JP2005312434A - Method for producing high-purity granulocyte colony-stimulating factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a high-purity G-CSF (granulocyte colony-stimulating factor) with which a recombinant baculovirus having the full-length cDNA of a natural type G-CSF and the high-purity G-CSF is prepared according to a method for column chromatographic purification in order to develop the G-CSF as a medicine. <P>SOLUTION: Several kinds of column chromatographic purifications are studied for the purpose of preparing a high-purity purified product having a physiological activity by paying attention to especially the G-CSF expressed/produced by an insect cell. As a result, a method for purifying the G-CSF preparable as a parenteral injection of 95% purity is established. The G-CSF is obtained as the result of the studies by using a cell culture supernatant containing the G-CSF produced by the insect cell according to the several kinds of column chromatographies. The method for high-purity production is carried out by combination of a cation exchange carrier with a hydroxyapatite column. Thereby, the high-purity G-CSF can be prepared and the G-CSF preparable as the parenteral injection can thereby be prepared. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a method for preparing granulocyte colony-stimulating factor produced in large quantities by gene recombination technology in high purity, and for the purpose of making it a pharmaceutical product, thereby producing high-purity granulocyte colony-stimulating factor. Regarding the law.

  Colony-stimulating factor (CSF) acts on blood cell progenitor cells in the bone marrow and has a function of regulating its differentiation and proliferation. In particular, granulocyte colony-stimulating factor (G-CSF) has the effect of differentiating and proliferating blood cell progenitor cells to neutrophils. The structure of human G-CSF is composed of 204 amino acids, of which 30 residues are a signal peptide, and the mature protein consists of 174 amino acids and undergoes some O-glycosylation. It is a glycoprotein. Initially, G-CSF was endotoxin administered to mice, and was separated and purified from the effluent in the lung (Non-patent Document 1). Human G-CSF was reported for cDNA cloning and expression from two groups (Non-patent Document 2.3). In the first report (Non-patent Document 2), human G-CSF was reported that the mature protein consists of 177 amino acids, but the biological activity was only one-tenth of the original activity. In the next report (Non-patent Document 3), cDNA cloning of 174-residue G-CSF, which has fewer 3 residue amino acids than the first report, was made, and G-CSF possesses the conventional biological activity. found. In addition, cDNA cloning of mouse G-CSF and rat G-CSF was performed (Non-patent Documents 4 and 5).

  Dogs and cats, which are companion animals, are reported to have many infections, and it is said that more than 1.5 million parvovirus infections in dogs worldwide, 20% of which will be fatal. Furthermore, this virus has been reported to induce severe neutropenia, and many dogs die further due to secondary infection. On the other hand, in cats, it is said that more than 500,000 to 1 million cats are infected with feline immunodeficiency virus (FIV) per year, and this virus induces neutropenia in about 30% of cats, making it susceptible to infection. It is supposed to be. In addition to FIV, feline leukemia virus (FeLV) is also said to become Neutropenia. Recently, it has been said that these companion animal tumors are susceptible to infectious diseases by inducing Neutropenia by administration of human antitumor drugs, radiation therapy, and the like.

  Recombinant human G-CSF has been used for Neutropenia induced by the prognosis of canine and cat infections and myelosuppression after administration of antitumor drugs. There is a report (Non-patent Document 6) that there is an improvement in symptoms when recombinant human G-CSF is administered to a dog in Newtropenia, and the improvement effect is similarly applied to a dog treated with radiation therapy. There is a report (Non-patent Document 7). However, it has been reported that, in the long-term administration, neutralizing antibody production against exogenous and endogenous G-CSF is observed, leading to severe neutropenia (Non-patent Document 8). The cause of the expression of neutralizing antibodies in the long-term administration is presumed to be due to the administration of G-CSF of different species, and is considered to be one of the serious clinical treatment problems in dogs and cats.

Cat G-CSF has been cloned by cDNA, and its expression in insect cells has also been confirmed (Patent Document 1). In this report, baculovirus was prepared and feline G-CSF was successfully produced using Sf21AE cells. The produced cat G-CSF promoted the growth of NFS60 cells, which is one of the bioassays for G-CSF. In addition, the prepared cat G-CSF is simplified by adding a tag to its 5 ′ end by gene recombination technology (Non-patent Document 9). On the other hand, canine G-CSF has been cloned by cDNA, and its expression in E. coli has also been confirmed (Patent Document 2). According to this report, the purification method of E. coli-produced canine G-CSF using a combination of ion exchange carriers has been reported.
Nicola et al .: Journal Biology of Chemistry (J. Biol. Chem), 258, 9017 (1983) Nagata et al .: Embo (EMBO), 5,575 (1986) Sucha et al .: Science, 232, 61 (1986) Tuchiya et al .: Proc. Natl. Acad. Sci, 83, 7633 (1986). Han et al .: Gene, 175, 101 (1996). Rosslop et al. Blood, 72, 1324 (1988). Shunin et al .: Blood, 74, 1308 (1989) Hammond et al .: Journal Clinical Investment (J. Clin. Invest), 87,704 (1991). Yamamoto et al .: Vet. Immunol. Immunopathol. 90.169-177 (2002) JP 11-196881 A U.S. Pat.No. 5,606,024

  There is no disclosure of a purification method for the above-mentioned methods for producing human G-CSF or cat G-CSF. In addition, although canine G-CSF has been described as a purification method using a combination of two ion exchange carrier columns, the purity is insufficient. Therefore, in order to develop G-CSF as a pharmaceutical, it is a problem to prepare high-purity G-CSF by a column chromatography purification method.

