CN111233999B - Method for extracting human chorionic gonadotrophin from human urine - Google Patents
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- CNLSPQQMAJCIFB-UHFFFAOYSA-N benzoic acid;ethanol Chemical compound CCO.OC(=O)C1=CC=CC=C1 CNLSPQQMAJCIFB-UHFFFAOYSA-N 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
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- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
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- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
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- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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Classifications
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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)
- 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)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for extracting human chorionic gonadotrophin from human urine, and relates to the technical field of biological pharmacy. The technical key points are as follows: adding anion exchange resin as adsorbent into pregnant woman urine, stirring, filtering, washing, eluting with ammonium acetate solution by chromatography, precipitating the eluate, collecting precipitate, and measuring titer to obtain human chorionic gonadotrophin. The method is used for extracting the human chorionic gonadotrophin in human urine, so that the purification efficiency is increased, the production cost is saved, and the pollution emission is reduced.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a method for extracting human chorionic gonadotrophin from human urine.
Background
Human Chorionic Gonadotrophin (HCG) is a glycoprotein secreted by trophoblast cells of the placenta and is composed of glycoproteins of alpha and beta dimers. Human Chorionic Gonadotrophin (HCG) is synthesized by syntrophic cells. Mature females form embryos after implantation by movement of fertilized ova into the uterine cavity, and during development to become fetuses, placental syngeneic trophoblast cells produce large amounts of Human Chorionic Gonadotrophin (HCG) which can be excreted into the urine by the blood circulation of the pregnant female. When gestation is carried out for 1-2.5 weeks, HCG level in serum and urine can rise rapidly, and the pregnancy reaches the peak at 8 weeks, and the pregnancy starts to fall to the medium level from 4 months and is maintained until the end of gestation.
At present, human Chorionic Gonadotrophin (HCG) is extracted from urine of pregnant women by people at home and abroad. Human Chorionic Gonadotrophin (HCG) has a medical important application, and the demand of Human Chorionic Gonadotrophin (HCG) is large in the international medical market. Human Chorionic Gonadotrophin (HCG) is a glycoprotein hormone having a molecular weight of about 30000-40000, which stimulates gonadal activity, promotes egg maturation and ovulation in females, promotes fine tubular function and interstitial cell activity in males, increases production of male elements, promotes testicular descent, and can treat neurasthenia, habitual abortion, severe uterine bleeding, amenorrhea, infertility, etc., and can be used for preparing gestational and radioimmunoassay reagents for pregnancy test and diagnosis of choriocarcinoma.
The invention discloses a high-purity Human Chorionic Gonadotrophin (HCG) and a preparation method thereof in Chinese patent application with application publication number of CN1302818A, and the invention relates to a separation and purification technology of the Human Chorionic Gonadotrophin (HCG) extracted from urine of pregnant women. Human Chorionic Gonadotrophin (HCG) is contained in urine of pregnant women, and crude products of the Human Chorionic Gonadotrophin (HCG) are prepared by adsorbing sodium benzoate or kaolin and ethanol.
The preparation method is characterized in that a high-salt extraction and low-salt reverse extraction technology of sodium acetate is used for obtaining a Human Chorionic Gonadotrophin (HCG) intermediate, the high-purity Human Chorionic Gonadotrophin (HCG) is prepared through a separation and purification technology of SP-sephadex, and a medical fine raw material of the Human Chorionic Gonadotrophin (HCG) for injection is prepared through aseptic pyrogen-free treatment.
1. And (3) manufacturing a crude product:
adsorbing, eluting, and drying, wherein the pregnant woman urine is fed according to the mixing ratio of benzoic acid ethanol saturated solution to ethanol=1:0.75:5.
Adjusting pH of urine to 4-5 with hydrochloric acid, adding saturated solution of benzoic acid and ethanol, stirring for 1 hr, standing for 2-3 hr, filtering, and removing supernatant to obtain adsorbate. Elution with 95% ethanol was carried out with stirring until the benzoic acid was completely dissolved, i.e. a precipitate formed. Standing overnight, centrifuging, washing the precipitate with 95% ethanol and acetone, and drying to obtain crude product with titer of more than 10iu per mg unit, and HCG content of more than 1000 ten thousand units per kg crude product.
