CN106117342A - A kind of preparation method of high-purity Urofollitropin - Google Patents
A kind of preparation method of high-purity Urofollitropin Download PDFInfo
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- CN106117342A CN106117342A CN201610528438.6A CN201610528438A CN106117342A CN 106117342 A CN106117342 A CN 106117342A CN 201610528438 A CN201610528438 A CN 201610528438A CN 106117342 A CN106117342 A CN 106117342A
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- urofollitropin
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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 77
- 108010047196 Urofollitropin Proteins 0.000 title claims abstract description 77
- 229960004371 urofollitropin Drugs 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 150000001875 compounds Chemical class 0.000 claims abstract description 14
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 13
- 229940088597 hormone Drugs 0.000 claims abstract description 11
- 239000005556 hormone Substances 0.000 claims abstract description 11
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 10
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 10
- 239000011265 semifinished product Substances 0.000 claims abstract description 7
- 210000002700 urine Anatomy 0.000 claims abstract description 7
- 108010057021 Menotropins Proteins 0.000 claims abstract description 6
- 238000011097 chromatography purification Methods 0.000 claims abstract description 3
- 230000031700 light absorption Effects 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 48
- 239000003480 eluent Substances 0.000 claims description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000000047 product Substances 0.000 claims description 21
- 238000011068 loading method Methods 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 16
- 238000004140 cleaning Methods 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229960004756 ethanol Drugs 0.000 claims description 8
- 230000033228 biological regulation Effects 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 229920000936 Agarose Polymers 0.000 claims description 6
- 229920001503 Glucan Polymers 0.000 claims description 6
- 230000008033 biological extinction Effects 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 6
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 claims description 6
- 239000001913 cellulose Substances 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 229920001661 Chitosan Polymers 0.000 claims description 4
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 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 claims description 3
- 238000002086 displacement chromatography Methods 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 claims description 2
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical group OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 claims description 2
- 229920002319 Poly(methyl acrylate) Polymers 0.000 claims description 2
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- 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 claims description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims 3
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims 1
- 239000005977 Ethylene Substances 0.000 claims 1
- 229920006389 polyphenyl polymer Polymers 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 238000005571 anion exchange chromatography Methods 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 abstract description 4
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 abstract description 4
- 229940028334 follicle stimulating hormone Drugs 0.000 abstract description 4
- 108010073521 Luteinizing Hormone Proteins 0.000 abstract description 3
- 102000009151 Luteinizing Hormone Human genes 0.000 abstract description 3
- 230000001294 luteotrophic effect Effects 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 22
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000000975 dye Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000018044 dehydration Effects 0.000 description 4
- 238000006297 dehydration reaction Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000006920 protein precipitation Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
- 108010076723 adducin Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- PZZYQPZGQPZBDN-UHFFFAOYSA-N aluminium silicate Chemical compound O=[Al]O[Si](=O)O[Al]=O PZZYQPZGQPZBDN-UHFFFAOYSA-N 0.000 description 1
- 229910000323 aluminium silicate Inorganic materials 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 230000004185 hypothalamic-pituitary-gonadal axis Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- NCYVXEGFNDZQCU-UHFFFAOYSA-N nikethamide Chemical group CCN(CC)C(=O)C1=CC=CN=C1 NCYVXEGFNDZQCU-UHFFFAOYSA-N 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002863 seminiferous tubule Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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]
Landscapes
- 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 present invention provides a kind of high-purity Urofollitropin (urine Follicle Stimulating Hormone, u FSH) preparation method, this preparation method comprises the following steps: (1) human menopausal gonadotropin (Human Menopausal Gonadotropin, HMG) semifinished product is through anion-exchange chromatography purification, obtains Urofollitropin intermediate compound I；(2) Urofollitropin intermediate compound I is through cation-exchange chromatography purification, obtains Urofollitropin intermediate II；(3) Urofollitropin intermediate II is through dye affinity chromatography purification, obtains Urofollitropin intermediate III；(4) Urofollitropin intermediate III is through hydrophobic chromatography purification, obtains high-purity Urofollitropin.This method have easy and simple to handle, production cost is low, in the Urofollitropin obtained, follicule-stimulating hormone (FSH) (Follicle Stimulating Hormone, FSH) purity is higher, lutropin (Luteotropic Hormone, LH) content is lower, the advantage polluted without foreign protein.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the extraction preparation method of a kind of Urofollitropin.
