WO1998030230A1

WO1998030230A1 - Protein-containing compositions and process for producing the same

Info

Publication number: WO1998030230A1
Application number: PCT/JP1998/000042
Authority: WO
Inventors: Koji Furushima; Katsuhiro Uriyu; Tuyoshi Takahashi; Motonori Hashimoto; Shoju Kameyama
Original assignee: Yoshitomi Pharmaceutical Industries, Ltd.
Priority date: 1997-01-09
Filing date: 1998-01-08
Publication date: 1998-07-16

Abstract

Compositions containing the desired proteins but being substantially free from any infectious viruses or modifications of the desired proteins. These compositions are produced by subjecting protein-containing compositions with a fear of the contamination with viruses to at least three procedures selected from among heating, UV-irradiation, treatment with a surfactant and treatment with a membrane for eliminating viruses. This production process makes it possible to efficaciously inactivate or eliminate viruses without damaging the activities of the proteins to thereby give compositions containing the desired proteins but being substantially free from any infectious viruses or modifications of the desired proteins. Thus, protein-containing preparations with a high safety and excellent qualities can be obtained.

Description

Specification

Protein-containing composition and method for producing the same

Technical field

The present invention relates to a composition containing an infectious virus and a desired protein substantially free of a denatured form of the desired protein. Furthermore, the present invention relates to a method for producing a composition containing a desired protein substantially free of an infectious virus and a denatured form of the desired protein from a protein-containing composition having a possibility of virus contamination. Background art

Pharmaceutical preparations containing proteins, especially plasma fraction preparations using human plasma as a raw material, may contain pathogens that can infect humans, and the problem of viral infection is particularly important. Up to now, after blood transfusion, infections with viruses such as human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) have occurred. Such accidents are not a thing of the past, and although the incidence of accidents has decreased dramatically with exponential numbers due to the introduction of various technologies, the occurrence cannot be ruled out today. Methods for inactivating or removing potentially contaminating viruses are known to prevent these virus transmissions. For example, a method of heating a protein-containing composition in a liquid state (Japanese Patent Application Laid-Open Nos. 554-145056, 56-1393942, and 56-1-1 No. 0 654 94), a method of heating in a dry state (Japanese Unexamined Patent Publication No. 58-500 48, Japanese Unexamined Patent Publication No. 58-213 3721, etc.) A method of contacting with an alkyl phosphate and a surfactant (Japanese Patent Application Laid-Open No. 60-51116), a method of irradiating with ultraviolet rays (Japanese Patent Application Laid-Open No. Hei 7-196331), virus A method using a removal film (Japanese Patent Application No. 7-2577771) is known.

However, heat treatment leaves heat-resistant viruses, surfactant treatment leaves non-enveloped viruses, and UV irradiation alone can inactivate proteins. Single treatment in the removal method, well-known union It has been difficult to completely inactivate or remove contaminating viruses without almost losing protein activity by the heat treatment. For example, it was difficult to inactivate or remove viruses, such as parvovirus, that are resistant to heat, are not inactivated by surfactants, and cannot be removed by 35-nm membrane treatment because of their small size.

As described above, many techniques for inactivating or removing contaminating viruses from protein-containing compositions are known, but even today, viruses that are unknown and viruses that are resistant to various treatments are used. There is a need to provide protein-containing preparations that may be present and that have the virus more completely inactivated or removed.

The present invention has been made to solve these problems, and in order to provide a protein preparation that is safe as a pharmaceutical, contaminant viruses were inactivated or removed efficiently with little loss of protein activity. It is an object of the present invention to provide a composition containing a desired protein and a method for producing the same. Disclosure of the invention

The present inventors have conducted various studies to solve the above-mentioned problems, and found that a protein-containing composition that may be contaminated with virus was subjected to heat treatment, ultraviolet irradiation treatment, treatment with a surfactant, and the like. It has been found that a composition containing a desired protein substantially free of infectious virus and a denatured form of the desired protein can be obtained by performing at least three types of treatment among the virus removal membrane treatments. The present invention has been completed.

That is, the present invention is as follows.

(1) Infectivity characterized by subjecting a protein-containing composition having the possibility of virus contamination to at least three treatments of heat treatment, ultraviolet irradiation treatment, treatment with a surfactant, and virus removal membrane treatment. A method for producing a composition containing a desired protein substantially free of a virus and a denatured form of the desired protein.

(2) The method according to (1), wherein at least a virus removal membrane treatment is performed. (3) The production method according to (1), wherein the heat treatment comprises treating the protein-containing composition in a liquid state at 30 to 65 ° C for 10 minutes to 20 hours.

(4) The production method according to (1), wherein the heat treatment comprises treating the protein-containing composition in a dry state at 60 to 100 ° C. for 20 to 100 hours.

(5) ultraviolet irradiation treatment, in ultraviolet light in the wavelength 1 8 0 to 3 5 0 nm, the irradiation energy-saving one weight 1~5 0 OmJ 0 u 1 e / cm 2 to protein-containing composition in liquid state (1) The production method according to (1), wherein the irradiation is performed.

(6) The method according to (1), wherein the treatment with a surfactant is a treatment with a polysorbate-based surfactant.