2. HCG raw material for injection:
1) Intermediate (500 u/mg. Shi) manufacturing section;
2) A pure HCG manufacturing section;
3) HCG manufacturing section for injection.
3. The preparation method of the HCG intermediate is a high-salt extraction method and a low-salt back extraction method, the quality of the intermediate product can reach 500iu/mg Shi 50iu, and the yield is more than 95%.
4. The HCG intermediate was purified using SP-sephadex c-50 and loaded and SP equilibrated with the wash prescription.
5. The purified refined product is subjected to secondary calcium phosphate to remove pyrogen, so that the endotoxin index of the product is stably controlled within the range specified by the injection, the purity of the product is improved by 15%, and the operation is Na 3 PO 4 CaAC (CaAC) 2 The concentration should be controlled at 0.1-0.3M, pH between 8.2-8.6, preferably 8.5, and the liquid temperature should be kept at 5 C.shi 1C.
The technical scheme has the following defects: the method has the advantages that the titer of the product obtained by the method is low, the used hydrochloric acid is a solvent with high volatility and corrosiveness, more pollution can be generated, the economic benefit is poor, and the environment is polluted. Therefore, a new solution is needed to solve the above-mentioned problems.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for extracting human chorionic gonadotrophin from human urine, which has the advantages of improving purification efficiency, saving production cost and reducing pollution emission.
In order to achieve the above purpose, the present invention provides the following technical solutions: a process for extracting human chorionic gonadotrophin from human urine includes such steps as adding anionic exchange resin as adsorbent to urine of pregnant woman, stirring, filtering, washing, eluting by ammonium acetate solution, depositing, collecting deposit and measuring titer.
By adopting the technical scheme, urine of pregnant women is collected, anion exchange resin is added into the urine of the pregnant women, then the urine of the pregnant women and the anion exchange resin are mixed and stirred, so that the anion exchange resin adsorbs human chorionic gonadotrophin in the urine, the adsorption function is to depend on Van der Waals attraction between the anion exchange resin and adsorbed molecules (adsorbates), and the adsorption function works through physical adsorption on the huge specific surface, so that organic compounds can be eluted and separated through a certain solvent according to adsorption force and molecular weight of the organic compounds, and different purposes such as separation, purification, impurity removal, concentration and the like are achieved. Ion exchange resins are a class of polymeric materials with ion exchange functionality. In the solution, the ion can exchange itself with the same number ion in the solution. Ion exchange resins can be classified into cation exchange resins and anion exchange resins according to the nature of the exchange groups. The anion exchange resin contains quaternary amino groups [ -N (CH) 3 ) 3 OH]Amino (-NH) 2 ) Or imino (-NH) 2 ) And an iso-basic group. They can generate OH-ions in water and can exchange with various anionsThe exchange principle is as follows: R-N (CH) 3 ) 3 OH+Cl - ——R—N(CH 3 ) 3 Cl+OH - In urine of pregnant women, OH in anion exchange resin - And (3) exchanging anions in the anion exchange resin with the human chorionic gonadotrophin to crosslink the anion exchange resin with the human chorionic gonadotrophin to realize adsorption, filtering a mixed solution of urine of a pregnant woman and the anion exchange resin, washing the anion exchange resin after adsorption to remove impurities in the urine, avoiding influencing the next experiment, eluting the human chorionic gonadotrophin adsorbed on the anion exchange resin by using ammonium acetate solution after washing, collecting eluent, precipitating to obtain precipitated solid, and measuring the titer to obtain the human chorionic gonadotrophin extracted from the urine of the pregnant woman.
Chromatography is a technique established by utilizing the differences in physicochemical properties of different substances, and all chromatographic systems consist of two phases: one is a stationary phase and the other is a mobile phase. When the mixture to be separated passes through the stationary phase with the mobile phase, the components are redistributed in the two phases continuously due to the difference in physicochemical properties of the components, the ability to interact (adsorb, dissolve, bind, etc.) with the two phases, the partition (content ratio) in the two phases being different and moving forward with the mobile phase. The effluent liquid is collected step by step, and each single component contained in the sample can be obtained, so that the aim of separating each component is fulfilled.
