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WO1998030230A1 - Compositions proteinees et procede de production - Google Patents

Compositions proteinees et procede de production Download PDF

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Publication number
WO1998030230A1
WO1998030230A1 PCT/JP1998/000042 JP9800042W WO9830230A1 WO 1998030230 A1 WO1998030230 A1 WO 1998030230A1 JP 9800042 W JP9800042 W JP 9800042W WO 9830230 A1 WO9830230 A1 WO 9830230A1
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WIPO (PCT)
Prior art keywords
treatment
protein
composition
virus
surfactant
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PCT/JP1998/000042
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English (en)
Japanese (ja)
Inventor
Koji Furushima
Katsuhiro Uriyu
Tuyoshi Takahashi
Motonori Hashimoto
Shoju Kameyama
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Yoshitomi Pharmaceutical Industries, Ltd.
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Publication of WO1998030230A1 publication Critical patent/WO1998030230A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person

Definitions

  • 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.
  • compositions containing proteins may contain pathogens that can infect humans, and the problem of viral infection is particularly important.
  • viruses such as human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) have occurred.
  • HAV human immunodeficiency virus
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • a method of heating a protein-containing composition in a liquid 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 Unexamined Patent Publication No. 60-51116
  • a method of irradiating with ultraviolet rays Japanese Unexamined Patent Publication No. Hei 7-196331
  • virus A method using a removal film Japanese Patent Application No. 7-2577771
  • heat treatment leaves heat-resistant viruses
  • surfactant treatment leaves non-enveloped viruses
  • 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.
  • 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.
  • 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.
  • the present invention is as follows.
  • 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.
  • virus removal membrane treatment is a filtration treatment using a porous hollow fiber membrane.
  • 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.
  • composition according to (11) which is substantially free of viral nucleic acid.
  • 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.
  • 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.
  • blood coagulation factors purinogen, prothrombin, thrombin, factor VI I, factor VI II, factor IX, factor X, factor XII I
  • Cofactor I II cofactor I II
  • 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 ⁇ 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.
  • 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.
  • 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.
  • viruses that may be contaminated are inactivated or removed.
  • the virus include vaccinia virus, mumbus virus, herpes simplex virus, echovirus, parvovirus, HIV, HAV, HBV, HCV and the like.
  • 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.
  • a dry state is a substantially anhydrous state.
  • 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%.
  • a stabilizer In the case of heat treatment, it is preferable to add a stabilizer.
  • stable It is preferable to add the agent before freeze-drying in order to reduce the effect of freeze-drying.
  • 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.
  • 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.
  • 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
  • octyl-3, D-dalcoviranoside and the like octyl-3, D-dalcoviranoside and the like.
  • 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%.
  • 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.
  • basic amino acids such as ⁇ - 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
  • 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.
  • the porous hollow fiber when using a porous hollow fiber membrane, 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.
  • 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.
  • 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 .
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a single or multiple bands are observed on the anode side or the cathode side of the desired protein migration position.
  • 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
  • Example 1 Coagulation factor VI II-containing composition
  • 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 Bood 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 ⁇ ⁇ ⁇ and ⁇ 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.
  • anion exchange resin DEAE-Sephadex
  • 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 ⁇
  • 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.
  • Example 3 Composition containing blood coagulation factor IX
  • Example 3 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.
  • DEAE-Sephadex anion exchange resin
  • 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
  • a blood coagulation factor II prothrombin
  • a surfactant treatment was performed at 30 ° C. for 6 hours.
  • 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.
  • SP-Sephadex a cation exchange resin
  • 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).
  • 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.
  • 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 .
  • 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%.
  • the pH of this HUTI solution was adjusted to 5.5, and a liquid heat treatment was performed at 60 ° C for 10 hours.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 hatchoglobin-containing composition
  • 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.
  • haptoglobin-containing A composition 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.
  • 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.
  • HCII Heparin Cofactor-II
  • HCII + III supernatant fraction obtained from cold ethanol fractionation of corn from normal human plasma was passed 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.
  • TNBP 0.1 M sodium citrate solution
  • Tween 80 1 w / v%
  • 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).
  • 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.
  • SP-Toyopearl a 0.1 M sodium citrate solution containing 0.15 M sodium chloride
  • the eluate was concentrated by ultrafiltration, followed by gel filtration to obtain a purified HC II solution.
  • 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 .
  • 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.
  • 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. ).
  • 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).
  • the composition finally obtained in each of the examples is a composition substantially free of HCV nucleic acid.
  • 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.
  • 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.
  • 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.