Background technology
Urofollitropin (urine Follicle Stimulating Hormone, u-FSH) is to be mainly composed of follicle
The compositions of stimulin (Follicle Stimulating Hormone, FSH), in high-purity Urofollitropin, FSH content can
To reach more than 95%, it is practically free of lutropin (Luteotropic Hormone, LH).FSH is a kind of glycoprotein hormones,
Molecular weight is about 30KD.FSH can promote granular cell hypertrophy, stimulates Steroidgenesis, regulates growth and the maturation of gametid,
It it is one of major hormone in hypothalamic pituitary gonadal axis.FSH has regulation and control menstrual cycle, ovulates and regulate male the mankind
The effect of testicular function, commonly uses its treatment infertility clinically and male's gonadotrophin secretion is not enough, Seminiferous tubule defect
Deng.
In the hypophysis cerebri of people and menopause with postclimacteric Women's Urine, both content is higher, is the most also to extract FSH
Main source.For producing raw material human menopausal gonadotropin (the Human Menopausal of u-FSH
Gonadotropin, HMG) mainly comprise follicule-stimulating hormone (FSH) and lutropin (Luteotropic Hormone, LH).FSH with
The ratio of LH titer is about 1:1.Preparation high-purity Urofollitropin needs to remove the LH in HMG and other urine proteins, protects
Stay the FSH of high-purity single-activity component.
At present, external highly purified u-FSH production technology is mainly grasped by Serono company, and its technological process is first
With ethanolic extraction HMG crude product, then adsorb through aluminium silicate, then through anion-exchange chromatography, re-use anti-FSH monoclonal antibody
Chromatographic column capture FSH, obtains high-purity u-FSH finally by reversed phase chromatography.Domestic highly purified u-FSH is mainly by beautiful pearl and sky
Wei Liang company produces, and all employs the absorption of anti-LH antibody and remove the processing step of LH in its production technology.Utilize antigen-antibody
Specific reaction removes LH, has purification efficiency advantages of higher.But current most of antibody is Mus source or rabbit source, and itself belongs to
Protein, in chromatography process, the antibody being coupled in substrate comes off and can cause the pollution of heterologous protein, affects product quality.
Meanwhile, antibody mediated immunity affinity column is expensive, and service life is short, safeguards and industry's enlarging production difficulty, and these deficiencies are all
Seriously hinder its application.Therefore, low content LH and high-purity can be prepared in the urgent need to developing one in this area
Easy and simple to handle, the production technology that production cost is low of FSH product.Inventor finds that the hydrophobicity of LH is better than FSH, can be by hydrophobic
Chromatographic technique is isolated, and hydrophobic chromatography therefore can be used to replace antibody mediated immunity affinity chromatograph, to solve the problems referred to above.
Summary of the invention
For the deficiency of process above, it is an object of the invention to provide the preparation method of a kind of Urofollitropin, with existing
Technique is compared, and this preparation method is easy and simple to handle, production cost is low, it is thus achieved that Urofollitropin purity higher, without foreign protein
Pollute.FSH specific activity is more than 10000IU/mg protein, and LH:FSH titer is less than 1:100.
The preparation method of the Urofollitropin that the present invention provides comprises the following steps:
(1) human menopausal gonadotropin's semifinished product is through anion-exchange chromatography purification, obtains Urofollitropin intermediate compound I；
(2) Urofollitropin intermediate compound I is through cation-exchange chromatography purification, obtains Urofollitropin intermediate II；
(3) Urofollitropin intermediate II is through dye affinity chromatography purification, obtains Urofollitropin intermediate III；
(4) Urofollitropin intermediate III is through hydrophobic chromatography purification, obtains the solution of high-purity Urofollitropin；
Preparation method in accordance with the above, it is characterised in that described preparation method also includes step (4) gained urine is promoted ovum
Bubble element carries out ultrafiltration and changes salt, and changing solution used by salt is 0.01M PB buffer solution, it is preferable that its pH is 5.5-7.0.
