CN106117342B - Preparation method of high-purity urofollitropin - Google Patents
Preparation method of high-purity urofollitropin Download PDFInfo
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- CN106117342B CN106117342B CN201610528438.6A CN201610528438A CN106117342B CN 106117342 B CN106117342 B CN 106117342B CN 201610528438 A CN201610528438 A CN 201610528438A CN 106117342 B CN106117342 B CN 106117342B
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- ZDRRIRUAESZNIH-BZGUUIOASA-N (2s)-1-[(4r,7s,10s,13s,16s,19r)-19-amino-7-(2-amino-2-oxoethyl)-13-[(2s)-butan-2-yl]-10-[(1r)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]-n-[(2s)-1-[(2-amino-2-oxoethyl)amino]- Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)[C@@H](C)O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZDRRIRUAESZNIH-BZGUUIOASA-N 0.000 title claims abstract description 70
- 108010047196 Urofollitropin Proteins 0.000 title claims abstract description 70
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims abstract description 43
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- 108010057021 Menotropins Proteins 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
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- QWXYZCJEXYQNEI-OSZHWHEXSA-N intermediate I Chemical compound COC(=O)[C@@]1(C=O)[C@H]2CC=[N+](C\C2=C\C)CCc2c1[nH]c1ccccc21 QWXYZCJEXYQNEI-OSZHWHEXSA-N 0.000 claims abstract description 9
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- 235000019441 ethanol Nutrition 0.000 claims 1
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- 102000004169 proteins and genes Human genes 0.000 abstract description 24
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- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- YKCWQPZFAFZLBI-UHFFFAOYSA-N cibacron blue Chemical compound C1=2C(=O)C3=CC=CC=C3C(=O)C=2C(N)=C(S(O)(=O)=O)C=C1NC(C=C1S(O)(=O)=O)=CC=C1NC(N=1)=NC(Cl)=NC=1NC1=CC=CC=C1S(O)(=O)=O YKCWQPZFAFZLBI-UHFFFAOYSA-N 0.000 description 2
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- YKTSYUJCYHOUJP-UHFFFAOYSA-N [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] Chemical compound [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] YKTSYUJCYHOUJP-UHFFFAOYSA-N 0.000 description 1
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- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical group OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a preparation method of high-purity urofolliculin (u-FSH), which comprises the following steps: (1) purifying a Human Menopausal Gonadotropin (HMG) crude product by anion exchange chromatography to obtain a urofollitropin intermediate I; (2) purifying the urofollitropin intermediate I by cation exchange chromatography to obtain a urofollitropin intermediate II; (3) purifying the urofollitropin intermediate II by dye affinity chromatography to obtain a urofollitropin intermediate III; (4) and (3) carrying out hydrophobic chromatography purification on the urofollitropin intermediate III to obtain the high-purity urofollitropin. The method has the advantages of simple operation, low production cost, higher purity of Follicle Stimulating Hormone (FSH) in the obtained urofollitropin, lower content of Luteinizing Hormone (LH) and no foreign protein pollution.
Description
Technical Field
The invention relates to the technical field of biology, and in particular relates to a method for extracting and preparing urofollitropin.
Background
Urofollitropin (u-FSH) is a composition containing Follicle Stimulating Hormone (FSH) as the main component, and the FSH content in the high-purity urofollitropin can reach more than 95 percent and hardly contains Luteinizing Hormone (LH). FSH is a glycoprotein hormone with a molecular weight of about 30 KD. FSH, which promotes granulosa cell proliferation, stimulates steroid production, regulates the development and maturation of gametocytes, is one of the major hormones in the hypothalamic-pituitary-gonadal axis. FSH has effects of regulating menstrual cycle, ovulation and regulating male testicular function, and is used for treating female infertility, male gonadotropin hyposecretion, seminal tubule defect, etc.
Both are present in the human pituitary and in the urine of menopausal and postmenopausal women and are therefore also the main source of FSH extraction. The starting material Human Menopausal Gonadotropin (HMG) for the production of u-FSH mainly comprises follicle stimulating Hormone and Luteinizing Hormone (LH). The ratio of FSH to LH titres is about 1: 1. The preparation of high purity urofollitropin requires removal of LH and other urine proteins in HMG, leaving high purity FSH as a single active ingredient.