The principle of the experimental process is that anion exchange resin which has absorbed crude human chorionic gonadotrophin is filled into a chromatographic system, and then passes through a mobile phase of 10% ammonium acetate solution and then passes through a stationary phase, and as the physical and chemical properties of the human chorionic gonadotrophin and the anion exchange resin have great difference, the two can be redistributed in the stationary phase and the mobile phase, and effluent liquid is collected step by step, so that the eluent containing single component human chorionic gonadotrophin can be obtained, and the purpose of mutual separation of the human chorionic gonadotrophin and the anion exchange resin is achieved.
The present invention may be further configured in a preferred example to: the dosage of the anion exchange resin is 1% -5% of the urine volume of pregnant women.
By adopting the technical scheme, the dosage of the anion exchange resin is 1-5% of the volume of the urine of the pregnant woman, so that the anion exchange resin is in a proper range, and is convenient for better absorbing human chorionic gonadotrophin in the urine of the pregnant woman.
Further preferably, the anion exchange resin is a Dow 550A (OH) anion exchange resin.
By adopting the technical scheme, the Dow 550A (OH) anion exchange resin is a first-line brand raw material imported from America by Shanghai Kaijin chemical industry Co., ltd. And is a high-quality resin with extremely high exchange capacity, and the resin has ultra-strong particle integrity and light color which is extremely easy to identify with the cationic resin. The device is particularly suitable for a high-speed mixed bed of a condensate polishing system of a thermal power plant and a nuclear power plant, has stronger adsorption capacity, and has good adsorption effect on human chorionic gonadotrophin in pregnant woman urine.
It is further preferred that the pregnant woman's urine is pregnant woman's fresh urine of 2-6 months of pregnancy.
It is further preferred that the pregnant woman's urine is preferably pregnant woman's fresh urine from 2 to 4 months of pregnancy.
By adopting the technical scheme, after the fertilized ovum of the mature female is transferred into the uterine cavity for implantation, an embryo is formed, and in the process of developing and growing into a fetus, placenta syngeneic trophoblast cells produce a large amount of human chorionic gonadotrophin, and the human chorionic gonadotrophin can be excreted into urine through the blood circulation of a pregnant woman. When gestation is carried out for 1-2.5 weeks, HCG level in serum and urine can be rapidly increased, the pregnancy reaches the peak at 8 weeks, the pregnancy is reduced to medium level from 4 months, and the pregnancy is maintained until the end of gestation, so that fresh urine of pregnant women selected for 2-4 months can extract more human chorionic gonadotrophin therefrom, and the extraction efficiency is improved.
More preferably, the anion exchange resin after adsorption is washed with purified water.
By adopting the technical scheme, the resin for adsorbing the human chorionic gonadotrophin is washed by purified water, so that the method is environment-friendly and pollution-free, and after washing, the next elution operation is convenient, the impurities contained on the adsorbed Dow 550A (OH) anion exchange resin are reduced, and experimental errors are reduced.
It is further preferable that the mass fraction of the ammonium acetate solution is 8% -12%.
By adopting the technical scheme, the ammonium acetate solution is used for eluting the human chorionic gonadotrophin on the Dow 550A (OH) anion exchange resin, the human chorionic gonadotrophin reacts with the ammonium acetate solution to form an eluting solution, the eluting solution is smoothly fallen off from the Dow 550A (OH) anion exchange resin, the mass fraction of the ammonium acetate solution can be a value between 8% and 12%, and the eluting solution is easier to react with the human chorionic gonadotrophin on the Dow 550A (OH) anion exchange resin.
It is further preferred that the volume of the ammonium acetate solution is 2% -10% of the urine volume of pregnant women.
By adopting the technical scheme, the ammonium acetate solution is obtained through conversion, and the dosage of the ammonium acetate solution is about 2 times of that of the anion exchange resin, so that the ammonium acetate solution can more easily wash off the human chorionic gonadotrophin on the anion exchange resin.
It is further preferred that the human chorionic gonadotrophin in the eluate is precipitated with ethanol.
By adopting the technical scheme, ethanol is added into the eluent mixed by the ammonium acetate solution and the human chorionic gonadotrophin, so that the ammonium acetate in the eluent is dissolved, the human chorionic gonadotrophin is smoothly precipitated, and the collection is convenient.
It is further preferred that the ratio of the eluent to ethanol is set to 1 (4-6).
