JP3005979B2 - Protein-containing composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a virus-inactivated protein-containing pharmaceutical composition or a protein-containing composition produced from a protein-containing composition having the possibility of virus contamination.
It relates to a reagent composition.

[0002]

2. Description of the Related Art Viruses such as hepatitis virus and AIDS virus may be contaminated in a protein-containing composition 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).

[0006] However, heat treatment may cause heat-resistant virus to remain, and trialkyl phosphate treatment may cause non-enveloped virus to remain.
In addition, there was a problem that the activity of the protein was reduced during the treatment with the trialkyl phosphate.

[0007]

The present invention invention is to solve the above is effectively contaminating viruses without losing little activity of the protein is inactivated processed <br/>, safer protein-containing pharmaceutical pharmaceutically or reagent
It is intended to provide a composition or a protein-containing reagent composition.

[0008]

In order to solve these problems, the present invention provides the following protein-containing pharmaceutical composition or protein- containing pharmaceutical composition.
A reagent composition is provided. (1) A protein-containing pharmaceutical composition or a protein-containing reagent composition which has been subjected to virus inactivation by a method comprising the following steps. (A) contacting a liquid protein-containing composition with potential viral contaminants with a trialkyl phosphate and a surfactant , and then removing the trialkyl phosphate and the surfactant from the protein-containing composition; removing the active agent,
(B) to a protein-containing composition was freeze-dried, heat-treating the dried form of the protein-containing composition.

[0009] (2) in the step of (a), the protein-containing liquid composition trialkyl phosphate transfection in the presence of protease inhibitors - Rukoto is contacted with bets and surfactant
The protein-containing pharmaceutical composition or protein according to (1) above,
White matter containing reagent composition.

[0010]

(3) The protein-containing pharmaceutical composition of (1) or (2) , wherein the protein is at least one selected from the group consisting of blood coagulation factor VIII, blood coagulation factor IX, thrombin, fibrinogen, and fibronectin. Thing or
A protein-containing reagent composition.

According to the present invention, protein-containing physician inactivated process efficiently virus without loss of activity of the protein
A drug composition or a protein-containing reagent composition is provided.

In the present invention, the protein is not particularly limited, and includes proteins derived from plasma, proteins derived from other tissues, proteins obtained by genetic recombination or tissue culture, and the like. 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,
Examples include fibrinogen, fibronectin, albumin, hemoglobin, interferon, plasminogen activator and the like. The protein-containing liquid composition (liquid protein-containing composition) is not particularly limited as long as it contains the protein as described above.

In the present invention, the protein-containing liquid composition (liquid protein-containing composition) is not particularly limited, and may be, for example, a plasma or tissue extract, or a plasma or tissue extract obtained by treating the plasma or tissue extract by various fractionation methods. Solution, a culture solution obtained by culturing a genetically modified host or tissue, a commercially available protein preparation (liquid) or a solution.

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.

Either the trialkyl phosphate treatment or the heat treatment may be performed first, but preferably the trialkyl phosphate treatment is performed first.

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.

Trialkyl phosphates can be used with or without surfactants. 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, suitably for 30 minutes or more, more preferably 1 to 30 minutes. It is carried out for a time, more preferably for 3 to 10 hours.

The trialkyl phosphate treatment is preferably performed in the presence of a protease inhibitor in order to prevent a decrease in protein activity.

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. Further, in addition to the trialkyl phosphate treatment, a known heat treatment can be further performed.

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

The protein-containing pharmaceutical composition or the protein-containing reagent composition of the present invention, wherein the contaminating virus has been inactivated , is characterized in that the protein activity in the trialkyl phosphate treatment step is lower than that in the trialkyl phosphate treatment step. Those treated under the condition that 90% or more of the activity is maintained are preferable. By performing the trialkyl phosphate treatment step in the presence of a protease inhibitor, a decrease in protein activity can be prevented, and 90% or more of the protein activity before the step can be maintained.

[0031]

According to the invention, and advantages of the present invention, without substantial loss of activity of the protein, effectively protein-containing pharmaceutical composition virus is inactivated processed or protein-containing trial
A drug composition is provided.