  The inventors of the present invention have studied the purification method of G-CSF, found that high-purity G-CSF is prepared by combining a cation exchange carrier and an affinity chromatography carrier, and completed the present invention. That is, the present invention is a high-purity production method of granulocyte colony stimulating factor comprising adding a surfactant to a culture supernatant containing granulocyte colony stimulating factor, contacting with a cation exchange carrier, and then contacting with an affinity chromatography carrier. In this method, granulocytes are characterized by containing 6000 or less insoluble fine particles having a particle size of 10 μm or less when containing 1 granule of colony-stimulating factor and surfactant and having a purity of 95% or more. An aqueous solution containing a colony stimulating factor can be obtained.

  According to the present invention, G-CSF can be prepared with high purity by adding a surfactant to the culture supernatant containing G-CSF and combining cation exchange carrier and affinity chromatographic carrier. G-CSF which can be prepared as an agent can be prepared.

  Examples of the G-CSF obtained by the production method of the present invention include human G-CSF and mammalian G-CSF, and the present invention is particularly applicable to the production of canine G-CSF and cat G-CSF. Furthermore, the present invention is preferably applicable to the production of cat G-CSF.

  A plasmid incorporating DNA encoding canine and feline G-CSF can be produced, for example, as follows. That is, after extracting poly (A) RNA from dog or cat cells, cDNA is synthesized, and polymerase chain reaction (hereinafter abbreviated as PCR) is performed using primers based on gene sequences encoding each subunit. By doing so, a gene encoding feline G-CSF can be obtained. Methods for obtaining RNA from canine and feline cells, such as dogs and feline lymphocytes stimulated with endotoxin, include conventional methods such as polysome isolation, sucrose density gradient centrifugation and electrophoresis. It is done. As a method for extracting RNA from the cells of dogs and cats, guanidine thiocyanate-cesium chloride method in which CsCl density gradient centrifugation is performed after guanidinium thiocyanate treatment (Chillin et al .: Biochemistry, 18, 5294 (1979)). Method of performing phenol extraction after treatment with surfactant in the presence of ribonuclease inhibitor using vanadium complex, guanidine thiocyanate-hot phenol method, guanidine thiocyanate-guanidine hydrochloride method, guanidine thiocyanate-phenol chloroform method, An appropriate method can be selected from a method in which RNA is precipitated by treatment with guanidine thiocyanate followed by treatment with lithium chloride.

  MRNA is isolated from dogs and cat lymphocytes by a conventional method, for example, lithium chloride / urea method, guanidine / isothiocyanate method, oligo dT cellulose method and the like, and a normal method such as the method of Gubler et al. Et al: Gene. 25, 236-269 (1983)), H. et al. CDNA is synthesized by the method of Okayama et al. (Okayama et al .: Molecular Cell Biology, 2, 161, (1982) & 3,280, (1983)). In order to synthesize cDNA from the obtained mRNA, basically a reverse transcriptase such as avian osteoblast virus (AMV) may be used, or a method using a DNA polymerase or the like using a one-part primer may be combined. It is convenient to use a commercially available synthesis or cloning kit. Obtaining DNA encoding canine G-CSF and feline G-CSF by performing PCR using this cDNA as a template and primers based on the nucleotide sequences of genes encoding canine G-CSF and feline G-CSF Can do.

  G-CSF can be produced using an insect cell expression system, for example, by producing a recombinant nuclear polyhedrosis virus that infects insect cells. Recombinant nuclear polyhedrosis virus is co-transfected into insect cells with a recombinant plasmid prepared by ligating DNA encoding G-CSF protein to a cloning vector and nuclear polyhedrosis virus DNA. Can be produced. Therefore, the recombinant virus can be prepared by an in vivo method. That is, a recombinant plasmid can be prepared according to a general genetic manipulation in which a DNA portion encoding a G-CSF protein is linked to an expression gene vector having a polyhedrin promoter. The recombinant plasmid and nuclear polyhedrosis virus DNA (Horiuchi et al .: Agricultural Biological Chemistry, 51, 1573-1580, (1987)) are prepared by methods such as those in the literature. After co-transfecting established cells such as BM-N strains and Sf cells, the culture is continued, and a non-recombinant (wild type) and recombinant virus appearing in the culture solution are limited dilution method, or Recombinant viruses can be cloned by common methods such as the plaque method. Recombinant viruses are easily distinguishable from wild type viruses because they lack the ability to form polyhedra. Production of G-CSF is performed by propagating the above-mentioned recombinant nuclear polyhedrosis virus in a silkworm organism.

  The produced G-CSF has an apparent molecular weight of about 20 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reduction.

  G-CSF is mainly characterized by its biological activity due to cell proliferation activity by NFS-60 in vitro.

  In the present invention, it is important to add a surfactant to the culture supernatant containing G-CSF and use a cation exchange carrier and an affinity chromatography carrier. By using both carriers, high-purity G-CSF can be obtained.

