CN105085660A - Method for extracting chorionic gonadotrophin crude product from large-volume pregnant woman urine on basis of ultrafiltration concentration - Google Patents
Method for extracting chorionic gonadotrophin crude product from large-volume pregnant woman urine on basis of ultrafiltration concentration Download PDFInfo
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- CN105085660A CN105085660A CN201510514368.4A CN201510514368A CN105085660A CN 105085660 A CN105085660 A CN 105085660A CN 201510514368 A CN201510514368 A CN 201510514368A CN 105085660 A CN105085660 A CN 105085660A
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- Prior art keywords
- urine
- crude product
- chorionic gonadotrophin
- sodium benzoate
- pregnant woman
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- 210000002700 urine Anatomy 0.000 title claims abstract description 36
- 229960004407 chorionic gonadotrophin Drugs 0.000 title claims abstract description 33
- 239000012043 crude product Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000000108 ultra-filtration Methods 0.000 title claims abstract description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims abstract description 18
- 235000010234 sodium benzoate Nutrition 0.000 claims abstract description 18
- 239000004299 sodium benzoate Substances 0.000 claims abstract description 18
- 238000003756 stirring Methods 0.000 claims abstract description 17
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 238000003828 vacuum filtration Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 10
- 238000001556 precipitation Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 239000004744 fabric Substances 0.000 claims description 5
- 244000144992 flock Species 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 10
- 238000001179 sorption measurement Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract 3
- 230000002708 enhancing effect Effects 0.000 abstract 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 206010000242 Abortion threatened Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 208000008899 Habitual abortion Diseases 0.000 description 1
- 206010058359 Hypogonadism Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000005985 Threatened Abortion Diseases 0.000 description 1
- MWOBKFYERIDQSZ-UHFFFAOYSA-N benzene;sodium Chemical compound [Na].C1=CC=CC=C1 MWOBKFYERIDQSZ-UHFFFAOYSA-N 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 210000002863 seminiferous tubule Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000021595 spermatogenesis Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 239000002699 waste material Substances 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)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (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)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for extracting a chorionic gonadotrophin crude product from large-volume pregnant woman urine on the basis of ultrafiltration concentration, which comprises the following steps: a. collecting fresh pregnant woman urine, and carrying out ultrafiltration concentration; b. adding sodium benzoate into the urine concentrated solution, and dissolving by stirring; c. acidifying with hydrochloric acid to precipitate abundant white insoluble substances, filtering to collect the insoluble substances, and carrying out centrifugal drying; d. adding precooled alcohol into the insoluble substances while stirring to precipitate abundant flocculent precipitate, and standing; and e. siphoning the solution supernate, carrying out vacuum filtration, collecting the precipitate, dewatering and drying. Compared with the traditional crude product preparation process, the method for producing the human urine chorionic gonadotrophin crude product is implemented by carrying out sodium benzoate adsorption after the ultrafiltration concentration, thereby reducing the consumption of the sodium benzoate, hydrochloric acid and ethanol and lowering the production cost; and the increase of the HCG (human chorionic gonadotrophin) concentration in the concentrated urine is beneficial to the adsorptivity of the sodium benzoate for the HCG, thereby enhancing the adsorption efficiency and enhancing the activity yield to 80%.
Description
Technical field
The invention belongs to biotechnology and extract natural drug, be specifically related to a kind of method extracting chorionic gonadotrophin crude product based on ultrafiltration and concentration from large volume pregnant woman urine.
Background technology
Chorionic gonadotrophin (ChorionicGonadotrophin) is also known as human chorionic gonadotrophin (HumanChorionicGonadotrophin), be called for short HCG, nineteen twenty-seven is found from urine of pregnant women by Aschhein and Zondek, chorionic gonadotrophin is a kind of glycoprotein secreted by the trophocyte of placenta, in the urine being mainly present in pregnant woman and blood.