The trialkyl phosphate treatment has a weak inactivating effect on viruses having no envelope.
However, according to the present invention, since there is a step of subjecting the dried protein-containing composition to a heat treatment, such a virus can also be significantly inactivated by passing through this step.

In the case of treatment with a trialkyl phosphate at 25 ° C. to 30 ° C., the protein may be decomposed by a protease mixed in the solution by prolonged storage at that temperature. The reduction in protein activity can be suppressed by treating with.

Accordingly, the present invention provides a protein-containing composition which has been subjected to virus inactivation treatment and which is safer as a medicine or a reagent .

[0035]

【Example】

Example 1 Blood Coagulation Factor VIII-Containing Composition Cryoprecipitate is dissolved in 20 mM Tris-HCl buffer, and aluminum hydroxide gel is added at 1% v / v to obtain a deprothrombin solution. TNBP (tri- (n-butyl) phosphate) was added to this solution at 0.3% w / v, Tw
een80 is added to a concentration of 1% w / v, and the virus is inactivated at 30 ° C. for 6 hours. And 2M glycine
, And centrifuged to remove fibrinogen by precipitation.
And recover Factor VIII as a precipitate. The collected factor VIII is dissolved in a 20 mM Tris-HCl buffer, and glycine fractionation and sodium chloride fractionation are performed again to remove the remaining TNBP and Tween 80. The precipitate of Factor VIII thus obtained is dissolved to a predetermined concentration, divided into vials and freeze-dried. The freeze-dried factor VIII preparation is subjected to a dry heat treatment at 60 ° C. for 72 hours or more to produce a preparation safe against virus.

Example 2 Blood Coagulation Factor IX-Containing Composition DEAE-Sephadex is charged into plasma, and Factor IX is adsorbed on a gel. After washing well with a buffer containing 0.15 M sodium chloride, factor IX is eluted with a buffer containing 0.5 M sodium chloride. 0.3% w / v of TNBP and 1% w / v of Tween 80 were added to this eluate.
And inactivated at 30 ° C. for 6 hours or more. The virus-inactivated factor IX solution was again charged with DEAE-Sephadex, adsorbed factor IX, and washed to obtain TNBP, Tween 80.
Can be removed in the washing liquid. Alternatively, instead of DEAE-Sephadex, factor IX can be bound to a gel to which a monoclonal antibody against factor IX has been bound, and the same treatment can be performed. After the factor IX is eluted from the gel, it is adjusted to a predetermined ionic strength and titer, aliquoted, and freeze-dried to obtain a factor IX preparation. Then, at 60 ° C., 72
Dry heating for more than an hour is performed to produce a safer formulation.

Example 3 Thrombin-Containing Composition 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 with SP-Sephadex, washed well with a 0.15 M sodium chloride solution, and then thrombin is eluted with a buffer containing 0.5 M sodium chloride. TNBP was added to this eluate in a volume of 0.1%.
3% w / v, 1% w / v Tween 80 was added, and 30 ° C, 6
Perform virus inactivation for more than an hour. After the virus inactivation treatment, thrombin is again adsorbed to SP-Sephadex or heparin-gel, washed well, and TNBP, Tw
After removing een 80, thrombin is eluted from the gel.
The thrombin thus obtained is adjusted in ionic strength and titer, divided into small portions, freeze-dried, and then subjected to a dry heat treatment at 60 ° C. for 72 hours or more.

Example 4 Fibrinogen-containing composition Corn Fr. I paste or cryoprecipitate is dissolved in physiological saline, and TNBP is dissolved in 0.3% w / v, Tw
een 80 is added to 1% w / v, and a virus inactivation treatment is performed at 30 ° C. for 6 hours or more. Then, fibrinogen is precipitated by glycine-sodium chloride fractionation or ethanol fractionation. The fibrinogen precipitate obtained is dissolved in a physiological saline solution and then subjected to glycine-sodium chloride fractionation or ethanol fractionation again to precipitate and collect fibrinogen. This fractionation operation is performed 2 to 5 times, and TNBP and Tween 80 are removed from the supernatant. The fibrinogen precipitate thus obtained was dissolved to a predetermined concentration,
After freeze-drying for a small amount, a drying heat treatment at 60 ° C. for 72 hours or more is performed.