  The cation exchange carrier used for the separation and fractionation of G-CSF in the present invention includes agarose, cellulose, sepharose, synthetic polymer gel, and the like through a hydrophilic spacer and a chemically stable ether bond. The thing which introduce | transduced the propyl group is mentioned. Preferably, sulfopropyl sepharose high performance (trademark) (Amersham) or the like is used. In addition, as an affinity chromatographic carrier, an antibody is used as a ligand to bind cellulose / agarose, sepharose, etc., and the target protein as an antigen is separated, or a lectin that recognizes and binds sugar or sugar chain is cellulose / agarose / sepharose. And the like for use in purification. Furthermore, a specific dye or heparin is bound to cellulose, agarose, sepharose or the like, and a specific protein existing in a living body is used to bind to the target protein and used for purification of the target protein. Hydroxyapatite is a kind of calcium phosphate and is known to bind to many proteins and nucleic acids by electrostatic interaction. Examples of affinity chromatographic carriers include crystalline hydroxyapatite and sintered ceramicized hydroxyapatite. Biogel HT (trademark) (manufactured by Bio-Rad) or the like is preferably used.

  In the method of the present invention, a method in which a solution containing G-CSF and a surfactant is brought into contact with a cation exchange carrier, an adsorbate adsorbed on the carrier is eluted with an eluent, and then brought into contact with a hydroxyapatite carrier.

  Elution from the cation exchange carrier can be separated and fractionated by gradually increasing the salt concentration to cancel the ionic interaction with the sulfopropyl group, which is a ligand, and by the difference in ionicity of the adsorbed protein. . When G-CSF is produced in various production systems, components derived from culture broth and production system are mixed. Some of these components have very similar properties as proteins, such as isoelectric point values, so the adsorption behavior to column chromatographic carriers is similar and each component must be completely separated. Is not easy. The cation exchange carrier used in the present invention can separate and fractionate a plurality of proteins having properties very similar to those of G-CSF depending on the concentration of the eluent. Next, it is brought into contact with an affinity chromatography carrier. For example, in the hydroxyapatite carrier, electrostatic interaction between the amine group on the protein surface and the phosphate group of hydroxyapatite causes a calcium complex between the carboxyl group on the protein surface and calcium of hydroxyapatite. The body is formed. The elution from the hydroxyapatite carrier can be separated and fractionated according to the difference in ionicity of the adsorbed protein by gradually increasing the salt concentration. In the solution containing G-CSF eluted with the cation exchange carrier, components derived from the culture solution and components derived from the production system remain, and these components have very similar protein properties such as isoelectric point values. Therefore, it is considered that it is not easy to completely separate each component. According to the hydroxyapatite carrier used in the present invention, G-CSF can be separated and fractionated from a plurality of other proteins by adding pH.

  In the purification operation using a cation exchange carrier and an affinity chromatography carrier, the composition of the eluent, the amount of liquid, etc. are not particularly limited, and the optimum separation conditions are the amount of contaminating proteins present, the amount of each component of useful protein, It is determined appropriately according to the dimensions of the column.

  Surfactants used in the present invention are ionic and nonionic surfactants, and nonionic surfactants are particularly suitable. G-CSF is a mineral oil-based polyoxyethylene hydrogenated castor oil (HCO60). ) Is optimal. The amount of surfactant added to the culture supernatant or column can be increased by adding in the range of 1% to 0.005%. For G-CSF, the amount added is 0.5%. A concentration of ~ 0.05% is optimal.

  In addition, insect-type sugar chains are bound to G-CSF produced in insect cells. Since natural G-CSF is modified with O-type sugar chain and protein stability is higher than G-CSF without sugar chain, G-CSF produced in insects undergoes glycosylation. Good material stability compared to those made with E. coli.

  For the injection, an endotoxin test, an insoluble foreign matter test, an insoluble fine particle test, a glass container test, and the like are prescribed. Endotoxins and fine particles produced in injections may be mixed from vials and lids, including during the manufacturing process. In particular, it is known that fine particles are produced by coagulation of protein in a protein preparation. Further, by using the purification method of the present invention, an aqueous solution containing G-CSF with few insoluble fine particles can be obtained. The present invention comprises granulocytes characterized by containing 6000 or less insoluble fine particles having a particle size of 8 to 12 μm when the injection is 1 mL of an injection with a purity of 95% or more, comprising a granulocyte colony stimulating factor and a surfactant. An aqueous solution containing a colony stimulating factor. More preferably, the aqueous solution is 600 or less insoluble fine particles having a particle size of 20 to 30 μm. There are several measurement methods (14th revised Japanese Pharmacopoeia) for measuring insoluble fine particles, but in the present invention, measurement is performed with a microscope. Operate in clean equipment. A membrane filter for measurement is attached to the filter holder, and 200 mL of purified water for fine particle test is suction filtered at a rate of 20 to 30 mL for 1 minute. Next, the membrane filter is placed in a Petri dish and sufficiently dried at 50 ° C. or lower. The Petri dish is set in a microscope and insoluble fine particles are measured. Similarly, the sample containing G-CSF for measurement is diluted to 50 mL, a membrane filter for measurement is attached to the filter holder, and 200 mL of purified water for fine particle test is suction filtered at a rate of 20 to 30 mL for 1 minute. Next, the membrane filter is placed in a Petri dish and sufficiently dried at 50 ° C. or lower. The Petri dish is set on a microscope, and the number of insoluble fine particles is measured. The number of insoluble fine particles is calculated by multiplying the obtained numerical value by the dilution factor. According to the present invention, an aqueous solution containing G-CSF with little endotoxin, insoluble foreign matter, insoluble fine particles and the like can be obtained.

  Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.

Example 1 Acquisition of Feline G-CSF Gene (1) Preparation of Feline cDNA After cat peripheral blood lymphocytes were stimulated with endotoxin for 4 hours at 37 ° C., total RNA was prepared using ISOGEN (Nippon Gene). The obtained RNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (hereinafter abbreviated as TE) containing 1 mM EDTA, treated at 70 ° C. for 5 minutes, and then the same amount of TE containing 1 M LiCl was added. . The RNA solution was applied to an oligo dT cellulose column equilibrated with TE containing 0.5 M LiCl and washed with the same buffer. Further, after washing with TE containing 0.3 M LiCl, the poly (A) RNA adsorbed with 2 mM EDTA (pH 7.0) containing 0.01% SDS was eluted. Single-stranded cDNA was synthesized using the poly (A) RNA thus obtained. That is, 5 μg of poly (A) RNA and 0.5 μg of oligo dT primer (12-18mer) were placed in a sterilized 0.5 ml microcentrifuge tube, and sterilized water treated with diethyl pyrocarbonate was added to 12 μl. Incubated for 10 minutes and then placed on ice for 1 minute. To this, 2 μl of 200 mM Tris-HCl (pH 8.4), 500 mM KCl solution, 2 μl of 25 mM MgCl ₂ , 1 μl of 10 mM dNTP and 2 μl of 0.1 M DTT were added and incubated at 42 ° C. for 5 minutes, and then 200 units of GibcoBRL 1 μl of SuperScript II RT manufactured by the company was added and incubated at 42 ° C. for an additional 50 minutes to carry out a cDNA synthesis reaction. The reaction was stopped by further incubation at 70 ° C. for 15 minutes and placed on ice for 5 minutes. 1 μl of E. coli was added to this reaction solution. E. coli RNase H (2 units / ml) was added and incubated at 37 ° C. for 20 minutes.

(2) Acquisition of feline G-CSF gene Based on the base sequences of N-terminal and C-terminal of feline G-CSF (Japanese Patent Laid-Open No. 11-196881),
Two kinds of primers of SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized with a DNA synthesizer. 2 μl of cDNA obtained from the cat peripheral blood lymphocyte of (1) above was added to a 0.5 ml microcentrifuge tube, and each primer was added at 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , Reagents are added to give 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP, 4 units Taq DNA polymerase to make a total volume of 100 μl. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of DNA denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, and primer extension at 72 ° C for 3 minutes. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 500 bp was prepared according to a conventional method.

  This DNA fragment was transferred to T-Vector of Novagen from Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment was inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) was used, and a Perterminmer dye terminator was used according to the attached protocol. Using a cycle sequencing kit, it was confirmed that the obtained DNA fragment was a sequence encoding feline G-CSF (SEQ ID NO: 1).

Example 3 Expression of feline G-CSF by Sf21 (1) Incorporation of feline G-CSF gene into gene vector for expression Based on the nucleotide sequence of N-terminal side and C-terminal of feline G-CSF shown in SEQ ID NO: 1 In addition, two kinds of primers of SEQ ID NO: 4 and SEQ ID NO: 5 were synthesized with a DNA synthesizer. Using the feline G-CSF cDNA obtained in Example 1 (2) as a template, the gene was amplified by PCR. Specifically, ₂ μl of cat G-CSF cDNA was added to a 0.5 ml microcentrifuge tube, and each primer was added at 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP. 4 units Each reagent is added to become Taq DNA polymerase to make a total volume of 100 μl. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of DNA denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, and primer extension at 72 ° C for 3 minutes. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 500 bp was prepared according to a conventional method.

  This DNA fragment was transferred to T-Vector of Novagen from Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment was inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) was used, and a Perterminmer dye terminator was used according to the attached protocol. Using a cycle sequencing kit, it was confirmed that the obtained DNA fragment was a sequence encoding feline G-CSF.

  The Sf21 expression gene vector pVL1392 (Pharmingen) and the cat G-CSF gene prepared above are treated with EcoRI and BglII. Next, electrophoresis is performed on a 1% agarose gel, and the target gene is excised by a conventional method. The obtained gene fragment was extracted with geneclean II kit (manufactured by Funakoshi), and ligation of pVL1392 and cat G-CSF gene was performed with DNA ligation kit ver II (manufactured by Takara Shuzo Co., Ltd.) at 16 ° C for 30 minutes. went. Next, the ligated gene was transformed into a DH5α competent cell (manufactured by TOYOBO) by a conventional method. Sf21 expression gene vector DNA containing a feline G-CSF gene fragment is prepared from the obtained transformant by the alkali method.

