JP2926728B2 - Method for producing protein-containing composition - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a highly active protein-containing composition from which a virus has been substantially removed from a protein-containing composition having the possibility of virus contamination. .

(Prior art)

A protein-containing composition derived from human blood may be contaminated with a virus, for example, a hepatitis virus or an AIDS virus.

A method of heating a liquid protein-containing composition to prevent such virus transmission is known (JP-A-55-145615).
JP-A-56-139422, JP-A-56-106594).

Also, a method of removing a virus by contacting a protein-containing composition with a trialkyl phosphate is known (Japanese Patent Application Laid-Open No. Sho 51511/1985).

However, heat treatment may leave heat-resistant virus, and non-enveloped virus may remain in trialkyl phosphate treatment.

[Problems to be solved by the invention]

An object of the present invention is to efficiently inactivate contaminating viruses such as heat-resistant viruses and non-enveloped viruses, as well as ordinary viruses, without losing protein activity. It is intended to provide.

[Means for solving the problem]

The object or object of the present invention is to provide a protein-containing liquid composition having a possibility of virus contamination by contacting the liquid composition with a trialkyl phosphate at a temperature of 50 ° C or higher to inactivate the virus, followed by affinity chromatography. Thus, there is provided a protein-containing composition in which a virus is inactivated efficiently without losing the activity of the protein.

The protein to which the method of the present invention can be applied is not particularly limited, and examples thereof include plasma-derived proteins, other tissue-derived proteins, and proteins obtained by genetic recombination or tissue culture. Examples of proteins include plasminogen, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor XIII, antithrombin III, haptoglobin,
Thrombin, immunoglobulin and the like. The protein-containing composition is not particularly limited as long as it contains the protein as described above.

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

In addition, 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. Protein separation,
It may be applied at any stage of the 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, tri- (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 0.
An amount ranging from 01 to 10 (w / v)%, preferably from about 0.1 to 3 (w / v).
v) Used in amounts in the% range.

Trialkyl phosphates can be used with or without surfactant. Preferably, a trialkyl phosphate is used in combination with a surfactant. The surfactant can be added at any stage before, at the same time as, or after the trialkyl phosphate contacts the protein-containing liquid composition. The action of the surfactant is to enhance the contact between the virus and the trialkyl phosphate in the protein-containing composition.

Surfactants include polyoxyethylene derivatives of fatty acids, esters of sorbitol anhydride moieties, such as Tween 80, Tween 20, and Polysorbate 80,
And nonionic oil-soluble detergents such as Triton X100 (oxyethylated alkylphenol). In addition, Zwit, a synthetic tubuiter ion detergent known as sodium deoxycholate and sulfobetaine
tergents, such as N-dodecyl-N, N-dimethyl-2
-Ammonio-1-ethanesulfonate, and homologs thereof, or non-ionic detergents such as octyl-β, D
-Glucopyranoside and the like.

If a surfactant is used, the amount is not critical, for example, from about 0.001% to about 10%, preferably about 0.01%.
Can be used in the range of ~ 1.5%.

Trialkyl phosphate treatment is particularly useful for inactivating envelope coat viruses such as hepatitis B virus, nonAnonB hepatitis virus. Also 50 more
By heat-treating at a temperature of not less than ℃ for a sufficient time, heat-resistant viruses can be inactivated at the same time.

Therefore, the treatment of the protein-containing composition with the trialkyl phosphate of the present invention is performed at a temperature of 50 ° C or higher, preferably 60 to 75 ° C.
C. for 30 minutes or more, preferably for 3 to 30 hours.
Optimally, the treatment is performed at about 60 ° C. for about 10 to 20 hours.

The heat treatment may be performed in the presence of a stabilizer to protect the protein from heat. As stabilizers, sugar,
Sugar alcohols, amino acids and the like can be mentioned.

After the treatment, the protein-containing composition is treated by affinity chromatography. By treating with affinity chromatography, it is possible to remove the trialkyl phosphate, the surfactant, and the stabilizer during the heat treatment. Furthermore, even if a non-envelope-coated virus and a virus resistant to special heat remains, it can be removed by the present affinity chromatography treatment.

Affinity chromatography can be appropriately selected depending on the type of protein. For example, in the case of plasminogen, it can be treated by chromatography on immobilized lysine.

The immobilized lysine is obtained by immobilizing lysine on an insoluble carrier. Examples of the insoluble carrier include agarose, cross-linked dextran, cellulose, polyacrylamide, and hydrophilic vinyl polymer.