By adopting the technical scheme, the ratio of the eluent to the ethanol is 1:4 to 1:6, and a large amount of ethanol is added, so that the ammonium acetate is more favorably dissolved by the ethanol, more human chorionic gonadotrophin is precipitated, the measured titer is improved, and the extraction efficiency of extracting the human chorionic gonadotrophin from human urine is improved.
It is further preferable that the dissolution stirring time of the eluent by the ethanol is 15 to 30 minutes.
By adopting the technical scheme, the ammonium acetate solution in the eluent is dissolved as much as possible by dissolving and stirring for 15-30 minutes, so that the human chorionic gonadotrophin is settled as much as possible, and the purification efficiency is improved.
In summary, compared with the prior art, the invention has the following beneficial effects:
(1) The method for extracting human chorionic gonadotrophin from human urine adopts anion exchange resin as adsorbent, and adds the adsorbent into pregnant woman urine to stir, filter and wash, then makes elution by ammonium acetate solution, then makes precipitation of eluent, and collects the precipitate and determines potency to obtain human chorionic gonadotrophin. The purification efficiency is increased, the production cost is saved, and the pollution emission is reduced.
(2) In the whole process of extracting human chorionic gonadotrophin from pregnant woman urine, some solvents such as resin, ammonium acetate and ethanol are used, so that the method is environment-friendly and pollution-free, can be repeatedly used, and reduces the whole experiment cost.
(3) The method adopts the American Dow brand and the model 550A (OH) anion exchange resin, the anion exchange resin is a high-quality resin with extremely high exchange capacity, the Dow 550A (OH) anion resin has good adsorption effect on human chorionic gonadotrophin, and the anion resin can be reused, thereby reducing the cost, having better economic benefit and being more friendly to the environment.
Detailed Description
The present invention will be described in detail with reference to examples.
1. Examples
Example 1: a method of extracting human chorionic gonadotrophin, a gonadotrophin present in urine of a pregnant woman, from human urine, the extraction method comprising the steps of:
step one, collecting 100 liters of pregnant urine from pregnant women who are pregnant for 2 months, adding 3 liters of Dow 550A (OH) anion exchange (the Dow brand and model 550A (OH) anion exchange resin imported from America by Kaijin chemical Co., ltd.) into 100 liters of pregnant urine, mechanically stirring for 30 minutes, and filtering the mixed solution by using a funnel with filter paper.
And step two, after the filtration is finished, washing the anion exchange resin of the filtered adsorbed crude human chorionic gonadotrophin product by purified water for 1 time, washing for 5 column volumes, and loading the adsorbed resin into a chromatographic system after impurity removal by washing.
And thirdly, adding 2.5L10% ammonium acetate solution to elute the adsorbed resin, wherein the elution flow rate is 0.5BV/h (namely, the flow rate per hour is 0.5 times of the filling volume of the resin) in the whole elution process, and in the elution process, the gonadotropin content in the pregnant woman urine in the previous 500 ml of eluent is very small (the gonadotropin content in the pregnant woman urine is less than or equal to 5%), so that the previous 500 ml of eluent (namely, the mixed solution of human chorionic gonadotropin and 10% ammonium acetate solution) is discarded, and then 2 liters of eluent are collected.
The specific operation steps of precipitation are as follows: according to the eluent: ethanol=1: 5L of ethanol was added to 2L of the eluent, and the 10% ammonium acetate solution in the eluent was dissolved for 25 minutes by ethanol, so that human chorionic gonadotrophin was precipitated, and after precipitation, the precipitate was dried, and 5.7g of a solid was obtained.
And fifthly, measuring according to the method of the 2015 edition of Chinese pharmacopoeia, and calculating the titer of 5.7g of the obtained solid.
Example 2: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the amount of the Dow 550A (OH) anion exchange resin used to adsorb human chorionic gonadotrophin in 100L of urine of a pregnant woman was 1 liter.
Example 3: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the amount of the Dow 550A (OH) anion exchange resin used to adsorb human chorionic gonadotrophin in 100L of urine of a pregnant woman was 5 liters.
Example 4: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the 10% ammonium acetate solution used to elute human chorionic gonadotrophin on a ceramic 550A (OH) anion exchange resin was used in a volume of 2 liters.