At present, the foreign high-purity u-FSH production technology is mainly mastered by Serono company, and the technological process comprises the steps of extracting an HMG crude product by ethanol, adsorbing by aluminum silicate, carrying out anion exchange chromatography, capturing FSH by using an anti-FSH monoclonal antibody chromatographic column, and finally obtaining the high-purity u-FSH by reversed phase chromatography. The domestic high-purity u-FSH is mainly produced by two companies, namely Lizhu and Tianwei, and the production process of the u-FSH adopts the process step of adsorbing and removing LH by an anti-LH antibody. The method for removing LH by using antigen-antibody specific reaction has the advantages of high purification efficiency and the like. However, most of the antibodies are of murine or rabbit origin and belong to proteins, and in the chromatography process, the antibody coupled to the matrix falls off to cause pollution of heterologous proteins, which affects the product quality. Meanwhile, the antibody immunoaffinity chromatography column has high price, short service life and difficult maintenance and industrial scale-up production, and the defects seriously hinder the application and popularization of the antibody immunoaffinity chromatography column. Therefore, there is a need in the art to develop a production process that can prepare low-content LH and high-purity FSH products with simple operation and low production cost. The inventors have found that LH is more hydrophobic than FSH and can be separated by hydrophobic chromatography techniques, and therefore hydrophobic chromatography can be used instead of antibody immunoaffinity chromatography to solve the above problems.
Disclosure of Invention
Aiming at the defects of the process, the invention aims to provide the preparation method of the urofollitropin, compared with the prior art, the preparation method has the advantages of simple and convenient operation, low production cost, higher purity of the obtained urofollitropin and no pollution of foreign protein. The specific activity of FSH is more than 10000IU/mg protein, and the potency of LH to FSH is less than 1: 100.
The preparation method of the urofollitropin provided by the invention comprises the following steps:
(1) purifying the crude product of human menopausal gonadotropin by anion exchange chromatography to obtain a urofollitropin intermediate I;
(2) purifying the urofollitropin intermediate I by cation exchange chromatography to obtain a urofollitropin intermediate II;
(3) purifying the urofollitropin intermediate II by dye affinity chromatography to obtain a urofollitropin intermediate III;
(4) carrying out hydrophobic chromatography purification on the urofollitropin intermediate III to obtain a high-purity urofollitropin solution;
the preparation method is characterized by further comprising the step of carrying out ultrafiltration and salt exchange on the urofollitropin obtained in the step (4), wherein the solution for salt exchange is 0.01M PB buffer solution, and the pH value is preferably 5.5-7.0.
Preferably, the preparation method further comprises the step of mixing the urofollitropin solution obtained in the step (4) with a freeze-drying protective agent and then freeze-drying, wherein the freeze-drying protective agent is lactose, and preferably, the addition amount of the lactose is 3% -6% (g/ml).
Preferably, the filler ligand of the anion exchange chromatography in the preparation method is triethylaminoethyl, diethylaminoethyl, aminoethyl or triethanolamino, and the matrix is cellulose, dextran, agarose or styrene; the cation exchange chromatography packing ligand is carboxymethyl, sulfonic group, phosphate group, phosphite group and phenol group, and the matrix is cellulose, glucan, agarose and polystyrene; the dye affinity chromatography filler ligand is Cibacron Blue, Red, Green and Orange, and the matrix is agarose, dextran or chitosan; the hydrophobic chromatographic packing is a copolymer of vinyl alcohol and methacrylate ester, agarose, cellulose, polystyrene, polymethyl acrylate or chitosan, wherein the copolymer takes butyl, octyl, phenyl or neopentyl as a ligand.