The immobilization method is not particularly limited, and may be performed by a method known per se.

As a treatment method, any of a batch method and a column method may be used. As the equilibrium solvent and the adsorption solvent, a buffer having a pH of 6 to 8 (preferably, a pH of 7 to 7.5) (for example, a phosphate buffer,
Glycine buffer). It is also possible to add sodium chloride or the like. Under the above conditions, the protein is adsorbed on the insoluble carrier. As an elution solvent, for example, EAC of 50 to 500 mM (preferably 100 to 300 mM) is used.
A solvent (pH 6 to 8) containing A (epsilonaminocaproic acid) or lysine is used. In the case of the column method, it is common knowledge that, after plasminogen adsorption, unadsorbed substances are washed away by washing with the buffer solution about 2.5 times the gel amount,
Washing of a trialkyl phosphate or the like in the present invention,
For removal, it is desirable to carry out washing with the above buffer at least 4 times (preferably 4 to 10 times).

Blood coagulation factor IX is an adsorbent composed of an anti-factor IX antibody,
Specifically, it can be treated by affinity chromatography using an immobilized anti-factor IX antibody. As the carrier, either a polyclonal antibody column or a monoclonal antibody column may be used.

Regarding the polyclonal antibody, an anti-factor IX antibody is obtained by immunizing an animal with highly purified factor IX, and recovering and purifying the obtained serum.

The production of the antiserum may be carried out by a known method. For example, a mixed emulsion of highly purified factor IX and Freund's complete adjuvant is prepared, injected intradermally into an animal two to three times, and determined several days after the final immunization. And then allowed to solidify at room temperature, followed by 4
The antiserum is obtained by leaving the mixture at ℃ overnight and centrifuging at 3,000 rpm for 20 minutes.

There is no particular need to select an animal species for the immunization, and examples thereof include rats, mice, rabbits, goats, and horses. Purification of the antiserum is described, for example, in J.
Am. Chem. Soc., 62 , 3386 (1940), Fed. Proc., 17 , 1161 (19
This is performed by the method described in 58).

For monoclonal antibodies, anti-factor IX antibodies are obtained by cell fusion. The cell fusion method is performed by a method known per se, and one example is to increase the proliferation by reacting proliferating cells with lymphocytes producing the target antibody in the presence of polyethylene glycol. This is to produce cells having both antibody-producing ability at the same time and to produce antibodies in these cells. The antibodies are single antibodies that react only with one antigenic determinant.

More specifically, mouse myeloma cells are fused as proliferative cells, and mouse spleen cells immunized with factor IX are used as antibody-producing lymph using (B cells) to produce a target antibody. Cells are screened to obtain a Factor IX monoclonal antibody from the cells.

As a method for immobilizing the anti-factor IX antibody thus obtained without losing its activity, the following method using an insoluble matrix can be mentioned. As such a method, for example, a method using a copolymer of amino acids (J. Biol. Chem., 236, 1970 (196)
1)), a method using cellulose (Nature, 189, 576 (1
961)), using agarose or Sephadex (Nature, 215, 1491 (1967), Nature, 245, 3059 (1
970)), using polyacrylamide (Bioche
m., 8, 4074 (1966)). By these methods, the anti-factor IX antibody can be immobilized efficiently. Also,
By using the adsorbent thus obtained, Factor IX with high yield and high purity can be obtained.

In the context of the present invention, treatment with immobilized anti-coagulation factor IX antibody is performed, for example, as follows. First,
The appropriately purified factor IX-containing aqueous solution is applied to an immobilized anti-factor IX antibody equilibrated with a suitable solvent [eg, 0.01 to 0.1 M Tris buffer (pH 6 to 8)]. After washing with the same solvent, the factor IX fraction eluted with the eluate is collected.

Blood coagulation factor VIII can also be treated, for example, by affinity chromatography using an immobilized anti-factor VIII antibody.

Antithrombin III can be treated, for example, by affinity chromatography using immobilized heparin (JP-A-48-35017).

Haptoglobin can be treated, for example, by affinity chromatography using immobilized globin (JP-A-55-111496).

In addition, affinity chromatography can be appropriately selected depending on the protein.

[Action and Effect of the Invention]

According to the production method of the present invention, a virus can be efficiently inactivated under a very small loss of protein activity, so that a protein-containing composition having very low virus infectivity and high activity is provided. can do.

More specifically, the present invention has the following effects.