Example 5: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the 10% ammonium acetate solution used to elute human chorionic gonadotrophin on a ceramic 550A (OH) anion exchange resin was used in a volume of 10 liters.
Example 6: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the collected urine of the pregnant woman is urine of the pregnant woman pregnant for 4 months.
Example 7: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the collected urine of the pregnant woman is urine of the pregnant woman pregnant for 6 months.
Example 8: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the collected urine of pregnant women is 10% (volume) urine of pregnant women pregnant for 6 months, 30% (volume) urine of pregnant women pregnant for 5 months, and 60% (volume) urine of pregnant women pregnant for 2 months.
Example 9: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the collected urine of pregnant women is 10% (volume) urine of pregnant women during pregnancy for 4 months, 30% (volume) urine of pregnant women during pregnancy for 5 months, and 60% (volume) urine of pregnant women during pregnancy for 3 months.
Example 10: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the mass fraction of ammonium acetate solution eluted from the Dow 550A (OH) anion exchange resin to which crude human chorionic gonadotrophin had been adsorbed was 8%.
Example 11: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the mass fraction of ammonium acetate solution eluted from the Dow 550A (OH) anion exchange resin to which crude human chorionic gonadotrophin had been adsorbed was 12%.
Example 12: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: in the precipitation of human chorionic gonadotrophin in the eluent, the ratio of eluent to ethanol is 1:4.
example 13: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: in the precipitation of human chorionic gonadotrophin in the eluent, the ratio of eluent to ethanol is 1:6.
example 14: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the dissolution time of the ethanol to the 10% ammonium acetate solution in the eluent is 15 minutes, so that the human chorionic gonadotrophin in the eluent is settled as much as possible.
Example 15: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the dissolution time of the ethanol to the 10% ammonium acetate solution in the eluent is 30 minutes, so that the human chorionic gonadotrophin in the eluent is settled as much as possible.
Example 16: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the same conditions were used as in the original method, but with the replacement of amberlite ira-400 anion exchange resin. Sample 7g was obtained under the same conditions. The potency was determined according to the method of the chinese pharmacopoeia 2015 edition.
2. Comparative example
Comparative example 1: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the same conditions were used as in the original method, but with sodium benzoate. A sample of 5.1g was obtained under the same conditions. The potency was determined according to the method of the chinese pharmacopoeia 2015 edition.
Comparative example 2: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the amount of the Dow 550A (OH) anion exchange resin used to adsorb human chorionic gonadotrophin in 100L of urine of pregnant women was 0.5L.
Comparative example 3: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the amount of the Dow 550A (OH) anion exchange resin used for adsorbing human chorionic gonadotrophin in 100L of urine of pregnant women was 6L.
Comparative example 4: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: in the precipitation of human chorionic gonadotrophin in the eluent, the ratio of eluent to ethanol is 1:3.
comparative example 5: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: in the precipitation of human chorionic gonadotrophin in the eluent, the ratio of eluent to ethanol is 1:7.
comparative example 6: a method for extracting human chorionic gonadotrophin from human urine, which differs from comparative example 1 in that: the pH of the urine of pregnant women is adjusted by hydrochloric acid under the same conditions as the original method.
Comparative example 7: a method for extracting human chorionic gonadotrophin from human urine, which differs from example 1 in that: the same conditions as in the original method were used, but the DEAE sepharose anion exchange resin was used instead. Sample 10g is obtained under the same condition, and the potency is measured according to the method of Chinese pharmacopoeia 2015 edition.
3. Performance detection analysis test one: effect of the species of adsorbent on the measured titers
Test object: example 1 was used as test sample 1, and example 16 was used as test sample 16; meanwhile, comparative examples 1 and 7 were respectively test comparative examples 1 and 7.
The test method comprises the following steps: four pregnant women urine of 2 months of pregnancy are prepared first, one hundred liters of the four pregnant women urine are added with the test sample 1, the test sample 16, the comparison sample 1 and the comparison sample 7 respectively, stirred for 30 minutes, and filtered by funnel filter paper. The resin was washed with purified water, loaded into a chromatography system, eluted with 10% ammonium acetate solution, the first 500 ml of eluent was discarded, and 2 l of eluent was collected. According to 1:5 (i.e. 10 litres of ethanol) and after precipitation, the precipitated solid is collected. The titers were determined according to the method of the Chinese pharmacopoeia 2015 edition and are shown in Table 1 below.