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

La présente invention concerne des compositions contenant les protéines désirées, mais sensiblement exemptes de tout virus infectieux ou de modifications des protéines désirées. Ces compositions sont produites en soumettant les compositions protéinées susceptibles de contamination par ces virus à au moins trois procédures choisies dans l'ensemble constitué du chauffage, de l'exposition aux UV, du traitement par un agent tensio-actif, et du traitement par une membrane pour éliminer les virus. Ce procédé de production permet d'inactiver ou d'éliminer efficacement les virus sans porter atteinte aux activités des protéines de façon à donner des compositions contenant les protéines désirées, mais sensiblement exemptes de tout virus infectieux ou de modification des protéines désirées. Il est ainsi possible d'obtenir des préparations protéinées présentant un haut degré de sûreté et d'excellentes qualités.
PCT/JP1998/000042 1997-01-09 1998-01-08 Compositions proteinees et procede de production WO1998030230A1 (fr)

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JP9/2155 1997-01-09
JP215597 1997-01-09

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003528803A (ja) * 1999-05-20 2003-09-30 アルファ・セラピューティック・コーポレーション 静注用免疫グロブリンの２回ウイルス不活化方法
WO2004089403A1 (fr) * 2003-04-09 2004-10-21 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Procede permettant d'eliminer un agregat d'albumine et/ou une proteine contaminee
WO2010001659A1 (fr) * 2008-07-02 2010-01-07 旭化成メディカル株式会社 Procédé permettant de produire une préparation contenant une substance utile unique avec un polypeptide
JP5080713B2 (ja) * 1999-12-20 2012-11-21 田辺三菱製薬株式会社 多孔性膜処理によるウイルスの除去された血漿蛋白組成物の製造方法、ウイルスの除去された血漿蛋白組成物およびウイルス除去方法
US9056896B2 (en) 2009-03-27 2015-06-16 Asahi Kasei Medical Co., Ltd. Method for removing viruses from high concentration monoclonal antibody solution
US11134669B2 (en) 2014-03-24 2021-10-05 The Chugoku Electric Power Co., Inc. Method for killing Pteriomorphia and barnacles using light irradiation
US11134671B2 (en) 2015-03-27 2021-10-05 The Chugoku Electric Power Co., Inc. Method for preventing settlement of sessile organisms
US11134670B2 (en) 2014-12-08 2021-10-05 The Chugoku Electric Power Co., Inc. Methods of suppressing settlement of barnacles
US11140893B2 (en) 2014-03-24 2021-10-12 The Chugoku Electric Power Co., Inc. Method for stopping swimming or crawling of adhesion-stage larvae
WO2022085606A1 (fr) * 2020-10-19 2022-04-28 一般社団法人日本血液製剤機構 Procédé pour éliminer des virus dans une solution protéique
US11517000B2 (en) 2012-08-14 2022-12-06 The Chugoku Electric Power Co., Inc. Method of stopping larva from swimming or crawling