(7) The production method according to (6), wherein a trialkyl phosphate is used in combination.

(8) The method according to (1) or (2), wherein the virus removal membrane treatment is a filtration treatment using a porous hollow fiber membrane.

(9) The production method according to (1), wherein the desired protein is fibrinogen, which is treated with a surfactant, irradiated with ultraviolet light, and heated in a dry state.

(10) The production method according to (1), wherein the desired protein is heparin cofactor-II.

(11) At least three types of heat treatment, ultraviolet irradiation treatment, treatment with surfactant, and virus removal membrane treatment are performed, and are substantially free of infectious virus and denatured form of desired protein. A composition containing the desired protein, wherein 90% or more of the effective desired protein is contained.

(1) The composition according to (11), which is substantially free of viral nucleic acid.

(13) The composition according to (11) or (12), wherein the three treatments are a treatment with a surfactant, an ultraviolet irradiation treatment, and a heat treatment in a dry state, and the desired protein is fibrinogen. (14) The composition according to (11) or (12), wherein the desired protein is heparin cofactor II.

The protein to which the production method of the present invention is applied is not particularly limited, and includes proteins derived from plasma, proteins derived from urine, proteins derived from other tissues, and proteins obtained by genetic recombination or tissue culture. No. Specific examples of proteins include, for example, blood coagulation factors (fipurinogen, prothrombin, thrombin, factor VI I, factor VI II, factor IX, factor X, factor XII I), heparin. Cofactor I II

, Albumin, hemoglobin, interferon, fibronectin, plasminogen, plasminogen activator, antithrombin III, C, inhibitor, α, protease inhibitor, immunoglobulin, haptoglobin, colonizing stimulator And the like. In particular, the production method of the present invention is suitable for fibrinogen and heparin cofactor 11.

Fibrinogen is a protein that exists in plasma with a molecular weight of about 340000, and is converted into fibrin by the action of thrombin in vivo, and the presence of factor XI11 and Ca ⁺⁺ activated by thrombin It forms a strong fibrin clot underneath, which causes the blood to clot. The composition containing fibrinogen is useful as a component of fibrin glue, a therapeutic agent for hypofibrinogenemia. The production method of the present invention is suitably applied to the fibrinogen-containing composition, and specifically, a combination of a treatment with a surfactant, an ultraviolet irradiation treatment, and a heat treatment in a dry state is particularly preferred.

Heparin 'cofactor で is a single-chain glycoprotein with a molecular weight of 72,000 and is present in normal plasma at about 1 O mg / dL. Heparin cofactor II inhibits only thrombin, a protease that is mainly produced in the liver and involved in blood coagulation. The heparin cofactor II-containing composition is suitably applied to the production method of the present invention. The protein-containing composition having the possibility of virus contamination applied to the production method of the present invention may be liquid or dry. The liquid protein-containing composition is not particularly limited. For example, a solution comprising a fraction obtained by treating plasma or tissue extract by various fractionation methods, a protein obtained by culturing a genetically modified host or tissue Containing solution, Examples thereof include a commercially available liquid protein preparation or a commercially available lyophilized protein preparation in the form of a solution. Further, the dry protein-containing composition is not particularly limited, and examples thereof include a liquid protein-containing composition obtained by freeze-drying in the presence of a stabilizer, and the like. The degree of purification of the product is not particularly limited, and the virus inactivation treatment or removal treatment of the present invention can be applied to both protein separation and purification steps.

In the production method of the present invention, almost all viruses that may be contaminated are inactivated or removed. Specific examples of the virus include vaccinia virus, mumbus virus, herpes simplex virus, echovirus, parvovirus, HIV, HAV, HBV, HCV and the like. In particular, viruses that may be contaminated include HIV, HBV, HCV and parvovirus.

The heat treatment may be either a liquid heat treatment for heating the liquid composition or a dry heat treatment for heating the freeze-dried composition, and is effective for viruses that are sensitive to heat, for example, HIV. The heat treatment is usually carried out at 30 to 100 ° C., usually for 10 minutes to 120 hours, preferably for 10 minutes to 100 hours. Preferred combinations of heating temperature and heating time are 30 minutes to 20 hours at 30 to 65 ° C for liquid heat treatment, and 20 hours at 60 to 100 ° C for dry heat treatment. The drying heat treatment can be performed under an inert gas atmosphere or under a reduced pressure stopper to further enhance the stability during heating. Examples of the inert gas include nitrogen, argon, and helium gas. In addition, a dry state is a substantially anhydrous state.

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%.

In the case of heat treatment, it is preferable to add a stabilizer.In the case of dry heat treatment, 100 mg to 3 g of disaccharides, sugar alcohols, amino acids, amino acids, or organic acid salts, etc., based on protein 1 , Preferably in a proportion of 1-2 g. In addition, stable It is preferable to add the agent before freeze-drying in order to reduce the effect of freeze-drying. In the case of liquid heat treatment, sugar (monosaccharide, disaccharide, sugar alcohol, etc.)

), Neutral salt, amino acid, organic acid salt, Z or albumin, etc. can be added as a stabilizing agent, 10 to 200 g of sugar per 100 Om of aqueous solution containing protein, 0 to 200 g of neutral salt It is preferable to add about 1 to 10 g, amino acids 1 to 30 g, organic acid salts 1 to 30 g, and albumin about 0.1 to 10 g.