TABLE 1
Test results: as can be seen from Table 1, the values of the measured titers of test sample 1, test sample 16, comparative sample 1 and comparative example 7 were highest, reaching 211.7IU/mg, indicating that the Dow 550A (OH) anion exchange resin had the best effect of adsorbing human chorionic gonadotropin in human urine, and the purification efficiency was highest, so that the use of the Dow 550A (OH) anion exchange resin for adsorbing human chorionic gonadotropin in human urine was preferred, whereas the amberlite ira-400 anion exchange resin had a poorer effect than the Dow 550A (OH) anion exchange resin, and was also useful as an anion exchange resin for adsorbing human chorionic gonadotropin in human urine.
And (2) testing II: effect of volume fraction of adsorbent on measured titers
Test object: example 1 was used as test sample 1, and example 2, example 3, comparative example 2 and comparative example 3 were used as test sample 2, test sample 3, comparative sample 2 and comparative sample 3, respectively.
The test method comprises the following steps: six pregnant women urine of 2 months of pregnancy were first prepared, and 0L, 0.5L, 1L, 3L, 5L and 6L of Dow 550A (OH) anion exchange resin were added to the six pregnant women urine, respectively, and stirred for 30 minutes, and filtered with funnel filter paper. The resin was washed with purified water, loaded into a chromatography system, eluted with 10% ammonium acetate solution, the first 500 ml of eluent was discarded, and 2 l of eluent was collected. According to 1:5 (i.e., 10 liters of ethanol) and after precipitation, the precipitated solids were collected to yield 0.1g, 3.5g, 5.7g, 5.72g, 5.73g, and 5.74g. The titers were determined according to the method of the Chinese pharmacopoeia 2015 edition and are shown in Table 2.
TABLE 2
Test results: as can be seen from table 2, test sample 2 showed a considerably smaller increase in solid mass and measured titer as compared with test sample 2, test sample 3, comparative example 2, comparative example 3 and blank as the amount of the dow 550A (OH) anion exchange resin increased continuously from 1L, whereas the amount of the dow 550A (OH) anion exchange resin added had increased from 1L, 3L, 5L to 6L,550A (OH) anion exchange resin at multiple times, and the measured titer had almost no increase, and in order to save resources and experimental costs, the experiment selected to add 1L of the dow 550A (OH) anion exchange resin to one hundred liters of pregnant urine, and measured titer reached 211.7IU/mg, and purification efficiency was high, while saving costs.
And (3) test III: effect of volume amount of ethanol on measured titers
Test object: examples 1, 12, 13, 4 and 5 are test sample 1, 12, 13, 4 and 5, respectively.
The test method comprises the following steps: five pregnant women urine of 2 months of pregnancy is prepared first, one hundred liters of urine is added into the five pregnant women urine, 1L of Dow 550A (OH) anion exchange resin is added into the five pregnant women urine respectively, the mechanical stirring is carried out for 30 minutes, and the filter paper is used for filtering. Washing resin with purified water, and loading into chromatographic systemSystem for managing a plurality of dataElution was performed with 10% ammonium acetate solution, the first 500 ml of eluent was discarded, and 2 l of eluent was collected. According to 1:3. 1:4. 1:51:6 and 1:7 (i.e., 6, 8, 10, 12 and 14 liters of ethanol) to produce a precipitate, and collecting the precipitated solids to yield 4.5g, 5.2g, 5.7g and 5.7g. The titers were determined according to the method of the Chinese pharmacopoeia 2015 edition and are shown in Table 3.
TABLE 3 Table 3
Test results: as can be seen from table 3, the titers of test sample 1, test sample 12, test sample 13, control sample 4 and control sample 5 were measured to be 212.7IU/mg, as eluent: ethanol=1: (3-4) the amount of ethanol is too small, and the ethanol cannot completely dissolve ammonium acetate in the eluent, so that the human chorionic gonadotrophin is completely precipitated, and when the eluent: ethanol=1: (6-7) since the amount of ethanol was relatively large, the human chorionic gonadotrophin was all precipitated, and the titer was measured to be 212.7IU/mg, and the eluate: ethanol=1: 5, the measured titers are equal, so that in order to save the experiment cost and reduce the experiment medicine dosage, the eluent is selected for use in the experiment: ethanol=1: 5, performing operation.