Specifically, the step (1) includes the steps of:
dissolving the crude HMG product with water, and adjusting the pH value of the solution to 5.5-8.0; loading the HMG crude product solution to a well balanced anion exchange chromatographic column; washing with 0.02M PB solution with pH of 5.5-8.0, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease; eluting with 0.02M PB solution containing 0.05-0.2M NaCl and having pH of 5.5-8.0, and detecting the absorbance of the eluate at 280nm until the absorbance does not decrease, to obtain eluate I;
specifically, the step (2) includes the steps of:
loading the solution of the follicle-stimulating hormone intermediate I to a well-balanced cation exchange chromatographic column; washing with 0.1M HAc-NaAc solution with pH of 4.0-5.5, and detecting absorbance of the washing solution at 280nm until absorbance does not decrease; eluting with 0.1M HAc-NaAc solution containing 0.1-0.5M NaCl and having pH of 4.0-5.5, and detecting the absorbance of the eluate at 280nm until the absorbance is not reduced, wherein the eluate is follicle stimulating hormone intermediate II;
specifically, the step (3) includes the steps of:
loading the solution of the follicle-stimulating hormone intermediate II to a well-balanced Cibacron Blue dye affinity chromatography column; washing with 0.1M NaAc solution with pH of 6.0-9.0, and detecting absorbance of the washing solution at 280nm until absorbance does not decrease; eluting with 0.02M PB solution containing 0.8-2.5M KCl with pH of 8.0-10.0, and detecting the absorbance of the eluate at 280nm until the absorbance does not decrease, to obtain eluate as intermediate III of urofollitropin;
specifically, the step (4) includes the steps of:
loading the solution of the follicle-stimulating hormone intermediate III to a well-balanced hydrophobic chromatographic column; washing with 0.02M PB solution containing 1.0-1.5M ammonium sulfate at pH 7.0-9.0, and detecting the absorbance of the washing solution at 280nm until the absorbance does not decrease; eluting with 0.02M PB solution containing 0.2-0.4M ammonium sulfate and having pH of 7.0-9.0, and detecting light absorption value of eluate at 280nm until the light absorption value is not reduced, wherein the eluate is high-purity urofollitropin solution;
the invention takes HMG as a starting material, and adopts anion exchange chromatography, cation exchange chromatography, dye affinity chromatography and hydrophobic chromatography processes in sequence to extract and purify to prepare the high-purity FSH. Wherein, the protein is charged with different charges by changing the pH value of the solution by utilizing the property of protein amphoteric ionization, and the FSH purification multiple can reach more than 50 times through anion exchange chromatography and cation exchange chromatography. By utilizing the affinity adsorption property of FSH and dye, impurity protein is further removed, and the FSH purification multiple reaches more than 150 times. And finally, removing LH by utilizing the hydrophobicity difference of LH and FSH to obtain the high-purity urofollitropin almost without LH.
Drawings
FIG. 1 is a process flow diagram of the present invention.
FIG. 2 is an HPLC chromatogram of a high purity urofollitropin finished product prepared in example 1.
Fig. 3 is an electropherogram of FSH prepared in example 1 and example 2. Wherein, 1: example 1 a finished product was prepared; 2: example 1 a finished 5% self control was prepared; 3: example 2 a finished 5% self control was prepared; 4: example 2 preparation of finished product;
FIG. 4 is an HPLC chromatogram of a high purity urofollitropin finished product prepared in example 2.
Detailed Description
The invention relates to a method for measuring and calculating biological potency, specific activity and purity.
And (3) measuring the biological potency:
the biological potency of FSH and LH is measured according to the rules 1216 and 1217 of the four Ministry of the pharmacopoeia of the people's republic of China 2015 edition.
Specific activity determination:
precisely weighing about 10mg, and precisely adding 1ml of water for dissolving; in addition, bovine serum albumin reference substances are precisely weighed, water is added to dissolve the bovine serum albumin reference substances to prepare solutions containing 1.0mg, 0.8mg, 0.6mg, 0.4mg, 0.2mg and 0mg in each 1ml, the absorbance is respectively measured at the wavelength of 280nm by an ultraviolet-visible spectrophotometry (pharmacopoeia 2015, four pharmacopoeia 0401), the concentration of the bovine serum albumin solution is taken as a horizontal coordinate, the absorbance at 280nm is taken as a vertical coordinate to draw a standard curve, and the relationship between the absorbance and the concentration is linear. And (3) obtaining the protein concentration of the test solution from the standard curve, and calculating the specific activity according to the following formula:
HPLC purity determination:
adding water into the product to obtain a solution containing about 500IU FSH per 1ml as a test solution. According to the high performance liquid chromatography of 0512 in the four ministry of communications in 2015 edition of pharmacopoeia of the people's republic of China. Gel chromatography column Superdex 75; adding phosphate buffer (85% H3PO46.74ml, adding water 800ml, adding Na2SO414.2g, adjusting pH to 6.7 with 50% NaOH, and diluting to 1000ml with water); the column temperature is 30 ℃; the flow rate is 0.5 ml/min; the detection wavelength is 214 nm; the amount of the sample was 100. mu.l.