Example 5 Composition Containing Blood Coagulation Factor VIII Cryoprecipitate obtained by freezing and thawing normal human / plasma was used as a starting material, and 20 mM Tris-10 mM
Extraction and dissolution were performed five-fold with a citrate buffer (pH 7.0), and aluminum hydroxide gel was added to cryoprecipitate at a weight of 1
/ 10 volume was added and stirred for 30 minutes. Thereafter, bentonite was added at a rate of 6 g / L and stirred for 1 hour.
Centrifuge at 0 rpm for 30 minutes to obtain supernatant, followed by detergent treatment for virus inactivation (0.3% tri-n-
After adding butyl phosphate to a concentration of 1% Tween 80, the mixture was stirred at 30 ° C. for 6 hours) and subjected to glycine fractionation. Glycine fractionation conditions were as follows: glycine was added to this solution at a rate of 150 g / L, stirred at 30 ° C. for 1 hour, and centrifuged (4000 rpm, 30 minutes, 30 ° C.) to obtain a supernatant. Sodium chloride (87g /
L) After stirring for 1 hour, the Factor VIII fraction was salted out to obtain a centrifugal precipitate (4000 rpm, 30 minutes, 30 ° C.).

Thereafter, the salted-out precipitate was dissolved in 20 mM Tris buffer (pH 7.0), and the absorbance at 280 nm was adjusted to 25 to 30.
After adjusting to, the sample was used as an injection sample of a gel filtration column. Sephacryl S-400 HR manufactured by Pharmacia was used as a packing material for the column, and a factor VIII active fraction discharged from the column was collected from the sample at an injection amount of 5 to 7% of the column bed capacity. The conditions are as follows: injection sample: salting-out precipitation solution column: Sephacryl S-400HR solvent: 20 mM Tris (HCL), 10 mM CaCl ₂ , 1 M NaCl buf
fer, pH 7.0

As a result, blood coagulation factor VIII was observed in the column void fraction, and contaminating proteins were highly separated. Thereafter, the mixture is adjusted to a predetermined ionic strength and titer, aliquoted, and freeze-dried to obtain a factor VIII preparation. Then, further drying and heating at 60 ° C. for 72 hours or more are performed,
Make safer formulations.

[0042]

[Experimental example]

Experimental Example 1 Virus Inactivating Effect The virus inactivating effect in the blood coagulation factor VIII-containing composition, the blood coagulation factor IX-containing composition, the thrombin-containing composition and the fibrinogen-containing composition was examined. (Experimental method) Cryoprecipitate lysate for factor VIII, DEAE-Sephadex eluate for factor IX,
For thrombin, SP-Sephadex eluate, for fibrinogen, Fr. 0.3% w / v TNBP and 1% w
/ V, as enveloped viruses, VSV, sindbis virus,
Echo virus (Ec
ho virus) was added as a monitor virus at 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 1. The results are as shown in Tables 2 to 4. In all the preparations, VSV and Sindbis virus were inactivated to below the detection limit by treatment at 30 ° C. for 1 hour. On the other hand, echo virus, which is a virus without a membrane, was hardly inactivated even after 30 hours at 30 ° C.

[0043]

[Table 1]

[0044]

[Table 2]

[0045]

[Table 3]

[0046]

[Table 4]

Experimental Example 2 Stability of Protein For Factor VIII, TNBP was added to crypaste extract at 0.3% w / v and Tween 80 at 1% w / v, and the mixture was heated at 30 ° C. Factor VIII activity was measured by sampling, and the remaining activity after 24 hours was 80% (FIG. 1).
Therefore, it was determined that there was almost no loss of factor VIII activity at 30 ° C. for 6 hours, and no screening for a stabilizer was performed. In addition, stability at the time of drying and heating is 60 ° C.
After 72 hours, the activity was maintained at 95% or more, and no screening for a stabilizer was performed.

For Factor IX, 0.3% w / v of TNBP and 1% w / v of Tween 80 were added to the DEAE eluate at 30 ° C.
The factor IX activity was measured by heating over time and sampling over time. The residual activity after 24 hours was 76%, and a slight decrease in activity was observed. No stabilizing agent was added because it was not a decrease. (See FIG. 2). In addition, stability during drying and heating is 60
The activity of 95% or more was maintained after 72 hours at 70 ° C., and the screening of the stabilizer was not performed.