The treatment with a surfactant is performed by adding a surfactant to a liquid protein-containing composition, and is effective against enveloped viruses such as mumps virus, herpes simplex virus, HIV, HBVs HCV, and the like. .

The treatment with the surfactant is carried out at a temperature of 0 to 70 ° C., preferably 20 to 60 ° C., preferably 30 minutes or more, more preferably 1 to 30 hours, more preferably Runs for 3 to 10 hours.

In the treatment with a surfactant, usable surfactants include a polyoxyethylene derivative of a fatty acid, a partial ester of sorbitol anhydride, for example, polysorbate 80 (trade name: Tween 80, etc.), polysorbate 2 Polysorbate-based surfactants such as No. 0 (trade name: Tween 20), non-ionic oil bath water detergents, for example, oxshethylated alkylphenols (trade name: Triton XI 00, etc.) It is. Furthermore, Zwitterters, which are synthetic zwitterionic detergents known as sodium dequincholate and sulfobetaine, such as N-dodecyl-N, N-dimethyl-2-ammonio-11-ethanesulfonate, and homologs thereof, or nonionic detergents For example, octyl-3, D-dalcoviranoside and the like. In particular, polycarbonate surfactants are preferred. The amount of the surfactant used is not particularly limited, but it can be used, for example, in the range of about 0.1 to 10 wZv%, preferably about 0.1 to 3 w "v%.

Further, a trialkyl phosphate may be used in combination with the surfactant. The trialkyl phosphate used is not particularly limited, but is preferably tri (n-butyl) phosphate, tri (tert-butyl) phosphate, or tree ( n-hexyl) phosphate, tri (2-ethylhexyl) phosphate, tri (n-decyl) phosphate, and the like. Particularly preferred is tree (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 is used in the range of 0.01 to 10 wZv ^, preferably in the range of about 0.01 to 3%.

The treatment with a surfactant is preferably performed in the presence of a protease inhibitor in order to prevent a decrease in the activity of the protein. The protease inhibitor is not particularly limited as long as it is a substance that substantially inhibits the activity of the protease. For example, basic amino acids such as _ε- aminocaproic acid (EACA), lysine and arginine, and proteins such as aprotinin and tranexamic acid are exemplified.

The ultraviolet irradiation treatment is performed by uniformly irradiating the liquid protein-containing composition with ultraviolet light, and is also effective against heat-resistant non-enveloped viruses such as parvovirus and HAV.

The temperature at the time of the ultraviolet irradiation treatment is 1 to 35 ° C, preferably 10 to 30 ° C, the wavelength is 180 to 350nm, preferably 200 to 320nm, the irradiation energy amount. is to reduce the thickness of the liquid layer, for example less than 3 mm, preferably not exceed 0. 5 mm, 1~ 5 0 OmJ ou 1 eZcm 2, preferably 5 0~2 0 OmJ ou 1 e / cm 2 And Irradiation energy is the product of UV intensity (WZcm ² ) and irradiation time (sec). The vessel used for the irradiation is practically a circulating vessel, and the thickness of the liquid layer is adjusted to 3 mm or less, preferably 0.5 mm or less. The membrane used for the virus removal membrane treatment may be a membrane membrane or a porous hollow fiber membrane.The virus removal membrane treatment uses these membranes to perform a filtration treatment. It is also effective in removing decomposed virus debris (virus nucleic acid, etc.) that has been inactivated by treatment with a surfactant or ultraviolet irradiation, and this treatment should be performed after the above three other treatments. preferable.

For example, when using a porous hollow fiber membrane, the porous hollow fiber is a tubular fiber. The peripheral wall has a large number of holes penetrating from the hollow portion inside the hollow fiber to the outside, and this peripheral wall becomes a membrane used for filtration. The average pore diameter of the pores on the peripheral wall of the porous hollow fiber used is 1 to 100 nm. The preferred average pore size depends on the type and concentration of the protein to be filtered.For example, haptoglobin or immunoglobulin is 35 ± 2 nm, thrombin and antithrombin III, and blood coagulation factor IX is 15 ± 2 nm.

C, oh

The material forming the porous hollow fiber is not particularly limited, but regenerated cellulose is preferably used. The porous hollow fiber made of regenerated cellulose is preferably prepared by a microphase separation method from a cell-mouth cuprous ammonia solution [Am. Chem. Soc., 9, 1997-228 (19985). ) Prepared by 3. The porous hollow fiber is preferably used in a module form. For example, there is an embodiment in which a large number of porous hollow fibers are bundled in parallel, filled into a force cartridge, and integrated using an adhesive. A commercially available product is BMM (Bemberg Microporus Membrane, manufactured by Asahi Kasei Corporation). BMM is a porous hollow fiber made from regenerated cellulose by the copper ammonia method. As the BMM module, Blanova 15, Blanova 35, and Blanova 75 (all of which are trade names, manufactured by Asahi Kasei Corporation) having a multi-layer structure having 100 or more peripheral walls serving as membranes can be used.