And (3) electrophoresis related substance determination:
the results were examined by SDS-polyacrylamide gel electrophoresis according to the fifth method of 0541, the fourth Community of the four Ministry of the pharmacopoeia of the people's republic of China 2015. The concentration of the sample solution was 30000IU FSH/ml, and the sample solution was diluted 20 times to give a control solution, and the amount of the sample was 10. mu.l.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1
As shown in FIG. 1, the crude HMG (manufactured by this company, lot No. Z0203150901) as a starting material had an FSH bio-potency of 30IU/mg, an LH bio-potency of 28IU/mg, and an FSH specific activity of 43IU/mg protein.
Capto DEAE anion exchange chromatography
Dissolving 8.0g HMG crude product in 200ml water, adjusting pH of the solution to 7.5, and loading the solution onto Capto DEAE chromatographic column (from GE) with column volume of 250ml, which is well balanced with balancing solution (0.02M PB, pH 7.5); after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.02M PB +0.15M NaCl), detecting the light absorption value of the eluent at 280nm until the light absorption value is not reduced, collecting the eluent 1.0L, concentrating with ultrafiltration membrane with cut-off molecular weight of 6KD to about 100ml, adding 95% ethanol pre-cooled to-20 deg.C until the ethanol concentration is 85%, stirring, standing overnight to precipitate protein, centrifuging the next day to collect precipitate, adding anhydrous ethanol to dehydrate for 3 times, and vacuum drying to obtain urofollitropin intermediate I0.73 g.
CM-Sepharose cation exchange chromatography
0.7g of the intermediate I of urofollitropin is dissolved by 30ml of a balancing solution (0.1M HAc-NaAc, pH4.5), and then the solution is loaded on a CM-Sepharose chromatographic column (purchased from GE) with the column volume of 20ml, wherein the chromatographic column is well balanced by the balancing solution in advance; after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.1M HAc-NaAc +0.5M NaCl, pH4.5), detecting absorbance of the eluent at 280nm until absorbance does not decrease, and collecting eluate 100ml to obtain urofollitropin intermediate II.
Capto Blue (high sub) dye affinity chromatography
98ml of the urofollitropin intermediate II is adjusted to have the pH value and the conductivity consistent with those of an equilibrium solution (0.1M NaAc, pH7.0), and is loaded to a Capto Blue (high sub) chromatographic column (purchased from GE company) with the well-balanced column volume of 15 ml; after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.02M PB +2.0M KCl, pH 9.0), detecting absorbance at 280nm of the eluent until absorbance does not decrease, and collecting eluate 70ml to obtain urofollitropin intermediate III.
Phenyl-650M hydrophobic chromatography
Adding solid ammonium sulfate into 68ml of urofollitropin intermediate III, stirring for dissolving, adjusting pH and conductivity, and balancing solution (0.02M PB +1.4M (NH)4)2SO4pH7.0), and loaded onto a equilibrated Phenyl-650M column (available from TOSOH corporation) having a column volume of 10 ml; after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; then eluent (0.02M PB +0.3M (NH)4)2SO4pH7.0), detecting the light absorption value of the eluent at 280nm until the light absorption value is not reduced any more, and collecting the elution effluent liquid of 40ml together to obtain the high-purity urofollitropin solution.
Ultrafiltering and concentrating the high-purity urofollitropin solution with ultrafiltration membrane with cut-off molecular weight of 6KD, changing salt with 0.01M PB buffer solution (pH 6.0) for 3 times, adding lactose according to the proportion of 4% (w/v) to prepare lyophilized stock solution, and lyophilizing to obtain 0.91g of high-purity urofollitropin finished product, wherein the related parameters of the finished product are shown in Table 1, and HPLC and electrophoretic purity maps of FSH are respectively shown in FIG. 2 and FIG. 3.
TABLE 1 parameters for the high purity urofollitropin product prepared in example 1
Example 2
HMG of low purity (manufactured by the same company, lot No. Z0203150901) was used as a starting material, and the biological potency of FSH was 30IU/mg, the biological potency of LH was 28IU/mg, and the specific activity of FSH was 43IU/mg protein.