For thrombin, 0.3% w / v of TNBP and 1% w / v of Tween 80 were added to the SP-Sephadex eluate.
After addition, the mixture was heated at 30 ° C. and sampled over time to measure thrombin activity. The activity loss after 30 hours was large, and it was judged that it was difficult to maintain 90% activity even after 30 hours at 30 ° C. Stabilizer screening was performed. As a result, addition of EACA (ε-aminocaproic acid) and arginine can prevent a decrease in thrombin activity,
In particular, EACA was able to maintain 90% or more of activity even after 30 hours at 30 ° C. with addition of 2% or more (see FIGS. 3 and 4).

Fibrinogen is obtained from Fr. 0.3% w / v of TNBP and 1% w / v of Tween 80 in the solution of I paste
The mixture was added, heated at 30 ° C., sampled with time, and the fibrinogen coagulation activity was measured. As a result, 30 ℃,
Twenty-four hours later, it was found that no coagulation activity was observed, and a stabilizing agent was screened. As a result, it was found that aprotinin and EACA had a stabilizing effect. 30 ° C., 2 by adding EACA of 5% or more.
After 4 hours, 90% of the coagulation activity could be maintained (see FIG. 5).

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of the addition of a surfactant on the residual activity of blood coagulation factor VIII at the time of treatment with a trialkyl phosphate over time.

FIG. 2 is a graph showing the effect of the addition of a surfactant on the residual activity of blood coagulation factor IX during trialkyl phosphate treatment over time.

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

FIG. 4 is a graph showing the effect of arginine addition on the residual activity of thrombin during trialkyl phosphate treatment over time.

FIG. 5 shows the effect of the addition of EACA on the residual activity of fibrinogen during trialkyl phosphate treatment over time.

──────────────────────────────────────────────────続 き Continued on the front page (72) Kazuo Takechi, Inventor 2-1,180-1, Hoshikata-Otani, Hirakata City, Osaka Prefecture 1 Inside the Midori Cross Research Institute Co., Ltd. (72) Takao Omura 2-1-1180, Invitation Otani, Hirakata City, Osaka Prefecture (1) Inside the Midori Cross Research Institute, Inc. (72) Inventor Yutaka Hirao 2-1-1, Shodai Otani, Hirakata City, Osaka Prefecture (1) Inside the Midori Cross Research Institute, Inc. (72) Yahiro Uemura, 2-Chome Otani, Hirakata City, Osaka Prefecture No. 1180, 1 inside the Midori Cross Research Institute, Inc. (72) Inventor Kazumasa Yokoyama 2-1-1, Shodai Otani, Hirakata City, Osaka Prefecture, 1st Inside the Midori Cross Research Institute, Inc. (56) References JP-A-60-511116 (JP) , A) JP-A-61-155332 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/16 A61K 38 / 43 C07K 14/745 C07K 14/78 CA (STN) MEDLINE (STN) REGISTRY (STN) WPIDS (STN)

Claims (4)

(57) [Claims] 1. A virus-free method comprising the following steps:
An activated protein-containing pharmaceutical composition. (A) a protein-containing liquid composition is the possibility of viral contamination trialkyl phosphate Fe - is contacted with bets and a surfactant, the
Thereafter, a trialkyl phosphate is obtained from the protein-containing composition.
Removing the door and a surfactant, (b) to a protein-containing composition was freeze-dried, heat-treating the dried form of the protein-containing composition. 2. A by a method comprising the following steps, virus inactivation
A protein-containing reagent composition that has been subjected to a chemical conversion treatment . (A) contacting a liquid protein-containing composition with potential virus contamination with a trialkyl phosphate and a surfactant ,
From the protein-containing composition, trialkyl phosphate and
Removing the surfactant and, (b) to a protein-containing composition was freeze-dried, heat-treating the dried form of the protein-containing composition. 3. A process of (a), trialkyl phosphine Fe a protein-containing liquid composition in the presence of protease inhibitors - wherein Rukoto is contacted with bets and surfactant
The composition according to claim 1 or 2, wherein 4. A protein factor VIII, blood coagulation factor IX, thrombin, fibrinogen, and compositions according to claim 1-3 is at least one selected from the group consisting of fibronectin.