(2) Cat G-CSF production at Sf21
A recombinant virus was prepared using Sf21 expression gene vector DNA and baculovirus DNA (AcNPV). That is, 2.5 ml of a DNA mixed solution (0. 5) was added to 2.5 ml of a solution composed of 50 mM HEPES buffer (pH 7.1), 0.28 M NaCl, 0.7 mM Na ₂ HPO ₄ and 0.7 mM NaH ₂ PO ₄ . 25M CaCl _2, nuclear polyhedrosis virus AcNPVDNA10myug, dropwise including) a recombinant vector 65μg for Sf21 expression, resulting in TC-10 medium the suspension 0.5ml supplemented with 10% FBS in 5ml was, 10 cm ² In addition to the culture medium of Sf21 cells, which are about 1 × 10 ⁷ insect cells planarly cultured in Dish, DNA was introduced into Sf21 cells. After 20 hours, the medium was replaced with a fresh medium, and further cultured at 27 ° C. for 5 days, and the culture solution was collected. Centrifuge the clarified supernatant and dilute and add to the culture medium of Sf21 cells cultured on a flat surface. After culturing for 1 hour, add 1% agarose gel containing the final concentration of 0.1 mg / mL X-gal. The culture was further performed at 27 ° C. for 5 days. Next, 1% agarose gel containing a final concentration of 0.1 mg / mL X-gal was overlaid and cultured at 27 ° C. for 5 days. Blue plaques, which are recombinant viruses, were picked up and treated overnight in 5 mL of TMN-FH insect medium (plaque assay method). The supernatant was diluted again and added to the culture medium of Sf21 cells cultured on a flat surface. After culturing for 1 hour, 1% agarose gel containing the final concentration of 0.1 mg / mL X-gal was added, and further at 27 ° C for 5 days. Culture was performed and the plaque assay was repeated three times to purify the recombinant virus.

  The recombinant virus containing DNA (SEQ ID NO: 1) encoding feline G-CSF produced here was designated as rAcNPV-FeG-CSF.

(3) Expression of feline G-CSF in insect cell Sf21 The recombinant virus obtained in (2) above was infected with Sf21 cultured at 27 ° C. After culturing at 27 ° C. for 5 days, the culture supernatant was collected and then centrifuged at 2,000 G for 10 minutes to collect the supernatant.

Example 4 Detection of Cat G-CSF Cat G-CSF obtained in Example 3 was detected by Western blotting. The culture supernatant was subjected to SDS-PAGE in a pagel made by Ato Corporation. Then, after blotting according to a conventional method on a clear blot membrane manufactured by Ato Co., Ltd., the membrane was reacted for 6 hours with a solution of Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing rabbit serum containing anti-human G-CSF polyclonal antibody. And washed 3 times with PBS containing 0.02% Tween 20, and further reacted with a block ace solution containing a peroxidase-labeled goat anti-rabbit IgG (Biorat Co., Ltd.) for 6 hours, washed in the same manner, and then Konica. Coloring was performed with Konica Immunostain HRP1000 manufactured by Co., Ltd. As a result, a band of about 20 kD was detected. Since the estimated molecular weight of cat G-CSF is approximately 20 kDa, it is considered that cat G-CSF was expressed in the culture supernatant of Example 3.

Example 5 Purification of Insect Cell Producing Cat G-CSF The cell culture supernatant containing the insect cell producing cat G-CSF obtained in Example 3 was desalted and 20 mM acetate buffer containing HCO60 by ultrafiltration. Buffer replacement was performed at (pH 5). Next, sulfopropyl sepharose (Amersham), which is a cation exchange carrier equilibrated with 20 mM acetate buffer (pH 5) containing HCO60, was prepared, and a solution containing cat G-CSF was introduced. After thoroughly washing the column, elution was performed with a solution containing 100 mM NaCl. The eluate containing cat G-CSF was subjected to desalting and buffer replacement with 20 mM acetate buffer (pH 5) containing HCO60 by ultrafiltration. Next, Biogel HT (manufactured by Bio-Rad) equilibrated with 20 mM acetate buffer (pH 5) containing HCO60 was prepared, and a solution containing cat G-CSF was introduced. The flow-through fraction and the fraction washed with 20 mM acetate buffer were collected, and finally desalted. The obtained sample was subjected to SDS-PAGE and subjected to gel staining by silver staining to confirm the unification of cat G-CSF. Furthermore, as a result of conducting a purity test using a gel stained with an image analyzer (manufactured by Bio-Rad), a purity of 95% could be confirmed. The endotoxin content was 0.9 ng / mg protein.

Example 6 Activity measurement of cat G-CSF The activity measurement of the cat G-CSF produced in Example 5 was performed as follows using NFS-60 cells (obtained from ATCC). NFS-60 was suspended in RPMI1640 / 10% FBS medium, adjusted to 2.2 × 10 ⁵ cells / mL, and 100 μL was added to a 96-well microplate. Further, 10 μL of a cat G-CSF sample was added, cultured for 24 hours under 5% CO 2 and 37 ° C., and the degree of cell proliferation was measured using cell counting kit-8. That is, 2 μL of each kit WST-8 was added, cultured for 2.5 hours, and then A450 was measured by ELISA Plate reader (Pharmacia). The result is shown in FIG. A significant proliferation of NFS-60 cells was observed as the sample amount increased, confirming the activity of the feline G-CSF.

