-containing reagent composition which has been subjected to a virus inactivation treatment , the method comprising:

[0009] By the method of the present invention, trialkyl phosphate Fe - DOO process efficiently inactivated treated protein-containing pharmaceutical group <br/> Narubutsu or protein-containing reagent composition of the virus without loss of activity of the protein in Products are manufactured.

[0010] The protein to which the method of the present invention is applied is not particularly limited, and examples thereof include a protein derived from plasma, a protein derived from another tissue, and a protein obtained by genetic recombination or tissue culture. Examples of proteins include plasminogen, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XIII, antithrombin III
Haptoglobin, thrombin, prothrombin, immunoglobulin, fibrinogen, fibronectin, albumin, hemoglobin, interferon, plasminogen activator and the like. If the protein-containing liquid composition contains a protein as described above,
There is no particular limitation.

The protein-containing liquid composition to which the method of the present invention is applied is not particularly limited. For example, a solution comprising a plasma or tissue extract, a fraction obtained by treating the plasma or tissue extract by various fractionation methods And a culture solution obtained by culturing a genetically modified host or tissue, a commercially available protein preparation (liquid form) or a solution.

Further, the degree of purification of the protein-containing liquid composition at the time of contact with the trialkyl phosphate of the present invention is not particularly limited, and can be applied to any degree of purification. May be applied to any stage of protein separation and purification.

The trialkyl phosphate used in the present invention is not particularly limited, but is preferably tri- (n-butyl) phosphate, tri- (tert-butyl) phosphate, tri- (n-hexyl) phosphate or tri- (n-hexyl) phosphate. (2
-Ethylhexyl) phosphate, tri- (n-decyl) phosphate and the like. A particularly preferred trialkyl phosphate is tri- (n-butyl) phosphate (hereinafter referred to as TNBP). It should be noted that a mixture of two or more different trialkyl phosphates can also be used.

The trialkyl phosphate used in the present invention is used in an amount ranging from 0.01 to 10 (w / v)%, preferably in an amount ranging from about 0.1 to 3 (w / v)%. Is done.

The trialkyl phosphate can be used with or without a surfactant. Preferably, a trialkyl phosphate is used in combination with a surfactant. The surfactant can be used before, simultaneously with, contacting the trialkyl phosphate with the protein-containing liquid composition,
Alternatively, it can be added at any later stage. The function of the detergent is to enhance the contact between the virus and the trialkyl phosphate in the protein-containing composition.

Examples of the surfactant include a polyoxyethylene derivative of a fatty acid, a partial ester of sorbitol anhydride,
For example, Polysorbate 80 (trade name: Tween 80)
Etc.), polysorbate 20 (trade name: Tween 20)
And nonionic oil bath water detergents such as oxyethylated alkylphenols (trade name: Triton X100)
Etc.). In addition, sodium deoxycholate and Zwittergents, a synthetic zwitterterionic detergent known as sulfobetaine, such as N-dodecyl-N, N-dimethyl-2-ammonio-1-ethanesulfonate and its homologs, or nonionics Detergents, for example, octyl-β, D-glucopyranoside.

When a surfactant is used, its amount is not critical and can be used, for example, in the range of about 0.001% to about 10%, preferably about 0.01% to 3%.

Trialkyl phosphate treatment is carried out using an envelope coat virus such as hepatitis B virus, n
It is particularly useful for inactivating hepatitis B virus, human immunodeficiency virus (HIV), vesicular stomatitis virus, Sindbis virus, and the like. Also,
Further heat treatment in a dry state for a sufficient time,
Heat-resistant viruses can also be inactivated.

The treatment of the protein-containing composition with the trialkyl phosphate of the present invention is carried out at a temperature of -5 ° C to 70 ° C, preferably 0 ° C to 60 ° C, preferably for 30 minutes or more, more preferably 1 to 30 It is carried out for a time, more preferably for 3 to 10 hours.

The protease inhibitor is not particularly limited as long as it is a substance that substantially inhibits the activity of the protease. For example, ε-aminocaproic acid (EACA),
Examples include basic amino acids such as lysine and arginine, and proteins such as aprotinin.

Usually, after the trialkyl phosphate treatment,
The trialkyl phosphate is removed. When a surfactant or a stabilizer is used, they are also removed. The removal may be performed by any method, and examples thereof include a method of adsorbing a protein by affinity chromatography and a method of recovering the protein by precipitation.

In the present invention, a known heat treatment may be performed before or after the trialkyl phosphate treatment in the presence of the protease inhibitor. The heat treatment may be performed in any of a liquid state and a dry state.

Dry composition (dry protein-containing composition)
May be performed before or after the trialkyl phosphate treatment, but is preferably performed after the trialkyl phosphate treatment. When the treatment is performed after the trialkyl phosphate treatment, the dried composition is obtained by removing the trialkyl phosphate and the like, recovering the protein, and freeze-drying by a known method.