Capto DEAE anion exchange chromatography
Dissolving 20g HMG with 500ml water, adjusting pH of solution 1 to 7.5, loading the solution onto Capto DEAE chromatographic column (from GE) with column volume of 630ml, which is well balanced with balancing solution (0.02M PB, pH 7.5); after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.02M PB +0.15M NaCl), detecting the light absorption value of the eluent at 280nm until the light absorption value is not reduced, collecting the elution effluent liquid 2.5L, concentrating with ultrafiltration membrane with cut-off molecular weight of 6KD to about 100ml, adding 95% ethanol pre-cooled to-20 deg.C until the ethanol concentration is 85%, stirring well, standing overnight to precipitate protein, centrifuging the next day to collect precipitate, adding anhydrous ethanol to dehydrate for 3 times, and vacuum drying to obtain urofollitropin intermediate I1.85g.
CM-Sepharose cation exchange chromatography
After dissolving the intermediate I1.8g of urofollitropin with 80ml of a balance solution (0.1M HAc-NaAc, pH4.5), loading the solution onto a CM-Sepharose chromatographic column (purchased from GE) with a column volume of 50ml, wherein the chromatographic column is well balanced by the balance solution in advance; after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.1M HAc-NaAc +0.5M NaCl, pH4.5), detecting absorbance of the eluent at 280nm until absorbance does not decrease, and collecting eluate 250ml to obtain urofollitropin intermediate II; concentrating with ultrafiltration membrane with molecular weight cutoff of 6KD to about 100ml, adding 95% ethanol pre-cooled to-20 deg.C until ethanol concentration is 85%, stirring, standing overnight to precipitate protein, centrifuging the next day, collecting precipitate, adding anhydrous ethanol, dehydrating for 3 times, and vacuum drying to obtain 0.24g of urofollitropin intermediate II dried product.
Capto Blue (high sub) dye affinity chromatography
Dissolving 0.21g of the intermediate II into a balanced solution (0.1M NaAc, pH7.0), and loading to a 40 ml-column Capto Blue (high sub) chromatography column (purchased from GE corporation); after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; eluting FSH with eluent (0.02M PB +2.0M KCl, pH 9.0), detecting absorbance at 280nm of the eluent until absorbance does not decrease, and collecting eluate 174ml to obtain urofollitropin intermediate III; adding 95% ethanol precooled to-20 ℃ until the ethanol concentration is 85%, uniformly stirring, standing overnight to precipitate protein, centrifuging the next day, collecting precipitate, adding absolute ethanol, dehydrating the precipitate for 3 times, and drying the precipitate in vacuum to obtain 0.13g of a urofollitropin intermediate III dried product.
Phenyl-650M hydrophobic chromatography
0.11g of urofollitropin intermediate III dried product was treated with equilibration solution (0.02M PB +1.4M (NH)4)2SO4pH7.0) and loaded onto a equilibrated Phenyl-650M column (from TOSOH); after the sample loading is finished, washing unadsorbed protein by using a balance solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease any more; then eluent (0.02M PB +0.3M (NH)4)2SO4pH7.0), detecting the light absorption value of the eluent at 280nm until the light absorption value is not reduced any more, and collecting the eluent 94ml together to obtain the high-purity urofollitropin.
Ultrafiltering and concentrating high-purity urofollitropin with ultrafiltration membrane with cut-off molecular weight of 6KD, replacing original buffer solution with 0.01M PB buffer solution (pH 6.0) for 3 times, adding lactose according to 4% (w/v) ratio to obtain lyophilized stock solution, and lyophilizing to obtain 2.31g high-purity urofollitropin product with relevant parameters shown in Table 2, and electrophoresis and HPLC purity maps of FSH shown in FIG. 3 and FIG. 4 respectively.