Example 7 Sugar chain modification of purified cat G-CSF The cat G-CSF purified in Example 5 was added with O-glycosidase in 50 mM acetate buffer, treated overnight at 37 ° C, and then subjected to SDS-PAGE. As a result, a decrease in molecular weight could be confirmed. That is, it is considered that the insect type O-type sugar chain was modified.

Example 8 Insoluble fine particles of purified feline G-CSF aqueous solution Formulation was examined using the feline G-CSF purified in Example 5. Desalinate feline G-CSF by ultrafiltration using a solution containing 20 mM citrate buffer (pH 7.0) and 0.15 M NaCl without adding HCO60 to a final concentration of 1%. The gum arabic was added. In other words, preparations with or without the addition of surfactant HCO60 were prepared, prepared to be 1 mL each in glass vials, and the residual activity of feline G-CSF when stored in a solution state at 4 ° C. for 1 week. Comparison was made according to the method shown in FIG. At the same time, insoluble fine particles were measured. As a result, when HCO60 was added, the residual activity was 90% or more, whereas when it was not added, the residual activity was below the detection limit. As for the insoluble fine particles, when HCO60 was added, the number of insoluble fine particles having a particle size of 8 to 12 μm was 6000 or less, whereas in the case of no addition, there were 100,000 insoluble fine particles having a particle size of 8 to 12 μm.

Example 9 Acute toxicity test using mice An acute toxicity test of the feline G-CSF prepared in Example 8 was performed. Crj: CD-1 (ICR) mice were administered subcutaneously to 1 group of 5 mice (female). The doses were set to 5 doses of 1 μg / head, 10 μg / head, 100 μg / head, 1 mg / head, and 10 mg / head. After the administration, the general condition was observed and the body weight was measured once a week for 14 days, and an autopsy was conducted after 14 days. No abnormal symptoms were observed.

Example 10 Acquisition of canine G-CSF gene (1) Preparation of canine cDNA Canine peripheral blood lymphocytes were stimulated with endotoxin for 4 hours at 37 ° C, and then total RNA was prepared using ISOGEN (Nippon Gene). The obtained RNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (hereinafter abbreviated as TE) containing 1 mM EDTA, treated at 70 ° C. for 5 minutes, and then the same amount of TE containing 1 M LiCl was added. . The RNA solution was applied to an oligo dT cellulose column equilibrated with TE containing 0.5 M LiCl and washed with the same buffer. Further, after washing with TE containing 0.3 M LiCl, the poly (A) RNA adsorbed with 2 mM EDTA (pH 7.0) containing 0.01% SDS was eluted. Single-stranded cDNA was synthesized using the poly (A) RNA thus obtained. That is, 5 μg of poly (A) RNA and 0.5 μg of oligo dT primer (12-18mer) were placed in a sterilized 0.5 ml microcentrifuge tube, and sterilized water treated with diethyl pyrocarbonate was added to 12 μl. And then incubated for 10 minutes on ice. To this, 2 μl of 200 mM Tris-HCl (pH 8.4), 500 mM KCl solution, 2 μl of 25 mM MgCl ₂ , 1 μl of 10 mM dNTP and 2 μl of 0.1 M DTT were added, incubated at 42 ° C. for 5 minutes, and then 200 units of GibcoBRL. 1 μl of SuperScript II RT manufactured by KK was added and incubated at 42 ° C. for an additional 50 minutes to carry out a cDNA synthesis reaction. The reaction was stopped by further incubation at 70 ° C. for 15 minutes and placed on ice for 5 minutes. In this reaction solution, 1 μl of E. coli was added. E. coli RNase H (2 units / ml) was added and incubated at 37 ° C. for 20 minutes.

(2) Acquisition of canine G-CSF gene Based on the nucleotide sequences of the N-terminal and C-terminal of canine G-CSF, two types of primers of SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized with a DNA synthesizer. 2 μl of cDNA obtained from the canine peripheral blood lymphocyte of (1) above was added to a 0.5 ml microcentrifuge tube, and each primer was added at 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , Reagents are added to give 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP, 4 units Taq DNA polymerase to make a total volume of 100 μl. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of DNA denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, and primer extension at 72 ° C for 3 minutes. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 500 bp was prepared according to a conventional method.

  This DNA fragment was transferred to T-Vector of Novagen from Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment was inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) was used, and a Perterminmer dye terminator was used according to the attached protocol. Using a cycle sequencing kit, it was confirmed that the obtained DNA fragment was a sequence encoding canine G-CSF (SEQ ID NO: 6).

Example 11 Expression of canine G-CSF by Sf21 (1) Incorporation of canine G-CSF gene into gene vector for expression Based on N-terminal and C-terminal nucleotide sequences of canine G-CSF shown in SEQ ID NO: 6 In addition, two kinds of primers of SEQ ID NO: 9 and SEQ ID NO: 10 were synthesized with a DNA synthesizer. Using the dog G-CSF cDNA obtained in Example 10 (2) as a template, PCR was used to amplify the gene. Specifically, ₂ μl of canine G-CSF cDNA was added to a 0.5 ml microcentrifuge tube, and each primer was 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP. 4 units Each reagent is added to become Taq DNA polymerase to make a total volume of 100 μl. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of DNA denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, and primer extension at 72 ° C for 3 minutes. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 500 bp was prepared according to a conventional method.