A kind of hormone that chorionic gonadotrophin is made up of the dimeric glycoprotein of α and β, its α subunit and β subunit are combined by the interaction of non covalent hydrophobic and ion, the molecular weight of chorionic gonadotrophin is approximately 32KD, the 25%-41% of its molecular weight comes from sugared side chain, and (conventional chorionic gonadotrophin is 25%-30%, the chorionic gonadotrophin of high-glycosylation is 35%-41%), α subunit has 92 amino-acid residues, and β subunit has 145 amino-acid residues.
Chorionic gonadotrophin is a kind of gonad-stimulating hormone of trophoblast secretion, and within 10-14 days after becoming pregnant, start secretion, in gestation initial 3 months, HCG level rises and doubles for every 2.2 days months, to maintain gestation.In non-pregnant woman urine, HCG is generally less than 25IU/L, and in pregnant 40 days urines, HCG content is greater than 5000IU/L.
Chorionic gonadotrophin impels follicle maturity and ovulation to female performance, and makes ruptured follicle change corpus luteum into, impels its secretion progestogen.To male, there is the effect of interstitial cell stimulating hormone(ICSH), the activity of convoluted seminiferous tubule function, particularly interstitial glands can be promoted, make it produce male sex hormone, impel sexual organ and secondary sex character to grow, ripe, impel testicular descent and promote spermatogenesis.Be mainly used in ovulation rate clinically, treat infertile, and First Trimester threatened abortion, habitual abortion and low short hypogonadism, recent research shows that chorionic gonadotrophin can be used for Therapeutic cancer etc.
The traditional preparation methods of chorionic gonadotrophin crude product directly adds Sodium Benzoate for collecting pregnant woman urine in urine, then adds hydrochloric acid reaction and generate phenylformic acid, the urine protein such as absorption chorionic gonadotrophin, then adds alcohol and make albumen precipitation, collects HCG precipitation, dry.The shortcoming of the method is that crude urine volume is too large, Sodium Benzoate adsorption rate is low, for ensureing that abundant absorption often adds excessive benzene sodium formiate (being generally about the 2-3% of crude urine liquid measure), and long reaction time needs whip attachment at least 2 hours, and in this process, HCG easily degrades; In crude urine, moisture is more, adjusts during pH and needs to waste a large amount of hydrochloric acid.
Method disclosed in CN103193880A, adopt ultrafiltration and concentration method urine to be concentrated into original weight amount 4-5%, direct acetone precipitation after adding a small amount of Calcium Chloride Powder Anhydrous, low to the deposition efficiency of target protein, product yield is only about 40%; In addition because foreign protein in urine is a lot, impurity protein various in urine easily precipitates by the method, and the product units activity obtained is low, is unfavorable for follow-up HCG purification process.
Summary of the invention
The object of the present invention is to provide a kind of method extracting chorionic gonadotrophin crude product based on ultrafiltration and concentration from large volume urine.By ultrafiltration and concentration, reduce the usage quantity of reagent, carry out adsorbing greatly promoting adsorption efficiency after liquid concentration simultaneously, improve the activity yield of product.
The present invention is achieved like this, and method steps is:
A. collect fresh pregnant woman urine, concentrate with molecular weight cut-off 10kDa ultra-filtration membrane after being cooled to temperature 2-8 DEG C, be concentrated into 10% of former volume of urine;
B., add the Sodium Benzoate of 6-8% to concentrated solution in a after, stir and dissolve completely to Sodium Benzoate for 15-30 minute;
C. add in b solution at hydrochloric acid adjusts pH to 3.8-4.0, temperature 2-8 DEG C and continue to stir 30-60 minute, separate out a large amount of white insolubles, solution becomes white suspension liquid; Pour in filter cloth bag by this suspension liquid, collecting by filtration white insolubles, dries;
D. stir in insolubles and add 2-8 DEG C of precooled edible ethanol (v/w95%) until alcohol final concentration is 80%, separate out a large amount of white flock precipitate in solution, at this being deposited in 2-8 DEG C, leave standstill 8-12 hour;
E. siphon removes the supernatant liquor in e, vacuum filtration collecting precipitation, and dewaters 2-3 time with acetone, and drying under reduced pressure obtains people and urinates chorionic gonadotrophin crude product.