The heat treatment of the dried composition is usually carried out at 30 ° C. to 1 ° C.
00 ° C., preferably 55 ° C. to 75 ° C., usually 3 to
It is carried out for 200 hours, preferably for 10 to 100 hours.
In order to protect the protein from heat, the reaction may be performed in the presence of a stabilizer. Examples of the stabilizer include sugar, sugar alcohol, amino acid and the like.

The dry composition is substantially free of moisture, and usually has a moisture content of 3% or less, preferably 1% or less.

The heat treatment in a liquid state may be performed before or after the trialkyl phosphate treatment, but is preferably performed after the trialkyl phosphate treatment. When the treatment is performed after the trialkyl phosphate treatment, the protein is heated after removing the trialkyl phosphate and the like.

When the heat treatment is performed in a liquid state,
It is carried out usually at 30 ° C to 100 ° C, preferably at 55 ° C to 75 ° C, for usually 1 to 100 hours, preferably 5 to 30 hours. In order to protect the protein from heat, the reaction may be performed in the presence of a stabilizer. Examples of the stabilizer include sugar, sugar alcohol, amino acid and the like.

[0028]

According to the production method of the present invention,
Trialkyl phosphate Fe - DOO process without almost loss activity of the protein in the process, efficiently protein-containing pharmaceutical composition virus is inactivated processed or protein-containing reagents
A composition is manufactured.

In the case of a trialkyl phosphate treatment at 25 ° C. to 30 ° C., the protein may be decomposed by a protease mixed in the solution by promoting the treatment at a temperature for a long time, but the treatment is carried out in the presence of a protease inhibitor. By doing so, a decrease in protein activity can be suppressed.

Incidentally, the trialkyl phosphate treatment has a weak inactivating effect on viruses having no envelope.
By subjecting the protein-containing composition to a heat treatment before or after the trialkyl phosphate treatment of the present invention, viruses without an envelope can also be significantly inactivated.

[0031] Thus, the production method of the present invention provides a highly preferred method for industrial preparation of safer protein-containing pharmaceutical composition or protein-containing reagent composition, the inactivation-treated protein preparation of the virus Particularly useful for manufacturing.

[0032]

【Example】

Example 1 Fr. I paste was added to 0.025M EDTA-
0.0 containing 2Na and 10 units / ml aprotinin
After dissolving in a 55 M sodium citrate buffer, pH 6.4, the mixture is heated at 50 ° C. for 30 minutes to denature and remove fibrinogen. After concentrating this solution, 30% aprotinin was added to the concentrate.
Then, 0.3% w / v of TNBP and 1% w / v of Tween 80 are added, and the virus is inactivated at 30 ° C. for 6 hours or more. After the virus inactivation treatment, fibronectin is adsorbed on DEAE-Sephadex, washed well to remove TNBP and Tween 80, and then fibronectin is eluted from the gel and concentrated.

Example 2 After preparing the fibronectin of Example 1 at a concentration of 30 mg / ml in 0.05 M Tris-phosphate buffer, pH 8.0, 1 kg of sucrose was added to 1 liter of the aqueous solution and stirred well. Thereafter, the mixture is heated at 60 ° C. for 10 hours to produce a virus-safe preparation.

Example 3 Cryoprecipitate was dissolved in a 20 mM Tris-HCl buffer, and aluminum hydroxide gel was added at 1% w / v to obtain a deprothrombin solution. 2M glycine in this solution
, Fibrinogen is removed as a precipitate, sodium chloride is added to the obtained supernatant to 1.5 M, and fibronectin is recovered in the supernatant. After desalting and concentrating this solution, 4% of EACA (ε-aminocaproic acid) was added to the concentrated solution, and TNBP was 0.3% w / v, Tween
80% is added at 1% w / v, and the virus is inactivated at 30 ° C. for 6 hours or more. After the virus inactivation treatment, TNBP and Tween 80 are removed in the same manner as in Examples 1 and 2, and the mixture is further heated at 60 ° C. for 10 hours to produce a virus-safe preparation.

[0035]

[Experimental example]

Experimental Example 1 Stability of Protein In thrombin, corn Fr. II + III paste or Fr. III After dissolving the paste with a 0.15 M sodium chloride solution, thromboplastin (placental extract) is added to convert prothrombin to thrombin. Then, thrombin is adsorbed by SP-Sephadex, and 0.15 M
After thorough washing with a sodium chloride solution, thrombin is eluted with a buffer containing 0.5 M sodium chloride. SP-
0.3% w / v of TNBP, Tw in Sephadex eluate
een 80 was added at 1% w / v, heated at 30 ° C. and sampled with time, and thrombin activity was measured. The activity loss after 30 hours was large, and 90% even after 6 hours at 30 ° C.
Was determined to be difficult to maintain the activity, and a stabilizer was screened. As a result, the addition of EACA (ε-aminocaproic acid) and arginine can prevent a decrease in thrombin activity. In particular, EACA can maintain an activity of 90% or more even after 30 hours at 30 ° C. after addition of 2% or more at 30 ° C. (See FIGS. 1 and 2).