This module is composed of the above-mentioned porous hollow fiber, a high-pressure steam sterilizable plastic container made of polycarbonate, and a polyurethane-based adhesive which integrates them. This module is autoclaved and filled with distilled water for injection. The safety of various materials that make up Branova has been confirmed by the method specified by the Japanese Pharmacopoeia (from BMM product description).

The temperature of the protein-containing solution at the time of filtration is 1 to 50 ° C, preferably 2 to 40 ° C. The filtration pressure is between 0.1 and 1 kgf Zcm ² , preferably between 0.1 and 0.9 kgf / cm ² . By setting the protein concentration of the protein-containing solution to 0.01 to 30 wZv%, preferably 0.05 to 28 w / v%, efficient filtration can be achieved. As a filtration method, there are a cross-floor single filtration method (circulation type) in which a solution is filtered while giving a strain rate, and a dead end filtration method (non-circulation type) in which a solution is filtered without giving a strain rate. Further, it is preferable that before the filtration treatment under the above conditions, the protein-containing aqueous solution is preliminarily subjected to a preliminary filtration treatment using a hollow fiber filtration or a flat membrane filtration filter other than the above conditions.

Each of the heat treatment, the ultraviolet irradiation treatment, the treatment with a surfactant, and the virus removal membrane treatment can be performed in any order in any production process regardless of the degree of protein purification.

The production method of the present invention can inactivate or remove even viruses that are resistant to heat, such as parvovirus, are not inactivated by a surfactant, and are not removed by a 35 nm membrane treatment. .

In the production method of the present invention, combinations of the above treatments include: liquid heat treatment, ultraviolet irradiation treatment, virus removal film treatment, treatment with a surfactant, ultraviolet irradiation treatment, dry heat treatment, treatment with a surfactant, and virus removal. Membrane treatment-dry heat treatment, treatment with surfactant-ultraviolet irradiation treatment—virus removal film treatment is recommended as the most suitable one. Note that these combinations do not limit the order of processing. According to the method of the present invention, after or before virus inactivation or removal treatment, purification treatment may be performed according to each desired protein. Purification is performed by appropriately selecting and using a method such as ion exchange chromatography, affinity chromatography, salt concentration fractionation technology, PEG (polyethylene glycol) fractionation, and alcohol fractionation.

The composition obtained by the production method of the present invention is substantially free of infectious virus and denatured form of the desired protein, and contains 90% or more of the effective desired protein in the total protein. It is. More preferably, the composition contains 95% or more of the effective desired protein in the total protein. The composition of the present invention is preferably a composition substantially free of viral nucleic acid.

An effective desired protein is not denatured and retains the same biological activity as before treatment. This is the protein we have. 90% or more of the effective desired protein means that the weight% of the effective desired protein per total protein is 90% or more. The effective protein weight percentage can be measured by high performance liquid chromatography (HPLC) analysis or cellulose acetate electrophoresis.

An effective desired protein has a peak in the same place as that before the treatment by HPLC analysis by absorbance at a wavelength of 280 nm, equipped with a TSK-G3000 SWXL column. In the cellulose acetate membrane electrophoresis or SDS polyacrylamide gel electrophoresis analysis, a band is observed at the same position as before the treatment.

The denatured protein of the desired protein is a protein that has been denatured by various treatments and has lost its activity. Various treatments, for example, change the molecular weight of the protein to produce dimers, polymers and degradants. When these variants are administered, undesirable effects such as the production of antibodies by new antigenic substances occur, so obtaining a composition that is substantially free of the variants is important in the production of pharmaceutical preparations. That is what.

The composition of the present invention substantially free of denatured form is obtained by adding a stabilizer to a virus inactivating treatment, a virus inactivating or removing treatment under conditions that do not produce a denaturing form, and before and after a virus inactivating or removing treatment. It can be obtained by combining various purification treatments.

The denatured form of the desired protein is observed by HPLC as a single or multiple peaks on the high or low molecular side of the peak development position of the desired protein. In addition, in electrophoresis, a single or multiple bands are observed on the anode side or the cathode side of the desired protein migration position.

In the composition of the present invention, a peak is observed only at a desired position in HPLC, and a band is observed only in a desired position in electrophoresis, so that substantially no denatured protein is contained.

The phrase "substantially free of viral nucleic acid" means that the nucleic acid is below the detection limit even if the detection of the nucleic acid is attempted by a method using PCR (polymerase chain reaction). PCR is described in Transfusion Vol. 32, p. 824-828 (1992). And the like. The PCR method is a very sensitive method for detecting nucleic acids, and if a nucleic acid is contaminated in a composition, the nucleic acid can be detected. The composition containing the desired protein of the present invention may be used as a preparation as it is, or may be made into a liquid preparation or a dry preparation by a conventional method. At that time, pharmacologically acceptable additives usually used for pharmaceuticals, for example, preservatives, chelating agents, thickeners, tonicity agents, etc., or pharmaceutically necessary ingredients are appropriately added. You may. Examples and experimental examples

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. Example 1 (Coagulation factor VI II-containing composition)

Cryoprecipitates obtained by freezing and thawing normal human plasma were dissolved with an aqueous solution of heparin, aluminum hydroxide gel was added at 2 vZv%, and centrifuged to obtain a deprothrombin solution. Then, TNB P is 0.3