Technique effect of the present invention is: method of the present invention comparatively traditional technology ratio have concentrated after extraction efficiency high; Sodium Benzoate and hydrochloric acid consumption are only 1/3rd of former consumption, reduce production cost; After urine is concentrated, moisture greatly reduces, and the liquid medium staple property urged fibroin concentration increases, and is more conducive to chorionic gonadotrophin and is combined with benzene first sodium, Product Activity yield improves nearly one times, up to more than 80%, obtain crude product units activity and be greater than 10IU/mg, specific activity is greater than 15IU/mg.pr.
Embodiment
Embodiment 1 one kinds extracts the method for chorionic gonadotrophin crude product from large volume pregnant woman urine based on ultrafiltration and concentration, step is as follows:
Collect 1000kg healthy pregnant women urine, concentrate with molecular weight cut-off 10kDa ultra-filtration membrane after being cooled to temperature 2-8 DEG C, be concentrated into 100kg and urinate concentrated solution; After adding the Sodium Benzoate of 6kg wherein, stir and dissolve completely to Sodium Benzoate for 15 minutes; Add at hydrochloric acid adjusts pH to 3.9, temperature 2-8 DEG C and continue stirring 30 minutes, separate out a large amount of white insolubles, solution becomes white suspension liquid; Pour in filter cloth bag by this suspension liquid, collecting by filtration white insolubles, dries; Stir in insolubles and add 2-8 DEG C of precooled edible ethanol (v/w95%) until alcohol final concentration is 80%, in solution, separate out a large amount of white flock precipitate, at this being deposited in 2-8 DEG C, leave standstill 9 hours; The supernatant liquor in e is removed in siphon, vacuum filtration collecting precipitation, and dewaters 2-3 time with acetone, and drying under reduced pressure obtains people and urinates chorionic gonadotrophin crude product 882g.
Embodiment 2 one kinds extracts the method for chorionic gonadotrophin crude product from large volume pregnant woman urine based on ultrafiltration and concentration, step is as follows:
Collect 1200kg healthy pregnant women urine, concentrate with molecular weight cut-off 10kDa ultra-filtration membrane after being cooled to temperature 2-8 DEG C, be concentrated into 120kg and urinate concentrated solution; After adding the Sodium Benzoate of 8.4kg wherein, stir and dissolve completely to Sodium Benzoate for 15 minutes; Add at hydrochloric acid adjusts pH to 3.9, temperature 2-8 DEG C and continue stirring 45 minutes, separate out a large amount of white insolubles, solution becomes white suspension liquid; Pour in filter cloth bag by this suspension liquid, collecting by filtration white insolubles, dries; Stir in insolubles and add 2-8 DEG C of precooled edible ethanol (v/w95%) until alcohol final concentration is 80%, in solution, separate out a large amount of white flock precipitate, at this being deposited in 2-8 DEG C, leave standstill 10 hours; The supernatant liquor in e is removed in siphon, vacuum filtration collecting precipitation, and dewaters 2-3 time with acetone, and drying under reduced pressure obtains people and urinates chorionic gonadotrophin crude product 1160g.
Embodiment 3 one kinds extracts the method for chorionic gonadotrophin crude product from large volume pregnant woman urine based on ultrafiltration and concentration, step is as follows:
Collect 1500kg healthy pregnant women urine, concentrate with molecular weight cut-off 10kDa ultra-filtration membrane after being cooled to temperature 2-8 DEG C, be concentrated into 150kg and urinate concentrated solution; After adding the Sodium Benzoate of 12kg wherein, stir and dissolve completely to Sodium Benzoate for 15 minutes; Add at hydrochloric acid adjusts pH to 3.9, temperature 2-8 DEG C and continue stirring 60 minutes, separate out a large amount of white insolubles, solution becomes white suspension liquid; Pour in filter cloth bag by this suspension liquid, collecting by filtration white insolubles, dries; Stir in insolubles and add 2-8 DEG C of precooled edible ethanol (v/w95%) until alcohol final concentration is 80%, in solution, separate out a large amount of white flock precipitate, at this being deposited in 2-8 DEG C, leave standstill 12 hours; The supernatant liquor in e is removed in siphon, vacuum filtration collecting precipitation, and dewaters 2-3 time with acetone, and drying under reduced pressure obtains people and urinates chorionic gonadotrophin crude product 1455g.