Preferably, above-mentioned preparation method also includes mixing step (4) gained Urofollitropin solution with freeze drying protectant
Rear lyophilizing, freeze drying protectant is lactose, it is preferable that its addition is 3%-6%(g/ml).
Preferably, described in above-mentioned preparation method, anion-exchange chromatography filler aglucon is TEAE, diethyl
Amino-ethyl, amino-ethyl or three ethanol base amino, substrate is cellulose, glucosan, agarose, styrene；Described cation
Displacement chromatography filler aglucon is carboxymethyl, sulfonic group, phosphate, phosphorous acidic group, phenolic group, and substrate is cellulose, glucosan, agar
Sugar, polystyrene；Described dye affinity chromatography filler aglucon is Cibacron Blue, Red, Green, Orange, and substrate is fine jade
Lipolysaccharide, glucosan or chitosan；Described hydrophobic chromatography filler is the vinyl alcohol with butyl, octyl group, phenyl or neopentyl as aglucon
With methacrylate copolymer, agarose, cellulose, polystyrene, polymethyl acrylate or chitosan.
Specifically, described step (1) comprises the following steps:
With water dissolution HMG semifinished product, the pH value of regulation solution to 5.5-8.0；By HMG solution of crude product loading to having balanced
Anion-exchange chromatography post；0.02M PB solution washing with pH value as 5.5-8.0, and detect cleaning mixture at 280nm
Light absorption value, until light absorption value no longer declines；The 0.02M PB solution containing 0.05-0.2M NaCl with pH value as 5.5-8.0 is washed
De-, and detect eluent light absorption value at 280nm, until light absorption value no longer declines, gained eluent is Urofollitropin
Intermediate compound I；
Specifically, described step (2) comprises the following steps:
By the solution loading of follicule-stimulating hormone (FSH) intermediate compound I to the cation-exchange chromatography post balanced；With pH value as 4.0-
The 0.1M HAc-NaAc solution washing of 5.5, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；
Again with the 0.1M HAc-NaAc eluant solution containing 0.1-0. 5M NaCl that pH value is 4.0-5.5, and detect eluent and exist
Light absorption value at 280nm, until light absorption value no longer declines, gained eluent is Urofollitropin intermediate II；
Specifically, described step (3) comprises the following steps:
By the solution loading of follicule-stimulating hormone (FSH) intermediate II to the Cibacron Blue dye affinity chromatography post balanced；With
PH value is the 0.1M NaAc solution washing of 6.0-9.0, and detects cleaning mixture light absorption value at 280nm, until light absorption value is not
Decline again；The 0.02M PB eluant solution containing 0.8-2.5M KCl with pH value as 8.0-10.0 again, and detect eluent and exist
Light absorption value at 280nm, until light absorption value no longer declines, gained eluent is Urofollitropin intermediate III；
Specifically, described step (4) comprises the following steps:
By the solution loading of follicule-stimulating hormone (FSH) intermediate III to the hydrophobic chromatography post balanced；It is 7.0-9.0's with pH value
0.02M PB solution washing containing 1.0-1.5M ammonium sulfate, and detect cleaning mixture light absorption value at 280nm, until extinction
Value no longer declines；The 0.02M PB eluant solution containing 0.2-0.4M ammonium sulfate with pH value as 7.0-9.0 again, and detect
Eluent light absorption value at 280nm, until light absorption value no longer declines, gained eluent is the molten of high-purity Urofollitropin
Liquid；
The present invention, with HMG as initiation material, uses anion-exchange chromatography, cation-exchange chromatography, dye affinity chromatography successively
And hydrophobic chromatography technique, extraction purification prepares high-purity FSH.Wherein, utilize the character that protein both sexes ionize, by changing
PH value of solution, makes different electric charges on protein belt, and through anion-exchange chromatography and cation-exchange chromatography, FSH purification can
Reach more than 50 times.Utilize FSH can the character of absorption affine with dyestuff, remove impurity protein further, make FSH purification reach
More than 150 times.Finally utilize LH Yu FSH hydrophobic difference, remove LH, it is thus achieved that high-purity Urofollitropin being practically free of LH.