TABLE 2 parameters for the high purity urofollitropin product prepared in example 2
Claims (4)
1. A preparation method of high-purity urofollitropin is characterized by comprising the following steps:
(1) purifying a Human Menopausal Gonadotropin (HMG) crude product by anion exchange chromatography to obtain a urofollitropin intermediate I; the method comprises the following steps: dissolving the crude HMG product with water, and adjusting the pH value of the solution to 5.5-8.0; loading the HMG solution to a well balanced anion exchange chromatographic column; washing with 0.02M PB solution with pH of 5.5-8.0, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease; eluting with 0.02M PB solution containing 0.05-0.2M NaCl and having pH of 5.5-8.0, and detecting the absorbance of the eluate at 280nm until the absorbance does not decrease, to obtain eluate I; the anion exchange chromatographic column is a CaptoDEAE anion exchange chromatographic column;
(2) purifying the urofollitropin intermediate I by cation exchange chromatography to obtain a urofollitropin intermediate II; the method comprises the following steps: loading the solution of the urofollitropin intermediate I to a balanced cation exchange chromatographic column; washing with 0.1M acetic acid-sodium acetate (HAc-NaAc) solution with pH of 4.0-5.5, and detecting absorbance of the washing solution at 280nm until absorbance does not decrease; eluting with 0.1M HAc-NaAc solution containing 0.1-0.5M NaCl and having pH of 4.0-5.5, and detecting the absorbance of the eluate at 280nm until the absorbance is not reduced, wherein the eluate is follicle stimulating hormone intermediate II; the cation exchange chromatographic column is a CM-Sepharose cation exchange chromatographic column;
(3) purifying the urofollitropin intermediate II by dye affinity chromatography to obtain a urofollitropin intermediate III; the method comprises the following steps: loading the solution of the urofollitropin intermediate II to a well-balanced dye affinity chromatographic column; washing with 0.1M NaAc solution with pH of 6.0-9.0, and detecting absorbance of the washing solution at 280nm until absorbance does not decrease; eluting with 0.02M PB solution containing 0.8-2.5M KCl with pH of 8.0-10.0, and detecting the absorbance of the eluate at 280nm until the absorbance does not decrease, to obtain eluate as intermediate III of urofollitropin; the dye affinity chromatographic column is a Capto Blue high sub dye affinity chromatographic column;
(4) carrying out hydrophobic chromatography purification on the urofollitropin intermediate III to obtain a high-purity urofollitropin solution; the method comprises the following steps: loading the solution of the urofollitropin intermediate III to a well-balanced hydrophobic chromatographic column; using a pH of 7.0-9.0 solution containing 1.0-1.5M (NH)4)2SO4Washing with 0.02M PB solution, and detecting the light absorption value of the washing solution at 280nm until the light absorption value does not decrease; further adding 0.2-0.4M (NH) at pH 7.0-9.04)2SO40.02M PB solution and detection of the eluate at 280nmThe light absorption value is not reduced until the light absorption value is not reduced any more, and the obtained eluent is the solution of the high-purity urofollitropin; the hydrophobic chromatographic column is Phenyl-650M chromatographic column.
2. The method for preparing high purity urofollitropin according to claim 1, further comprising subjecting the urofollitropin solution obtained in step (4) to ultrafiltration, and replacing the buffer solution with a disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) buffer solution, wherein the pH of PB is 5.5 to 7.0.
3. The method for preparing high-purity urofollitropin according to claim 1, further comprising mixing the urofollitropin solution with a lyoprotectant, wherein the lyoprotectant is lactose, and the addition amount of the lyoprotectant is 3% -6% g/ml.
4. The method for preparing urofollitropin with high purity according to claim 1, wherein 95% ethanol is added to the product obtained in the step (1) or (2) or (3) for precipitation; then dehydrated by absolute ethyl alcohol and dried.
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| CN102448979A (en) * | 2009-04-01 | 2012-05-09 | 拜奥吉耐里克斯股份公司 | Method for purifying recombinant FSH |
| CN102924588A (en) * | 2012-10-26 | 2013-02-13 | 日照新康生物科技有限公司 | Preparation method of high-specific-activity urofollitropin |
| CN103059125A (en) * | 2012-12-27 | 2013-04-24 | 浙江海正药业股份有限公司 | Purification method for recombinant human follicle-stimulating hormone |
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| CN102448979A (en) * | 2009-04-01 | 2012-05-09 | 拜奥吉耐里克斯股份公司 | Method for purifying recombinant FSH |
| CN102924588A (en) * | 2012-10-26 | 2013-02-13 | 日照新康生物科技有限公司 | Preparation method of high-specific-activity urofollitropin |
| CN103059125A (en) * | 2012-12-27 | 2013-04-24 | 浙江海正药业股份有限公司 | Purification method for recombinant human follicle-stimulating hormone |
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| Title |
|---|
| 采用染料亲和层析的方法纯化卵泡刺激素;刘志勇等;《江西科学》;20040215;第22卷(第1期);摘要，第11页左栏第1段至右栏第1段，第12页左栏最后一段 * |
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