And 6 en 80 was added so that the concentration became 1 wZv%, and a surfactant treatment was performed at 30 ° C for 6 hours.o

Glycine was added to a concentration of 2M, and centrifuged to remove fiprinogen by precipitation. To the resulting supernatant, sodium chloride was added to a concentration of 1.75M, and Factor VIII was recovered as a precipitate. The collected precipitate was dissolved in 20 mM Tris-HCl buffer containing 1 M sodium chloride, the thickness of the liquid layer was adjusted to 0.5 mm, the wavelength was 200 to 320 nm, and the irradiation energy was 50 mJou 1 e /. An ultraviolet irradiation treatment of cm ² was performed. The solution was subjected to gel filtration chromatography to purify factor VIII and to remove the remaining TNBP and Tween 80. The factor VIII thus obtained was precipitated again by glycine fractionation and sodium chloride fractionation, and after lysis dialysis

The mixture was adjusted to a predetermined concentration, divided into vials, and freeze-dried. The freeze-dried factor VIII-containing composition was subjected to a dry heat treatment at 60 ° C for 72 hours. A composition containing blood coagulation factor VII I was obtained.

The specific activity of the obtained blood coagulation factor VI11-containing composition was 30 units or more of Zmg protein, and substantially did not contain denatured blood coagulation factor VIII. Example 2 (Blood coagulation factor IX-containing composition)

Normal human plasma is loaded with anion exchange resin (DEAE-Sephadex), and Factor IX is adsorbed to the gel. After washing well with a buffer containing 0.15 M sodium chloride, 0.5 Factor IX was eluted with a buffer containing M sodium chloride. Then, TNB P was added to 0.3 ^^ ノ ^ and 丁 e en 80 to 1 wZv%, and a surfactant treatment was performed at 30 ° C for 6 hours. The treated factor IX solution was again charged with an anion exchange resin (DEAE-Sephadex), and the factor IX was adsorbed and washed to remove TNBP and Tween 80 from the washing solution.

Then, the thickness of the liquid layer of the Factor IX solution was to 0. 5 mm, wavelength 20 from 0 to 320 nm, the ultraviolet irradiation treatment of the irradiation energy amount 1 50 m J ou 1 e / cm 2 was performed ο

Adjust the protein concentration of the factor IX solution to 0.1 to 1.0 w / v% with a buffer containing 0.5 M sodium chloride, and use a BMM membrane (15 ± 2 nm) to adjust the temperature to 2 to 2 w / v%. The virus removal membrane treatment was performed by dead end filtration at 10 ° C and a filtration pressure of 0.5 kgf Zcm ² .

The membrane-treated factor IX solution is adjusted to the prescribed ion intensity and titer, divided, freeze-dried, and dried and heat-treated at 60 ° C for 72 hours to contain blood coagulation factor IX. A composition was obtained.

The weight% of the effective blood coagulation factor IX complex obtained by HPLC analysis of the obtained blood coagulation factor IX-containing composition was 95%, and substantially contained a denatured blood coagulation factor IX complex. Did not. Example 3 (Composition containing blood coagulation factor IX) Same as Example 2 except that instead of treating with an anion exchange resin (DEAE-Sephadex) after surfactant treatment, factor IX was bound to a resin bound with a monoclonal antibody against factor IX According to the method described above, a blood coagulation factor IX-containing composition was obtained.

The weight percent of effective blood coagulation factor IX by HP LC analysis of the obtained blood coagulation factor IX-containing composition was 95%, and was substantially free of denatured blood coagulation factor IX. . Example 4 (Thrombin-containing composition)

Thromboplastin (placental extract) was added to a blood coagulation factor II (prothrombin) lysate prepared by dissolving the Fr. II + III or Fr. Ill fraction of corn derived from normal human plasma with 0.15 M sodium chloride solution. ) Was added to convert prothrombin to thrombin. Next, 0.3 wZv% of TNB P and 1 v% of Tween 80 were added, and a surfactant treatment was performed at 30 ° C. for 6 hours.

Then, thrombin was adsorbed with a cation exchange resin (SP-Sephadex), washed well with a 0.15 M sodium chloride solution, and then thrombin was eluted with a buffer containing 0.5 M sodium chloride.

The thrombin concentration was adjusted to 0.5 wZv% with a buffer containing 0.5 M sodium chloride, and the temperature was 2 to 10 using a BMM membrane (15 ± 2 nm). C. A virus removal membrane treatment was performed by a dead end filtration method at a filtration pressure of 0.5 kgf / cm ² . The thrombin solution thus obtained was adjusted to a predetermined ionic strength and titer, subdivided, freeze-dried, and then subjected to a dry heat treatment at 60 ° C for 72 hours to obtain a thrombin-containing composition. .

The thrombin-containing composition obtained had a weight percentage of effective thrombin of 95% by HPLC analysis, and was substantially free of modified thrombin. Example 5 (Fibrinogen-containing composition) The Fr.I fraction of corn derived from normal human plasma was dissolved in saline, TNBP was added to 0.3 wZv% and Tween 80 to 1 wZv%, and the mixture was incubated at 30 for 6 hours. Surfactant treatment was performed. Then, TNBP and Tween.en80 were removed from the supernatant by fractionation with glycine sodium monochloride or ethanol, and fibrinogen was collected by precipitation.