Accompanying drawing explanation
Fig. 1 is the process chart of the present invention.
Fig. 2 is the HPLC collection of illustrative plates of the high-purity Urofollitropin finished product of embodiment 1 preparation.
Fig. 3 is the electrophoresis pattern of the FSH implementing row 1 and embodiment 2 preparation.Wherein, 1: embodiment 1 prepares finished product；2: real
Execute example 1 and prepare finished product 5% own control；3: embodiment 2 prepares finished product 5% own control；4: embodiment 2 prepares finished product；
Fig. 4 is the HPLC collection of illustrative plates of the high-purity Urofollitropin finished product of embodiment 2 preparation.
Detailed description of the invention
The mensuration of biological value, specific activity and the purity that the present invention relates to and computational methods.
Estimation of biological potency:
FSH, LH estimation of biological potency is according to Pharmacopoeia of the People's Republic of China version four general rules 1216 and 1217 detection in 2015.
Specific activity measures:
Precision weighs this product about 10mg, the accurate water 1ml that adds and makes dissolving；Another precision weighs bovine serum albumin reference substance, adds water
Dissolve and make the solution containing 1.0mg, 0.8mg, 0.6mg, 0.4mg, 0.2mg, 0mg in every 1ml respectively, according to UV-vis spectroscopy
Photometry (Pharmacopoeia of the People's Republic of China four general rules 0401 of version in 2015), measures extinction respectively at the wavelength of 280nm
Degree, with the concentration of bovine serum albumin solution as abscissa, the absorbance at 280nm is that vertical coordinate draws standard curve, extinction
Degree and concentration relationship should be linearly.From standard curve, try to achieve the protein concentration of need testing solution, be calculated as follows than work:
HPLC purity testing:
Take this product appropriate, add water and make in every 1ml the solution containing about 500IU FSH as need testing solution.According to " the China people are altogether
With state's pharmacopeia " four the general rule 0512 high performance liquid chromatography tests of 2015 years versions.Use gel chromatographic columns Superdex 75；With
Phosphate buffer (taking 85%H3PO46.74ml, add water 800ml, then adds Na2SO4 14.2g, adjusts pH to 6.7 with 50%NaOH,
It is 1000ml with water constant volume)；Column temperature 30 DEG C；Flow velocity 0.5ml/min；Detection wavelength 214nm；Sample size 100 μ l.
The relevant substance-measuring of electrophoresis:
According to (the Pharmacopoeia of the People's Republic of China four general rule 0541 the 5th method SDS-polyacrylamide gel electrophoresis of version in 2015
Method) check.Need testing solution concentration is 30000IU FSH/ml, diluted 20 times as contrast solution, applied sample amount is 10 μ l.
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for explaining
The bright present invention rather than in order to limit the scope of the present invention.
Embodiment 1
As it is shown in figure 1, by HMG semifinished product (our company produces, lot number Z0203150901) as initiation material, its FSH biology is imitated
Valency be 30IU/mg, LH biological value be 28IU/mg, FSH specific activity be 43IU/mg protein.
1. Capto DEAE anion-exchange chromatography
With 200ml water dissolution 8.0g HMG semifinished product, this solution loading, to 7.5, to column volume is by the pH value of regulation solution
The Capto DEAE chromatographic column (purchased from GE company) of 250ml, chromatographic column balances with balance liquid (0.02M PB, pH7.5) in advance；
With the albumen that balance liquid washing is unadsorbed after end of the sample, and detect cleaning mixture light absorption value at 280nm, until light absorption value is not
Decline again；Again with eluent (0.02M PB+0.15MNaCl) eluting FSH, and detect eluent light absorption value at 280nm, directly
No longer decline to light absorption value, collect eluting effluent 1.0L altogether, be concentrated into about 100ml with the ultrafilter membrane that molecular cut off is 6KD,
It is 85% to concentration of alcohol that addition is cooled to 95% ethanol of-20 DEG C in advance, stands overnight protein precipitation after stirring, and is centrifuged next day and receives
Collection precipitation, adds dehydrated alcohol 3 final vacuums of dehydration and is dried, obtain Urofollitropin intermediate compound I 0.73g.