  This DNA fragment was transferred to T-Vector of Novagen from Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment was inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) was used, and a Perterminmer dye terminator was used according to the attached protocol. Using a cycle sequencing kit, it was confirmed that the obtained DNA fragment was a sequence encoding canine G-CSF.

  The Sf21 expression gene vector pVL1392 (Pharmingen) and the canine G-CSF gene prepared above are treated with EcoRI and BglII. Next, electrophoresis is performed on a 1% agarose gel, and the target gene is excised by a conventional method. The obtained gene fragment was extracted with geneclean II kit (manufactured by Funakoshi Co., Ltd.). went. Next, the ligated gene was transformed into a DH5α competent cell (manufactured by TOYOBO) by a conventional method. Sf21 expression gene vector DNA containing a canine G-CSF gene fragment is prepared from the obtained transformant by the alkaline method.

(2) Production of canine G-CSF with Sf21
A recombinant virus was prepared using Sf21 expression gene vector DNA and baculovirus DNA (AcNPV). That is, 2.5 ml of a DNA mixed solution (0. 5) was added to 2.5 ml of a solution composed of 50 mM HEPES buffer (pH 7.1), 0.28 M NaCl, 0.7 mM Na ₂ HPO ₄ and 0.7 mM NaH ₂ PO ₄ . 25M CaCl _2, nuclear polyhedrosis virus AcNPVDNA10myug, dropwise including) a recombinant vector 65μg for Sf21 expression, resulting in TC-10 medium the suspension 0.5ml supplemented with 10% FBS in 5ml was, 10 cm ² In addition to the culture medium of Sf21 cells, which are about 1 × 10 ⁷ insect cells planarly cultured in Dish, DNA was introduced into Sf21 cells. After 20 hours, the medium was replaced with a fresh medium, and further cultured at 27 ° C. for 5 days, and the culture solution was collected. Centrifuge the clarified supernatant and dilute and add to the culture medium of Sf21 cells cultured on a flat surface. After culturing for 1 hour, add 1% agarose gel containing the final concentration of 0.1 mg / mL X-gal. The culture was further performed at 27 ° C. for 5 days. Next, 1% agarose gel containing a final concentration of 0.1 mg / mL X-gal was overlaid and cultured at 27 ° C. for 5 days. Blue plaques, which are recombinant viruses, were picked up and treated overnight in 5 mL of TMN-FH insect medium (plaque assay method). The supernatant was diluted again and added to the culture medium of Sf21 cells cultured on a flat surface. After 1 hour of culture, 1% agarose gel containing 0.1 mg / mL X-gal at a final concentration was added, and further at 27 ° C for 5 days. Culture was performed and the plaque assay was repeated three times to purify the recombinant virus.

  The recombinant virus containing the canine G-CSF-encoding DNA (SEQ ID NO: 6) prepared here was designated as rAcNPV-CaG-CSF.

(3) Expression of canine G-CSF in insect cells Sf21 The recombinant virus obtained in (2) above was infected with Sf21 cultured at 27 ° C. After culturing at 27 ° C. for 5 days, the culture supernatant was collected and then centrifuged at 2,000 G for 10 minutes to collect the supernatant.

Example 12 Detection of Dog G-CSF The dog G-CSF obtained in Example 11 was detected by Western blotting. The culture supernatant was subjected to SDS-PAGE in a pagel made by Ato Corporation. Then, after blotting according to a conventional method on a clear blot membrane manufactured by Ato Co., Ltd., the membrane was reacted for 6 hours with a solution of Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing rabbit serum containing anti-human G-CSF polyclonal antibody. And washed 3 times with PBS containing 0.02% Tween 20, and further reacted with a block ace solution containing peroxidase-labeled goat anti-rabbit IgG (manufactured by Biorat Co., Ltd.) for 6 hours. Coloring was performed with Konica Immunostain HRP1000 manufactured by Co., Ltd. As a result, a band of about 20 kD was detected. Since the estimated molecular weight of canine G-CSF is approximately 20 kDa, it is considered that canine G-CSF was expressed in the culture supernatant of Example 11.

Example 13 Purification of insect cell-producing canine G-CSF The cell culture supernatant containing the insect cell-producing canine G-CSF obtained in Example 11 was desalted and 20 mM acetate buffer containing HCO60 by ultrafiltration. Buffer replacement was performed at (pH 5). Next, sulfopropyl sepharose (manufactured by Amersham), which is a cation exchange carrier equilibrated with 20 mM acetate buffer (pH 5), was prepared, and a solution containing canine G-CSF was introduced. After thoroughly washing the column, elution was performed with a solution containing 100 mM NaCl. The eluate containing canine G-CSF was subjected to desalting and buffer replacement with 20 mM acetate buffer (pH 5) containing HCO60 by ultrafiltration. Next, Biogel HT (manufactured by Bio-Rad) equilibrated with 20 mM acetate buffer (pH 5) containing HCO60 was prepared, and a solution containing canine G-CSF was introduced. The flow-through fraction and the fraction washed with 20 mM acetate buffer were collected, and finally desalted. The obtained sample was subjected to SDS-PAGE and subjected to gel staining by silver staining to confirm the unification of canine G-CSF. Furthermore, as a result of conducting a purity test using a gel stained with an image analyzer (manufactured by Bio-Rad), a purity of 95% could be confirmed. The endotoxin content was 0.9 ng / mg protein.