Next, the thickness of the liquid layer of the recovered fibrinogen solution was set to 0.5 mm, and ultraviolet irradiation was performed at a wavelength of 200 to 320 nm and an irradiation energy of 10 OmJou 1 e / cm ² .

Glycine-sodium chloride fractionation or ethanol fractionation was performed again, and fibrinogen was precipitated and recovered. The fibrinogen precipitate thus obtained was dissolved in a predetermined concentration, divided into small portions, freeze-dried, and dried and heated at 60 ° C. for 72 hours to obtain a fibrinogen-containing composition.

The weight percent of effective fipurinogen in the obtained fipurinogen-containing composition was 90%, and it did not substantially contain denatured fibrinogen. Example 6 (Human urinary trypsin inhibitor (HUT I) -containing composition)

A HUT I solution was prepared using human fresh urine obtained according to the method described in J. Lab. Clin. Med., 79, 491, (1972) as a raw material. The concentration of the HUTI solution was about 3 wZv%. Next, the pH of this HUTI solution was adjusted to 5.5, and a liquid heat treatment was performed at 60 ° C for 10 hours.

Using a BMM membrane (15 ± 2 nm), the virus was subjected to dead-end filtration at a temperature of 10 to 15 ° C and a filtration pressure of 0.8 kgf Zcm ^{2 for the} liquid heat-treated HUT I solution. A removal film treatment was performed.

Next, the thickness of the liquid layer of HUTI solution 0. 5 mm, wavelength 200 to 3 20 nm, the ultraviolet irradiation treatment of the irradiation energy amount 1 O OmJ ou l eZcm ² was carried out. After titration and sterilization filtration, the HUT I solution was dispensed to obtain a HUT I-containing composition. The effective HUT I weight% of the obtained HUT I-containing composition by HP LC analysis was 95%, and it was substantially free from denatured HUT I. Example 7 (Human urinary trypsin inhibitor (HUT I) -containing composition)

A HUT I-containing composition was obtained in the same manner as in Example 6, except that the BMM film treatment in the production method of Example 6 was performed after the ultraviolet irradiation treatment.

The effective HUT I weight percent by HPLC analysis of the resulting HUT I-containing composition is

95%, which was substantially free of denatured HUT I. Example 8 (Albumin-containing composition)

Fraction V obtained from cold plasma fractionation of corn from normal human plasma was purified to obtain an albumin fraction with a purity of 96% or more. This was adjusted to an albumin concentration 5 w / v% solution, and subjected to a liquid heat treatment at 60 ° C. for 10 hours.

For the liquid heat-treated solution, use a BMM membrane (35 ± 2 nm) at a temperature of 2 to 10 ° C and a filtration pressure of 0.5 kgf / cm ^{2 to} remove the virus by dead-end filtration. Processing was performed.

After the completion of the filtration treatment, the thickness of the liquid layer of the filtrate was set to 0.5 mm, and ultraviolet irradiation treatment with a wavelength of 200 to 320 nm and an irradiation energy of 50 mJou 1 e / cm ² was performed. This solution was concentrated to obtain an albumin-containing composition.

The effective albumin weight% of the obtained albumin-containing composition by HPLC analysis was 95%, and it did not substantially contain denatured albumin. Example 9 (Antithrombin-containing composition)

10 kg of Fr. IV—1 fraction obtained from the cold ethanol fractionation of corn from normal human plasma was suspended in 100 L of physiological saline, and this solution was adjusted to pH 6.5. To 12.5 wZv%, remove the resulting precipitate by centrifugation, add PEG4000 to 25 w / v%, and collect the resulting precipitate by centrifugation. I

This precipitate was dissolved in about 20 L of cold physiological saline, injected into a column of heparin sepharose prepared in advance with physiological saline, and antithrombin III was adsorbed on the column. After washing the column with a 0.4 M sodium chloride solution, a 2.0 M sodium chloride solution was passed through the column to collect an eluted portion.

Then, sucrose (1 g per 1 mL of solution) and sodium citrate (0.3 g per 1 mL of solution) are added to adjust the pH to 7.8, and then the solution is kept at 60 ° C for 10 hours. Heat treatment is performed, followed by concentration while dialysis against 0.9 w / v% sodium chloride solution overnight to obtain an lw / v% aqueous solution of antithrombin 、, and filtration or centrifugation as necessary. And a clear liquid.

Add 2 wZv% of mannitol and 0.52 wZv% of sodium citrate to a 1 w / v% aqueous solution of antithrombin III, and add a small amount of cold distillation so that sodium chloride becomes 0.5 w / v%. Diluted with water and adjusted to pH 7.6 with 1 N sodium hydroxide. Using a BMM membrane (15 ± 2 nm), virus removal membrane treatment was performed by a dead end filtration method at a temperature of 2 to 10 ° C and a filtration pressure of 0.5 kgf / cm ² . After filtration finished, the thickness of the filtrate of the liquid layer to 0. 5 mm, wavelength 2 0 0 to 320 nm, the ultraviolet irradiation treatment of the irradiation energy amount 50 m J 0 u 1 e / cm 2 was performed. The solution was sterilized and filtered through a sterilized 0.2 / m membrane filter, dispensed in 500 units, and freeze-dried to obtain an antithrombin III-containing composition.