2. CM-Sepharose cation-exchange chromatography
Urofollitropin intermediate compound I 0.7g with 30ml balance liquid (0.1M HAc-NaAc, pH4.5) dissolve after loading to cylinder
Amassing the CM-Sepharose chromatographic column (purchased from GE company) for 20ml, chromatographic column balances with balance liquid in advance；End of the sample
Afterwards with the albumen that balance liquid washing is unadsorbed, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；
Again with eluent (0.1M HAc-NaAc+0.5M NaCl, pH4.5) eluting FSH, and detect eluent extinction at 280nm
Value, until light absorption value no longer declines, collects eluting effluent 100ml altogether, is Urofollitropin intermediate II.
3. Capto Blue (high sub) dye affinity chromatography
By consistent with balance liquid (0.1M NaAc, pH7.0) to Urofollitropin intermediate II 98ml regulation pH and electrical conductivity, loading
To Capto Blue (high sub) chromatographic column (purchased from GE company) that the column volume balanced is 15ml；After end of the sample
Wash unadsorbed albumen with balance liquid, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；Again
With eluent (0.02M PB+2.0M KCl, pH 9.0) eluting FSH, and detect eluent light absorption value at 280nm, until
Light absorption value no longer declines, and collects eluting effluent 70ml altogether, is Urofollitropin intermediate III.
4. Phenyl-650M hydrophobic chromatography
Solid ammonium sulfate, stirring and dissolving, regulation pH and electrical conductivity and balance is added in Urofollitropin intermediate III 68ml
Liquid (0.02M PB+1.4M (NH4)2SO4, pH7.0) and consistent, loading to the column volume balanced is the Phenyl of 10ml
Phenyl-650M chromatographic column (purchased from TOSOH company)；With the albumen that balance liquid washing is unadsorbed after end of the sample, and detection is washed
Wash liquid light absorption value at 280nm, until light absorption value no longer declines；Again with eluent (0.02M PB+0.3M (NH4)2SO4,
PH7.0) eluting FSH, and detect eluent light absorption value at 280nm, until light absorption value no longer declines, collect eluting altogether and flow out
Liquid 40ml, is high-purity Urofollitropin solution.
After being concentrated by ultrafiltration by the ultrafilter membrane that high-purity Urofollitropin solution molecular cut off is 6KD, use 0.01M PB
Buffer solution (pH 6.0) changes salt 3 times, according to 4%(w/v) ratio add lactose and be configured to lyophilizing stock solution, after lyophilization
To 0.91g high-purity Urofollitropin finished product, finished product relevant parameter is shown in Table 1, and the HPLC of FSH and electrophoresis purity collection of illustrative plates are respectively such as figure
Shown in 2 and Fig. 3.
The high-purity Urofollitropin product parameters of table 1 embodiment 1 preparation
Embodiment 2
Low-purity HMG(our company is produced, lot number Z0203150901) as initiation material, its FSH biological value is 30IU/
Mg, LH biological value be 28IU/mg, FSH specific activity be 43IU/mg protein.
1. Capto DEAE anion-exchange chromatography
With 500ml water dissolution 20g HMG, this solution loading to column volume, to 7.5, is 630ml's by the pH value of regulation solution 1
Capto DEAE chromatographic column (purchased from GE company), chromatographic column balances with balance liquid (0.02M PB, pH7.5) in advance；Loading is tied
The Shu Houyong balance liquid unadsorbed albumen of washing, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer under
Fall；Again with eluent (0.02M PB+0.15M NaCl) eluting FSH, and detect eluent light absorption value at 280nm, until
Light absorption value no longer declines, and collects eluting effluent 2.5L altogether, is concentrated into about 100ml with the ultrafilter membrane that molecular cut off is 6KD, adds
Entering pre-95% ethanol being cooled to-20 DEG C is 85% to concentration of alcohol, stands overnight protein precipitation after stirring, centrifugal collection next day
Precipitation, adds dehydrated alcohol 3 final vacuums of dehydration and is dried, obtain Urofollitropin intermediate compound I 1.85g.