Heat-resistant viruses can be inactivated by trialkylphosphate treatment, but trialkylphosphate treatment has a weaker inactivation effect on non-enveloped coat viruses, and inactivates viruses at 50 ° C or higher. This has the effect of supplementing it. And
If any virus still remains, it can be removed by a subsequent affinity chromatography treatment.

Further, in the case of the trialkyl phosphate treatment at 25 ° C. to 30 ° C., if the temperature is long, decomposition of the protein by a protease such as plasmin mixed in the solution may be promoted. However, when the treatment is performed at 50 ° C. or higher, degradation of the protein by the mixed protease is not considered.

Therefore, the production method of the present invention provides a very preferable method as an industrial production method of a protein-containing composition which is safer as a pharmaceutical.

〔Example〕

Example 1 Fraction II + III obtained by cold ethanol fractionation of corn
The paste extraction residue (100 g) was mixed with Tris-HCl buffer (pH 8.10) containing 10 units / ml of aprotinin and 0.1 M sodium chloride.
After suspending in 3) and stirring for a while, 5% ammonium sulfate was added and stirred, and the supernatant was separated by centrifugation. Furthermore,
30% ammonium sulfate was added to the supernatant, stirred, and 1 g of precipitate was separated by centrifugation. This precipitate is used for aprotinin
The suspension was suspended in a 0.9% glycine solution (pH 7.2) containing 40 KIE / ml and 9% sodium chloride.

A solution was prepared by adding 1.75 g of sucrose, 0.3% TNBP (tri- (n-butyl) phosphate) and 1% Tween 80 per 1 ml of the plasminogen-containing solution,
Heated for hours.

After heating, the plasminogen solution was added to Deutsch, DG et al. (Scin
ce, 170, 1095, (1970)] and injected into a lysine-sepharose column method (100 ml) equilibrated with a 0.9% glycine solution (pH 7.2) containing 0.9% sodium chloride to allow plasminogen to adsorb. After washing with 0.9% glycine solution (pH 7.2) containing 0.9% sodium chloride, and further washing with 500 ml of 0.9% glycine solution (pH 7.2) containing 1M sodium chloride, 0.9% glycine solution containing 0.25M lysine ( pH 7.
The adsorbed plasminogen was eluted using 2).

Example 1 The stability of plasminogen when the plasminogen-containing solution obtained in Example 1 was liquid-heated at 60 ° C. for 10 hours in the presence of sucrose or in the presence of sucrose and TNBP / Tween 80 was examined.

(Experimental method) After adding aprotinin 40KIE / ml to the plasminogen-containing solution, 1.5 g of sucrose, 1.75 or 1.75 ml / ml
2.0 g was added, the mixture was heated at 60 ° C. for 10 hours, and the stability of PLG (plasminogen) was examined. In the system containing 1.5 g and 1.75 g of sucrose, a solution containing 0.3% TNBP / 1% Tween 80 was further prepared, and the effect of the surfactant on PLG stability was examined. The PLG activity was measured using an MCA (methylcoumarylamide) synthetic substrate, and the activity was converted based on a calibration curve of a plasminogen standard.

(Results) The stability of plasminogen is shown in Table 1 as the activity recovery rate of plasminogen.

As shown in Table 1, 1 ml of the solution was heated at 60 ° C. for 10 hours with a solution containing 1.5 g or more of sucrose, and 70% or more of the activity was recovered. Further, even when a surfactant was further added to the sucrose-added solution, no decrease in the activity recovery rate was observed. This result suggested that PLG was not inactivated by the surfactant even after heating at 60 ° C for 10 hours.

Example 2-Virus inactivating effect Using the plasminogen-containing solution obtained in Example 1, the virus inactivating effect of the surfactant was examined at 30 ° C and 60 ° C.

(Experimental method) Sucrose 1.5 g per 1 ml of plasminogen-containing solution
, And 0.3% TNBP,
A solution to which% Tween 80 was added was prepared, and a certain amount of virus was added, followed by heating at 60 ° C. On the other hand, for examination of virus inactivation by heating at 30 ° C., a solution in which TNBP / Tween 80 was added to the plasminogen-containing corn fraction, and a solution in which TNBP / Tween 80 was added after diluting the above sucrose solution by 4 times, were used. After adjusting and adding a certain amount of virus, the mixture was heated at 30 ° C. Thereafter, sampling was performed at 5, 10, 30, 60, and 120 minutes, and the virus infection titer was measured by the method described below.

[Method for measuring viral infectious titer]

(Virus and cells) The test virus, its host cell, and the virus infectivity were measured using the system shown in Table 2.