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JPS6051116A (ja) * 1983-07-14 1985-03-22 ニユ−ヨ−ク　ブラツド　センタ−，インコ−ポレイテイド 脂質含有ウイルスを含まない蛋白質含有組成物及びその製造方法
JPH01143835A (ja) * 1987-10-15 1989-06-06 Biotest Ag フィブリノゲンおよび因子ｘ３を含有する無菌の血漿−タンパク質を調整する方法
JPH02167232A (ja) * 1988-12-21 1990-06-27 Asahi Chem Ind Co Ltd ウイルス除去方法
JPH02180833A (ja) * 1988-12-29 1990-07-13 Green Cross Corp:The 蛋白質含有組成物の製造方法
JPH03218322A (ja) * 1989-01-13 1991-09-25 Green Cross Corp:The 蛋白質含有組成物の製造方法
JPH06279297A (ja) * 1993-03-25 1994-10-04 Asahi Chem Ind Co Ltd ウィルス感染性除去方法およびその装置
JPH07215891A (ja) * 1994-01-31 1995-08-15 Green Cross Corp:The 筋注用グロブリン製剤の製造方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6051116A (ja) * 1983-07-14 1985-03-22 ニユ−ヨ−ク　ブラツド　センタ−，インコ−ポレイテイド 脂質含有ウイルスを含まない蛋白質含有組成物及びその製造方法
JPH01143835A (ja) * 1987-10-15 1989-06-06 Biotest Ag フィブリノゲンおよび因子ｘ３を含有する無菌の血漿−タンパク質を調整する方法
JPH02167232A (ja) * 1988-12-21 1990-06-27 Asahi Chem Ind Co Ltd ウイルス除去方法
JPH02180833A (ja) * 1988-12-29 1990-07-13 Green Cross Corp:The 蛋白質含有組成物の製造方法
JPH03218322A (ja) * 1989-01-13 1991-09-25 Green Cross Corp:The 蛋白質含有組成物の製造方法
JPH06279297A (ja) * 1993-03-25 1994-10-04 Asahi Chem Ind Co Ltd ウィルス感染性除去方法およびその装置
JPH07215891A (ja) * 1994-01-31 1995-08-15 Green Cross Corp:The 筋注用グロブリン製剤の製造方法

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003528803A (ja) * 1999-05-20 2003-09-30 アルファ・セラピューティック・コーポレーション 静注用免疫グロブリンの２回ウイルス不活化方法
JP5080713B2 (ja) * 1999-12-20 2012-11-21 田辺三菱製薬株式会社 多孔性膜処理によるウイルスの除去された血漿蛋白組成物の製造方法、ウイルスの除去された血漿蛋白組成物およびウイルス除去方法
WO2004089403A1 (fr) * 2003-04-09 2004-10-21 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Procede permettant d'eliminer un agregat d'albumine et/ou une proteine contaminee
WO2010001659A1 (fr) * 2008-07-02 2010-01-07 旭化成メディカル株式会社 Procédé permettant de produire une préparation contenant une substance utile unique avec un polypeptide
US9056896B2 (en) 2009-03-27 2015-06-16 Asahi Kasei Medical Co., Ltd. Method for removing viruses from high concentration monoclonal antibody solution
US11517000B2 (en) 2012-08-14 2022-12-06 The Chugoku Electric Power Co., Inc. Method of stopping larva from swimming or crawling
US11134669B2 (en) 2014-03-24 2021-10-05 The Chugoku Electric Power Co., Inc. Method for killing Pteriomorphia and barnacles using light irradiation
US11140893B2 (en) 2014-03-24 2021-10-12 The Chugoku Electric Power Co., Inc. Method for stopping swimming or crawling of adhesion-stage larvae
US11134670B2 (en) 2014-12-08 2021-10-05 The Chugoku Electric Power Co., Inc. Methods of suppressing settlement of barnacles
US11134671B2 (en) 2015-03-27 2021-10-05 The Chugoku Electric Power Co., Inc. Method for preventing settlement of sessile organisms
WO2022085606A1 (fr) * 2020-10-19 2022-04-28 一般社団法人日本血液製剤機構 Procédé pour éliminer des virus dans une solution protéique

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