2. CM-Sepharose cation-exchange chromatography
Urofollitropin intermediate compound I 1.8g with 80ml balance liquid (0.1M HAc-NaAc, pH4.5) dissolve after loading to cylinder
Amassing the CM-Sepharose chromatographic column (purchased from GE company) for 50ml, chromatographic column balances with balance liquid in advance；End of the sample
Afterwards with the albumen that balance liquid washing is unadsorbed, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；
Again with eluent (0.1M HAc-NaAc+0.5M NaCl, pH4.5) eluting FSH, and detect eluent extinction at 280nm
Value, until light absorption value no longer declines, collects eluting effluent 250ml altogether, is Urofollitropin intermediate II；With retaining molecule
The ultrafilter membrane that amount is 6KD is concentrated into about 100ml, and it is 85% that addition is cooled to 95% ethanol to the concentration of alcohol of-20 DEG C in advance, stirs
After stand overnight protein precipitation, centrifugal collecting precipitation, added dehydrated alcohol 3 final vacuums of dehydration and was dried, obtain urine and promote ovum next day
Bubble element intermediate II dry product 0.24g.
3. Capto Blue (high sub) dye affinity chromatography
Urofollitropin intermediate II dry product 0.21g balance liquid (0.1M NaAc, pH7.0) dissolves, and loading is to balancing
Good Capto Blue (high sub) chromatographic column (purchased from GE company) that column volume is 40ml；Balance liquid is used after end of the sample
Wash unadsorbed albumen, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；Use eluent again
(0.02M PB+2.0M KCl, pH 9.0) eluting FSH, and detect eluent light absorption value at 280nm, until light absorption value is not
Decline again, collect eluting effluent 174ml altogether, be Urofollitropin intermediate III；Add 95% ethanol being cooled to-20 DEG C in advance
Being 85% to concentration of alcohol, stand overnight protein precipitation after stirring, next day, centrifugal collecting precipitation, added dehydrated alcohol dehydration 3
Secondary final vacuum is dried, and obtains Urofollitropin intermediate II dry product 0.13g.
4. Phenyl-650M hydrophobic chromatography
Urofollitropin intermediate II dry product 0.11g balance liquid (0.02M PB+1.4M (NH4)2SO4, pH7.0) dissolve,
Loading is to the Phenyl-650M chromatographic column (purchased from TOSOH company) that the column volume balanced is 25ml；With flat after end of the sample
Weighing apparatus liquid washes unadsorbed albumen, and detects cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；Use eluting again
Liquid (0.02M PB+0.3M (NH4)2SO4, pH7.0) and eluting FSH, and detect eluent light absorption value at 280nm, until inhaling
Light value no longer declines, and collects eluting effluent 94ml altogether, is high-purity Urofollitropin.
After the ultrafilter membrane that high-purity Urofollitropin molecular cut off is 6KD is concentrated by ultrafiltration, buffer with 0.01M PB
Solution (pH 6.0) replaces former buffer solution 3 times, according to 4%(w/v) ratio add lactose and be configured to lyophilizing stock solution, freezing dry
Obtaining 2.31g high-purity Urofollitropin finished product after dry, finished product relevant parameter is shown in Table 2, the electrophoresis of FSH and HPLC purity collection of illustrative plates
The most as shown in Figures 3 and 4.
The high-purity Urofollitropin product parameters of table 2 embodiment 2 preparation
。
Claims (9)
1. a preparation method for high-purity Urofollitropin, this preparation method comprises the following steps:
(1) human menopausal gonadotropin (Human Menopausal Gonadotropin, HMG) semifinished product is through anion
Displacement chromatography purification, obtains Urofollitropin intermediate compound I；
(2) Urofollitropin intermediate compound I is through cation-exchange chromatography purification, obtains Urofollitropin intermediate II；
(3) Urofollitropin intermediate II is through dye affinity chromatography purification, obtains Urofollitropin intermediate III；
(4) Urofollitropin intermediate III is through hydrophobic chromatography purification, obtains the solution of high-purity Urofollitropin.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described preparation method
Also include carrying out step (4) gained Urofollitropin solution ultrafiltration, then with disodium hydrogen phosphate-sodium dihydrogen phosphate (PB) buffer
Replace former buffer solution, it is preferable that the pH of PB is 5.5-7.0.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described preparation method
Lyophilizing after also including mixing Urofollitropin solution with freeze drying protectant, freeze drying protectant is lactose, it is preferable that its addition
For 3%-6%(g/ml).