Adopt the method in above embodiment to prepare people and urinate chorionic gonadotrophin crude product, yield and Activity determination result as follows:
Claims (4)
1. from large volume pregnant woman urine, extract a method for chorionic gonadotrophin crude product based on ultrafiltration and concentration, it is characterized in that method steps is:
A. collect fresh pregnant woman urine, concentrate with molecular weight cut-off 10kDa ultra-filtration membrane after being cooled to temperature 2-8 DEG C;
B., after adding Sodium Benzoate to concentrated solution in a, stir and dissolve completely to Sodium Benzoate for 15-30 minute;
C. add in b solution at hydrochloric acid adjusts pH to 3.8-4.0, temperature 2-8 DEG C and continue to stir 30-60 minute, separate out a large amount of insolubles and become white suspension liquid; Pour in filter cloth bag by above-mentioned suspension liquid, collecting by filtration white insolubles, dries;
D. in insolubles, stir that to add 2-8 DEG C of precooled weightmeasurement ratio be 95% edible ethanol, until alcohol final concentration is 80%, in solution, separate out a large amount of white flock precipitate, at this being deposited in 2-8 DEG C, leave standstill 8-12 hour;
E. siphon removes the supernatant liquor in e, vacuum filtration collecting precipitation, and dewaters 2-3 time with acetone, drying under reduced pressure.
2. people as claimed in claim 1 urinates the preparation method of chorionic gonadotrophin crude product, and it is characterized in that, in described step a, ultra-filtration membrane molecular weight cut-off is 10kDa.
3. people as claimed in claim 1 urinates the preparation method of chorionic gonadotrophin crude product, it is characterized in that, in described step a, the volume of the urine concentrated solution after concentrated is 10% of crude urine volume.
4. people as claimed in claim 1 urinates the preparation method of chorionic gonadotrophin crude product, it is characterized in that, in described step b, the amount adding Sodium Benzoate to urine concentrated solution is urinate the 6-8% of concentrated solution.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510514368.4A CN105085660A (en) | 2015-08-21 | 2015-08-21 | Method for extracting chorionic gonadotrophin crude product from large-volume pregnant woman urine on basis of ultrafiltration concentration |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510514368.4A CN105085660A (en) | 2015-08-21 | 2015-08-21 | Method for extracting chorionic gonadotrophin crude product from large-volume pregnant woman urine on basis of ultrafiltration concentration |
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| CN105085660A true CN105085660A (en) | 2015-11-25 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58157720A (en) * | 1982-03-15 | 1983-09-19 | Nippon Chem Res Kk | Preparation of placental gonadotropic hormone in high purity |
| CN101792481A (en) * | 2010-03-12 | 2010-08-04 | 丽珠医药集团股份有限公司 | Purification method of human chorionic gonadotropin (HCG) |
| CN103193880A (en) * | 2013-04-09 | 2013-07-10 | 邹平县滨渤生物科技有限公司 | Preparation method of HCG (Human Chorionic Gonadotropin) crude product |
| CN103739700A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Method using sodium benzoate for extraction of chorionic gonadotrophin |
-
2015
- 2015-08-21 CN CN201510514368.4A patent/CN105085660A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58157720A (en) * | 1982-03-15 | 1983-09-19 | Nippon Chem Res Kk | Preparation of placental gonadotropic hormone in high purity |
| CN101792481A (en) * | 2010-03-12 | 2010-08-04 | 丽珠医药集团股份有限公司 | Purification method of human chorionic gonadotropin (HCG) |
| CN103193880A (en) * | 2013-04-09 | 2013-07-10 | 邹平县滨渤生物科技有限公司 | Preparation method of HCG (Human Chorionic Gonadotropin) crude product |
| CN103739700A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Method using sodium benzoate for extraction of chorionic gonadotrophin |
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