Each cell was cultured in Eagle's MEM medium (Nissui Pharmaceutical) supplemented with 10% inactivated fetal bovine serum (Flow), and used a maintenance medium in which the amount of serum was reduced to 2% during virus infection. Was.

(Results) TNBP / Tween 80 was added to the plasminogen-containing solution, and 30
When heated at ℃, the virus infectivity fell below the detection limit in 30 minutes. (See Table 3).

On the other hand, in order to examine the effect of sucrose on the virus inactivating effect of TNBP / surfactant, the sucrose-added plasminogen-containing corn fraction solution was diluted 4-fold (since the stock solution is too viscous at 30 ° C) Later, TNBP
The virus inactivating effect was examined for the solution to which / Tween 80 was added. As a result, the virus infection titer fell below the detection limit within 5 minutes after heating at 30 ° C. This result suggests that the dilution of the plasminogen-containing solution enhances the virus inactivating effect of the TNBPK detergent, and at the same time, indicates that at least the coexistence of sucrose does not affect the virus inactivating effect of TNBP / surfactant. it was thought.

On the other hand, when a solution obtained by adding sucrose to the plasminogen-containing solution was heated at 60 ° C., a virus infection titer was observed even after 30 minutes, but the solution further contained TNBP / Tween.
When 80 was added and the mixture was heated at 60 ° C, the virus infectious titer fell below the detection limit within 5 minutes, and a strong virus inactivating effect of the surfactant at 60 ° C was confirmed.

Experimental Example 3-Removal effect of TNBP / Tween 80 500 mg of purified plasminogen (250 CU / ml) containing 1% Tween 80 and 0.3 TNBP was added to Deutsch, DG et al. [Science, 17
0, 1095, (1970)], injecting 1 ml of a lysine-sepharose column equilibrated with a 0.9% glycine solution (pH 7.2) containing 0.9% sodium chloride, adsorb plasminogen, and then 0.9% chloride After washing with 0.9% glycine solution containing sodium (pH 7.2), 0.9% containing 0.25M lysine
The adsorbed plasminogen was eluted using a glycine solution (pH 7.2). Then, the amount of Tween 80 and the amount of TNBP were measured for the starting material, the washing solution, and the eluate, respectively.

Tween 80 assay is based on complex formation of barium and iodine (So
og et al., Vox Sang 37, 345-349 (1979)).

 Quantification of TNBP was performed by gas chromatography.

(Experimental results) Tween 80 and TNBP added for virus inactivation
Was not detected in the eluted fraction of lysine-sepharose (see Tables 4 and 5).

Continuing on the front page (72) Inventor Kenji Tanaka 2-1180-1 Invitation Otani, Hirakata City, Osaka Prefecture, Japan Inside the Midori Cross Research Laboratory (72) Inventor, Yahiro Uemura 2-1180-1 Invitation Otani, Hirakata City, Osaka Prefecture (72) Inventor Hideyuki Ishikawa 2-1180-1 Invited Otani, Hirakata City, Osaka Prefecture, Japan Incorporated (72) Inventor, Naoko Tojo 2-1180-1 Invited Otani, Hirakata City, Osaka Prefecture (72) Inventor Motonori Hashimoto 2-1180-1 Invitation Otani, Hirakata City, Osaka Prefecture (72) Inventor Yuriko Shinobu 2 Invitation Otani, Hirakata City, Osaka Prefecture ―1180-1 Inside Midori Cross Research Institute, Inc. (72) Inventor Kazuo Takechi Invitation to Otani, Hirakata City, Osaka Prefecture 2-1180-1 Inside Midori Cross Research Institute, Ltd. (72) Inventor Kiyoshi Tsutsui, invited from Hirakata City, Osaka Prefecture 2-1180-1 Otani Midori Cross Research Center In-house (72) Inventor Ryoichiro Murashima 2-1180-1 Hyokata-Otani, Hirakata-shi, Osaka Inside Midori-Cross Central Research Institute, Inc. (56) References JP-A-60-5116 (JP, A) JP-A-58-2223 ( JP, A) JP-A-62-240623 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 38/00-38/58

Claims (2)

(57) [Claims] 1. A method for producing a protein-containing composition, comprising heat-treating a protein-containing liquid composition having the possibility of virus contamination in the presence of trialkyl phosphate and sucrose to inactivate the virus. 2. A protein-containing composition obtained by removing a trialkyl phosphate and a stabilizer from the protein-containing composition obtained by the production method according to claim 1.