And (3) testing four: effect of volume amount of ethanol on measured titers
Test object: example 1 was used as test sample 1, and example 2, example 3 and comparative example 6 were used as test sample 2, test sample 3 and comparative sample 6, respectively.
The test method comprises the following steps: four pregnant women urine of 2 months of pregnancy is prepared first, one hundred liters of urine of five pregnant women is added with 1L, 3L and 5L of Dow 550A (OH) anion exchange resin and sodium benzoate respectively, the pH of the urine of the pregnant women needs to be regulated by hydrochloric acid to facilitate the absorption of the sodium benzoate, and then the urine of the four pregnant women is mechanically stirred for 30 minutes and filtered by funnel filter paper. Washing resin with purified water, and loading into chromatographic systemSystem for managing a plurality of dataElution was performed with 10% ammonium acetate solution, the first 500 ml of eluent was discarded, and 2 l of eluent was collected. According to 1:5 (i.e. 10 litres of ethanol) and after precipitation, the precipitated solid is collected. The potency was measured according to the method of "Chinese pharmacopoeia" 2015 edition. The volatile gases generated in the whole process of extracting human chorionic gonadotrophin in four groups of experiments are respectively introduced into four parts of sodium hydroxide solution with the same PH=12, and compared with the prior art, the PH of the sodium hydroxide solution is changed.
TABLE 4 Table 4
Test results: as is clear from Table 4, in the course of the present experiment (examples 1, 2 and 3) for extracting human chorionic gonadotrophin, the generated volatile gas did not change the pH of the sodium hydroxide solution, whereas comparative example 6 lowered the pH of the sodium hydroxide solution from 12 to 5, indicating that a large amount of hydrochloric acid volatilized during the experiment of comparative example 6, causing environmental pollution. Therefore, compared with comparative example 6, hydrochloric acid is not used in the experiment, so that the pollution to the environment is reduced, and the experiment process is more environment-friendly.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (4)
1. A method for extracting human chorionic gonadotrophin from human urine, characterized by: adding anion exchange resin as adsorbent into pregnant woman urine, stirring, filtering, washing, eluting with ammonium acetate solution by chromatography, precipitating human chorionic gonadotrophin with ethanol, collecting precipitate, determining titer to obtain human chorionic gonadotrophin,
wherein, the anion exchange resin adopts a Dow 550A (OH) anion exchange resin;
the volume of the anion exchange resin is 1% -5% of the urine volume of pregnant women;
the mass fraction of the ammonium acetate solution is 8% -12%, and the volume of the ammonium acetate solution is 2% -10% of the urine volume of pregnant women;
the ratio of the eluent to the ethanol is set to be 1 (4-6).
2. A method for extracting human chorionic gonadotrophin from human urine according to claim 1, wherein: the pregnant woman urine is pregnant woman fresh urine which is pregnant for 2-6 months.
3. A method for extracting human chorionic gonadotrophin from human urine according to claim 1, wherein: and washing the adsorbed anion exchange resin by purified water.
4. A method for extracting human chorionic gonadotrophin from human urine according to claim 1, wherein: the dissolution and stirring time of the ethanol on the eluent is 15-30 minutes.
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| CN1084858A (en) * | 1993-02-15 | 1994-04-06 | 唐山市妇幼保健院 | Extract the method for chorionic-gonadotropin hormone |
| CN1404485A (en) * | 2000-02-22 | 2003-03-19 | 应用研究系统Ars股份公司 | Process of purification of hCG and recombinant hCG purified by that method |
| WO2015124759A1 (en) * | 2014-02-24 | 2015-08-27 | Altergon Sa | Extraction process to obtain hcg having a high biological activity |
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| CN1084858A (en) * | 1993-02-15 | 1994-04-06 | 唐山市妇幼保健院 | Extract the method for chorionic-gonadotropin hormone |
| CN1404485A (en) * | 2000-02-22 | 2003-03-19 | 应用研究系统Ars股份公司 | Process of purification of hCG and recombinant hCG purified by that method |
| WO2015124759A1 (en) * | 2014-02-24 | 2015-08-27 | Altergon Sa | Extraction process to obtain hcg having a high biological activity |
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