The obtained antithrombin III-containing composition had an effective antithrombin III weight% of 95% by HPLC analysis, and did not substantially contain a modified antithrombin III. Example 10 (Antithrombin III-containing composition)

An antithrombin III-containing composition was obtained in the same manner as in Example 9, except that the BMM film treatment in the production method of Example 9 was performed after the ultraviolet irradiation treatment. Efficient anti-thrombin III-containing composition obtained by HP LC analysis The weight% of thrombin II was 95%, and it did not substantially contain a modified antithrombin III. Example 11 (hatoglobin-containing composition)

30 kg of Fr. IV fraction obtained from corn by the low-temperature ethanol method from normal human plasma was suspended in 120 L of physiological saline, and this solution was adjusted to pH 8.0. After adjusting the pH to 6.5, PEG4000 was added to 25 w / v% and the resulting precipitate was centrifuged. Collected. To 1 kg of this precipitate, 4 L of water for injection was added for extraction and dissolution, and then ammonium sulfate was added to a concentration of 24 wZv%, and the resulting precipitate was adjusted to pH 4.6. Under a condition of 0, ammonium sulfate was added to a concentration of 30 w / v%, and the resulting precipitate was collected.

The obtained precipitate was dissolved in water for injection, and after dialysis, glycine was added to 20 w / v% and dissolved. Next, a liquid heat treatment was performed at 60 ° C. for 10 hours. The heat-treated solution is permeated with 0.05 M sodium phosphate buffer (pH 7.0), and then anion-exchange resin (DEAE-Sephadex A-50) is used in the same solution. Haptoglobin is adsorbed to the equilibrated solution, washed with the same solution as the equilibrating solution, and containing 0.13 M NaC1 0.05 M sodium phosphate buffer (pH 7.0) Eluted haptoglobin. After the eluate was treated with an ultrafiltration membrane, it was dialyzed against a physiological saline solution and clarified and filtered through a membrane filter to obtain a haptoglobin solution.

This haptoglobin solution (5 wZv%) was subjected to dead-end filtration using a BMM membrane (35 ± 2 nm) at a temperature of 30 to 40 ° C and a filtration pressure of 0.5 kgf Zcm ² by dead-end filtration. A removal film treatment was performed.

After filtration finished, the thickness of the filtrate of the liquid layer to 0. 5 mm, perform wavelength 2 0 0~ 3 2 0 nm, ultraviolet irradiation treatment of the irradiation energy amount 1 0 OmJ ou 1 e / cm 2, haptoglobin-containing A composition was obtained. The obtained haptoglobin-containing composition was analyzed by cellulose acetate electrophoresis. As a result, the effective weight percentage of haptoglobin was 95%, and it did not substantially contain denatured haptoglobin. Example 12 (habglobin-containing composition)

The haptoglobin-containing composition was prepared in the same manner as in Example 11 except that the BMM film treatment in the production method of Example 11 was performed after the ultraviolet irradiation treatment, and the BMM film (35 ± 2 nm) was used. Obtained.

The obtained haptoglobin-containing composition was analyzed by cellulose acetate electrophoresis. As a result, the effective weight percentage of haptoglobin was 95%, and it did not substantially contain a denatured haptoglobin. Example 13 (Heparin Cofactor-II (HCII) -containing composition)

After passing the Fr.II + III supernatant fraction obtained from cold ethanol fractionation of corn from normal human plasma through a heparin affinity resin column at 16 ° C to 14 ° C, HCII was eluted using a 0.1 M sodium citrate solution (pH 6.8) containing sodium chloride. To the HCII solution desalted and concentrated by ultrafiltration, add TNBP to 0.3 w / v% and Tween 80 to 1 w / v%, and bring to 30 ° C. For 6 hours.

Then, the HCII solution with adjusted salt concentration is passed through an anion exchange resin (DEAE-Sephadex) column to adsorb HCII, and a 0.1 M sodium citrate solution containing 0.1 M sodium chloride ( It was eluted at pH 7.2). After desalting and concentration, the HCII solution is passed through a cation exchange resin (SP-Toyopearl), and a 0.1 M sodium citrate solution containing 0.15 M sodium chloride (pH 7.2) is added. Was eluted. The eluate was concentrated by ultrafiltration, followed by gel filtration to obtain a purified HC II solution.

Adjust this purified HCII solution to 1 w7v% and use a BMM membrane (15 ± 2 nm). The virus removal membrane treatment was performed by a dead end filtration method at a temperature of 30 to 40 ° C. and a filtration pressure of 0.5 kgf / cm ² .

After completion of the filtration treatment, the thickness of the liquid layer of the filtrate was set to 0.5 mm, and an ultraviolet irradiation treatment with a wavelength of 200 to 320 nm and an irradiation energy of 150 mJ 0 u1 e / cm was performed. , HC II containing composition.