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described anion is handed over
Changing chromatographic stuffing aglucon is TEAE, diethylamino ethyl, amino-ethyl or three ethanol base amino, and substrate is fine
Dimension element, glucosan, agarose, styrene；Described cation-exchange chromatography filler aglucon is carboxymethyl, sulfonic group, phosphate, Asia
Phosphate, phenolic group, substrate is cellulose, glucosan, agarose, polystyrene；Described dye affinity chromatography filler aglucon is
Cibacron Blue, Red, Green, Orange, substrate is agarose, glucosan or chitosan；Described hydrophobic chromatography filler is
Vinyl alcohol with butyl, octyl group, phenyl or neopentyl as aglucon and methacrylate copolymer, agarose, cellulose, polyphenyl
Ethylene, polymethyl acrylate or chitosan.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described step (1)
Comprise the following steps:
With water dissolution HMG semifinished product, the pH value of regulation solution to 5.5-8.0；By HMG solution loading to the anion balanced
Displacement chromatography post；0.02M PB solution washing with pH value as 5.5-8.0, and detect cleaning mixture light absorption value at 280nm,
Until light absorption value no longer declines；The 0.02M PB eluant solution containing 0.05-0.2M NaCl with pH value as 5.5-8.0, and examine
Surveying eluent light absorption value at 280nm, until light absorption value no longer declines, gained eluent is follicule-stimulating hormone (FSH) intermediate compound I.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described step (2)
Comprise the following steps:
By the solution loading of Urofollitropin intermediate compound I to the cation-exchange chromatography post balanced；With pH value as 4.0-
0.1M Acetic acid-sodium acetate (HAc-NaAc) the solution washing of 5.5, and detect cleaning mixture light absorption value at 280nm, until inhaling
Light value no longer declines；Again with the 0.1M HAc-NaAc eluant solution containing 0.1-0.5M NaCl that pH value is 4.0-5.5, and examine
Surveying eluent light absorption value at 280nm, until light absorption value no longer declines, gained eluent is Urofollitropin intermediate
II。
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described step (3)
Comprise the following steps:
By the solution loading of Urofollitropin intermediate II to the dye affinity chromatography post balanced；With pH value as 6.0-9.0
The washing of 0.1M NaAc solution, and detect cleaning mixture light absorption value at 280nm, until light absorption value no longer declines；Again with pH
Value is the 0.02M PB eluant solution containing 0.8-2.5M KCl of 8.0-10.0, and detects eluent extinction at 280nm
Value, until light absorption value no longer declines, gained eluent is Urofollitropin intermediate III.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that described step (4)
Comprise the following steps:
By the solution loading of Urofollitropin intermediate III to the hydrophobic chromatography post balanced；It is 7.0-9.0's with pH value
Containing 1.0-1.5M (NH4)2SO4The washing of 0.02M PB solution, and detect cleaning mixture light absorption value 280nm at, until suction
Light value no longer declines；Again with pH value as 7.0-9.0 containing 0.2-0.4M (NH4)2SO40.02M PB eluant solution, and
Detection eluent light absorption value at 280nm, until light absorption value no longer declines, gained eluent is high-purity urine and promotees follicle
The solution of element.
The preparation method of a kind of high-purity Urofollitropin, it is characterised in that to described step
(1) or add ethanol in (2) or (3) product of obtaining, such as 95% ethanol precipitates；Then it is dehydrated with dehydrated alcohol, then enters
Row is dried, such as, be vacuum dried.
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| CN107698676A (en) * | 2017-11-07 | 2018-02-16 | 江西浩然生物医药有限公司 | A kind of extraction preparation method of high-purity menotropins |
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