Fibrinogen is obtained from Fr. TNBP was added to a solution obtained by dissolving I paste in a physiological saline solution at 0.3% w /
v, Tween 80 was added at 1% w / v, heated at 30 ° C., sampled with time, and the fibrinogen coagulation activity was measured. As a result, it was found that no coagulation activity was observed after 24 hours at 30 ° C., and a screening for a stabilizer was performed. As a result, it was found that aprotinin and EACA had a stabilizing effect. By adding 0.5 M or more of EACA, 90% of the coagulation activity could be maintained even after 24 hours at 30 ° C. (see FIG. 3).

Experimental Example 2 TNB was added to the desalted concentrated solution of fibronectin obtained in Example 3.
0.3% w / v of P and 1% w / v of Tween 80 were added, and 3
After 3 hours, 6 hours, and 30 hours after heating at 0 ° C., the gelatin binding activity of fibronectin was measured.
The activity retention rate after 85 hours was 85%, and 5 hours after 30 hours.
Since it decreased to 5%, a screening for a stabilizer was performed. As a result, by adding 2% or more of EACA and 10 units / ml of aprotinin, it was possible to prevent a decrease in fibronectin activity (see Tables 1 and 2).

[0038]

[Table 1]

[0039]

[Table 2]

Experimental Example 3 Virus Inactivating Effect The virus inactivating effect in the fibronectin-containing composition was examined. (Experimental method) Aprotinin was added at 30 units / ml to the desalted concentrated solution of fibronectin obtained in Example 3, and TNBP was added to 0.1%.
3% w / v and Tween 80 were added to 1% w / v, and VSV and Sindbis virus as enveloped viruses and Echo virus as non-enveloped virus were monitored. 10 ⁶ to 10 ⁷ cells / ml, heated at 30 ° C., sampled with time, and the remaining virus was measured by the method shown in Table 3. The results are as shown in Table 4. In each preparation, VSV and Sindbis virus were inactivated to below the detection limit by treatment at 30 ° C. for 30 hours. On the other hand, the echovirus, which is a virus without a membrane, was hardly inactivated even after 6 hours at 30 ° C.

[0041]

[Table 3]

[0042]

[Table 4]

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of addition of EACA on the residual activity of thrombin at the time of trialkyl phosphate treatment over time.

FIG. 2 is a graph showing the effect of arginine addition on the residual activity of thrombin during the treatment with trialkyl phosphate over time.

FIG. 3 shows the effect of the addition of EACA on the residual activity of fibrinogen during trialkyl phosphate treatment over time.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Kazuo Takechi, Inventor 2-1,180-1, Odani, Hirakata City, Osaka Prefecture 1 Inside the Midori Cross Research Institute, Inc. (72) Takao Omura 2,1,180, Invited Otani, Hirakata City, Osaka (1) Inside the Midori Cross Research Institute, Inc. (72) Inventor Yutaka Hirao 2-1-1, Shodai Otani, Hirakata City, Osaka Prefecture 1180, 1 Inside Midori Cross Research Institute, Inc. (72) Inventor Kazumasa Yokoyama 2-1-1, Shodai Otani, Hirakata City, Osaka Prefecture, 1 Inside Midori Cross Research Institute, Inc. (56) References Table 2-504587 (JP) , A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 38/16 A61K 38/43 CA (STN) REGI STRY (STN) WPIDS (STN)

Claims (4)

(57) [Claims] Claims: 1. A virus-inactivated treatment , comprising the step of contacting a liquid composition containing a protein with the possibility of virus contamination with a trialkyl phosphate in the presence of a protease inhibitor. A method for producing a protein-containing pharmaceutical composition. 2. A protein-containing solution having the possibility of virus contamination.
Composition in the presence of a protease inhibitor
Characterized by including a step of contacting with kilf phosphate
Of a protein-containing reagent after inactivation of a virus to be used
Method of manufacturing a product. The 3. A protein-containing liquid composition, in the presence of a protease inhibitor and a surfactant, trialkyl phosphate Fe - claim 1 or 2 method characterized by comprising the step of contacting with and. 4. A protein factor VIII, blood coagulation factor IX, thrombin, fibrinogen, and methods of claim 1-3 is at least one selected from the group consisting of fibronectin.