The effective HC II weight% of the obtained HC II-containing composition by HP LC analysis was 97%, and was substantially free of denatured HC II. Example 14 (Heparin cofactor II (HCII) -containing composition)

An HCII-containing composition was obtained in the same manner as in Example 13 except that the BMM film treatment in the production method of Example 13 was performed after the ultraviolet irradiation treatment.

According to the HPLC analysis of the obtained H C II-containing composition, the effective weight percentage of H C II was 9 7

%, And contained substantially no denatured form of HC II. Experimental Example 1 (Confirmation of virus inactivation or removal effect)

Samples before virus inactivation treatment were prepared in the same manner as in Examples 1 to 14. To these respective samples, vaccinia Hee § virus as a monitor one virus, Munpusuui Angeles, herpes simplex virus, echovirus, and added to a parvovirus and HIV each 1 0 ⁵ infectivity or, in each embodiment described Virus inactivation or removal treatment was performed, and the virus infectivity was measured for the sample after each treatment.

The virus infectivity is measured by the plaque formation method (PFU measurement method) for vaccinia virus, mumbus virus and herpes simplex virus, and the CPE formation method (TCI Dso measurement method) for echovirus, parvovirus and HIV. ).

For each virus, the reduction rate of the virus infectious titer before and after each virus inactivation or removal treatment was calculated, the virus infectious titer was calculated for all steps in each example, and the virus infectious titer for each final composition was calculated. Inactivation or removal effect The result was confirmed. As a result, the infectious titer of each virus was below the detection limit, and the virus inactivating or removing effect was confirmed. Experimental Example 2 (Detection of viral nucleic acid)

It was confirmed that hepatitis C virus (HCV) nucleic acid was not contained in the composition containing the desired protein obtained in each example. Nucleic acid was detected by the polymerase chain reaction (PCR) measurement method. The PCR measurement method followed the method described in Transfusion on Vol. 32, p. 824-828 (1992).

As a result, in each of the compositions, the nucleic acids of HCV were all below the detection limit. Therefore, the composition finally obtained in each of the examples is a composition substantially free of HCV nucleic acid. Industrial applicability

According to the production method of the present invention, the virus is efficiently inactivated or removed without losing the activity of the protein, and contains the desired protein substantially free of infectious virus and denatured product of the desired protein. A composition can be obtained. For example, viruses which are resistant to heat, resistant to surfactant treatment, and which cannot be removed by ordinary membrane treatment due to small viruses can be efficiently inactivated or removed by the production method of the present invention. Therefore, it is a very preferable method as an industrial production method of a highly safe protein-containing preparation. In particular, it is useful for producing a preparation containing a desired protein from a protein-containing composition in which unknown virus contamination is feared.

Further, the composition containing the desired protein of the present invention is substantially free of infectious virus and denatured form of the desired protein, and contains 90% or more of the effective desired protein in the total protein. Therefore, it is possible to provide a protein-containing preparation having higher safety and quality as a pharmaceutical than the composition of the present invention. The present invention is based on Japanese Patent Application No. 2,155 / 1997 filed in Japan, The contents of which are all incorporated herein.

Claims

The scope of the claims

1. an infectious virus, which comprises subjecting a protein-containing composition that may have virus contamination to at least three types of treatment: heat treatment, ultraviolet irradiation treatment, treatment with a surfactant, and virus removal membrane treatment. And a method for producing a composition containing a desired protein substantially not containing a denatured form of the desired protein.

2. The production method according to claim 1, wherein at least a virus removal membrane treatment is performed.

3. The production method according to claim 1, wherein the heat treatment comprises treating the protein-containing composition in a liquid state at 30 to 65 ° C for 10 minutes to 20 hours.

4. The production method according to claim 1, wherein the heat treatment comprises treating the protein-containing composition in a dry state at 60 to 100 ° C for 20 to 100 hours.

5. ultraviolet irradiation treatment, irradiation with ultraviolet light in the wavelength 1 8 0~3 5 0 nm, the irradiation energy formic one weight 1~5 0 OmJ ou 1 eZcm ² against protein-containing composition in liquid state processing The production method according to claim 1, wherein:

6. The method according to claim 1, wherein the treatment with a surfactant is a treatment with a polysorbate-based surfactant.

7. The production method according to claim 6, wherein a trialkyl phosphate is used in combination.

8. The production method according to claim 1, wherein the virus removal membrane treatment is a filtration treatment using a porous hollow fiber membrane.

9. The method according to claim 1, wherein the desired protein is fibrinogen, and the treatment is carried out with a surfactant, ultraviolet irradiation, and drying and heating.

10. The method according to claim 1, wherein the desired protein is heparin cofactor II.

1 1. Heat treatment, UV irradiation treatment, surfactant treatment, and virus removal membrane treatment are performed for at least three kinds of treatments to transform infectious viruses and desired proteins. A composition containing a desired protein, which is substantially free of a sex substance and contains 90% or more of an effective desired protein among total proteins.

12. The composition according to claim 11, wherein the composition is substantially free of viral nucleic acids.

13. The composition according to claim 11, wherein the three kinds of treatments are a treatment with a surfactant, an ultraviolet irradiation treatment, and a drying and heating treatment, and the desired protein is fibrinogen.

14. The composition according to claim 11 or 12, wherein the desired protein is heparin 'cofactor II.