Example 14 Activity Measurement of Canine G-CSF The activity measurement of canine G-CSF produced in Example 13 was performed as follows using NFS-60 cells (obtained from ATCC). NFS-60 was suspended in RPMI1640 / 10% FBS medium, adjusted to 2.2 × 10 ⁵ cells / mL, and 100 μL was added to a 96-well microplate. Furthermore, 10 μL of a canine G-CSF sample was added, cultured under 5% CO 2 and 37 ° C. for 24 hours, and the degree of cell proliferation was measured using cell counting kit-8. That is, 2 μL of each kit WST-8 was added, cultured for 2.5 hours, and then A450 was measured by ELISA Plate reader (Pharmacia). The result is shown in FIG. Significant proliferation of NFS-60 cells was observed with increasing sample volume, confirming the activity of canine G-CSF.

Example 15 Sugar Chain Modification of Purified Canine G-CSF SDS-PAGE was performed after adding canine G-CSF purified in Example 13 with O-glycosidase in 50 mM acetate buffer overnight at 37 ° C. As a result, a decrease in molecular weight could be confirmed. That is, it is thought that the insect type O sugar chain was modified.

Example 16 Insoluble fine particles of purified canine G-CSF aqueous solution Formulation was examined using canine G-CSF purified in Example 13. Canine G-CSF is desalted by ultrafiltration using a solution containing 20 mM citrate buffer (pH 7.0) and 0.15 M NaCl without adding HCO60, so that the final concentration is 1%. The gum arabic was added. That is, preparations with or without the addition of the surfactant HCO60 were prepared and prepared in glass vials to 1 mL each, and the residual activity of canine G-CSF when stored in a solution state at 4 ° C. for 1 week was determined as Example 14. Comparison was made according to the method shown in. At the same time, insoluble fine particles were measured. As a result, when HCO60 was added, the residual activity was 90% or more, whereas when it was not added, the residual activity was below the detection limit. As for the insoluble fine particles, when HCO60 was added, the number of insoluble fine particles having a particle size of 8 to 12 μm was 6000 or less, whereas in the case of no addition, 90000 insoluble fine particles having a particle size of 8 to 12 μm existed.

Example 17 Acute toxicity test using mice Acute toxicity test of canine G-CSF prepared in Example 16 was performed. Crj: CD-1 (ICR) mice were administered subcutaneously to 1 group of 5 mice (female). The doses were set to 5 doses of 1 μg / head, 10 μg / head, 100 μg / head, 1 mg / head, and 10 mg / head. After the administration, the general condition was observed and the body weight was measured once a week for 14 days, and an autopsy was conducted after 14 days. No abnormal symptoms were observed.

Example 18 Toxicity test using dogs The dog G-CSF prepared in Example 16 was administered to dogs to conduct toxicity tests. Subcutaneous administration was performed on 3 beagle dogs. The dose was set at 5 μg / head and 25 μg / head and administered twice a week for one month. Blood was collected before and after the first administration and immediately after the last administration, and the number of leukocytes and neutrophils were increased and decreased, and other general conditions were observed. As a result, the increase in white blood cell count and neutrophil, which are the drug effects of G-CSF, was confirmed, and no abnormality was observed in the general state.

  According to the invention of the present application, a protein having G-CSF activity can be produced with high purity, and a pharmaceutical such as an injection can be provided.

It is a figure which shows the result of having measured the cell growth ability using the mouse | mouth NFS-60 cell about the cat G-CSF expressed in the insect cell in Example 6. FIG. It is a figure which shows the result of having measured the cell growth ability using the mouse | mouth NFS-60 cell about the dog G-CSF expressed by the insect cell in Example 14. FIG.

Claims (7)

A high-purity production method of granulocyte colony stimulating factor comprising adding a surfactant to a culture supernatant containing granulocyte colony stimulating factor, bringing the surfactant into contact with a cation exchange carrier, and then contacting with an affinity chromatography carrier. The method for producing a granulocyte colony stimulating factor according to claim 1, wherein the granulocyte colony stimulating factor is a cat or canine granulocyte colony stimulating factor. 3. The method for producing a granulocyte colony-stimulating factor with high purity according to claim 1, wherein the cation exchange carrier is a sulfopropyl exchange carrier. The method for producing a granulocyte colony-stimulating factor with high purity according to any one of claims 1 to 3, wherein the affinity chromatography carrier is a hydroxyapatite carrier. The high-purity production method of granulocyte colony stimulating factor according to any one of claims 1 to 4, wherein the culture supernatant containing granulocyte colony stimulating factor is derived from insects. The method for producing a granulocyte colony stimulating factor according to any one of claims 1 to 5, wherein the granulocyte colony stimulating factor is a natural type and can be prepared as an injection with a purity of 95% or more. A granulocyte colony stimulating factor comprising 6000 or less insoluble fine particles having a particle size of 8 to 12 μm when containing 1 mL of an injection with a purity of 95% or more, comprising granulocyte colony stimulating factor and surfactant An aqueous solution containing.

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