CN1958603A - Method for purifying human chorionic gonadotropin - Google Patents
Method for purifying human chorionic gonadotropin Download PDFInfo
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- CN1958603A CN1958603A CN 200510110035 CN200510110035A CN1958603A CN 1958603 A CN1958603 A CN 1958603A CN 200510110035 CN200510110035 CN 200510110035 CN 200510110035 A CN200510110035 A CN 200510110035A CN 1958603 A CN1958603 A CN 1958603A
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- ion exchange
- human chorionic
- chorionic gonadotropin
- exchange chromatography
- purifying
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Abstract
This invention provides a method for purifying human chorionic gonadotropin. The method comprises: purifying through SP-agarose ion exchange chromatography, and purifying through Q-agarose ion exchange chromatography. The method has such advantages as high product biological value and high yield.
Description
Technical field
The present invention relates to a kind of protein purification method, relate to the purification process of human chorionic gonadotropin particularly.
Background technology
Human chorionic gonadotropin (for for simplicity, hereinafter referred to as HCG) is a kind of hormone that is produced by placenta, and generally the urine from the pregnant woman extracts.
HCG is a kind of polymer glycoprotein, is made up of α chain and two subunits of β chain, and the α chain has 89～92 amino acid, molecular weight 14500～18000D; The β chain has 145～147 amino acid, molecular weight 22200～39000D.Article two, polypeptide chain combines with carbohydrate chain with covalent linkage respectively, and carbohydrate chain accounts for 30% of the full molecular weight of HCG, is made up of seminose, semi-lactosi, Fucose and sialic acid etc.The iso-electric point of HCG is pH3.0, is amphoteric substance.HCG has identical α subunit with follicular stimulating hormone (FSH), lutropin (LH), the thyrotropin (TSH) in hypophysis source, and its β subunit has specificity, has determined biological activity and the immunocompetence of HCG.HCG a kind ofly is mainly used in metropathia hemorrhagica and habitual abortion that treatment is caused by luteal phase defect clinically to keeping the essential glycoprotein hormones of early pregnancy, share with HMG and can bring out ovulation and treatment infertility.HCG also can be used for treating the male sex and causes sterile disease because of the not enough hypogonadism of gonadotropin.The another kind of important use of HCG is preparation Clearblue Pregnancy Test and radioimmunity reagent, in order to the diagnosis of gestation check and choriocarcinoma.
The existing processing step that extracts HCG from pregnant woman urine is as follows: at first from pregnant woman urine with Sodium Benzoate or kaolin absorption, make HCG urine enriched material.Carry out the ethanol extracting then, obtain the HCG raw material.Then, be further purified again, obtain final finished product.Some traditional HCG purifying process all use the pure aluminium silicate resin chromatography method at present, technological process is loaded down with trivial details relatively, and yield is low, thereby makes production cost higher, quality product only can reach about standards of pharmacopoeia 2500IU/mg simultaneously, can't satisfy the demand of domestic and international market to high purity HCG product.
Chinese patent CN1302818A provides another technology, has wherein used sulfonic acid propyl group dextran (SP-Sephadex C-50) ion exchange chromatography, and yield is increased to 80%～90%, but it is tired and still is lower than 5000IU/mg.
Recently, develop and a kind of technology with monoclonal antibody affinity chromatography chromatography purification HCG, though can obtain the HCG of higher degree, but its yield all very low [that mother respects is strongly fragrant, Lv Yancheng etc., and the monoclonal antibody affinity chromatography is produced the live foundation of HCG method of height ratio. Norman Bethune Medical University's journal, 1995,21 (1): 96～98], and monoclonal antibody affinity column manufacturing process complexity also may be introduced the biotic pollution in host source, and suitability for industrialized production is subjected to certain restriction.
In sum, this area lacks a kind of biological value height, the high HCG purification process of while yield.This area presses for this kind of exploitation biological value height, the high HCG purification process of while yield.
Summary of the invention
The objective of the invention is to obtain biological value height, the high HCG purification process of while yield.
In a first aspect of the present invention, a kind of purification process of human chorionic gonadotropin is provided, comprise the steps:
(a) solution that will contain human chorionic gonadotropin carries out purifying with sulfonic acid propyl group agarose ion exchange chromatography, obtains containing first purified product of human chorionic gonadotropin;
(b) first purified product that step (a) is obtained carries out purifying with diethyl quaternary amine agarose ion exchange chromatography, obtains the human chorionic gonadotropin of purifying.
In a preference of purification process of the present invention, carry out purifying with sulfonic acid propyl group agarose ion exchange chromatography in the step (a), comprising: (i) sulfonic acid propyl group agarose ion exchange resin equilibrium step; (ii) go up the sample step; (iii) elution step.Preferably, wherein said step (i) and step are (ii) carried out in the damping fluid of pH4～6.Described (iii) elution step is to carry out in the damping fluid of 0.1～0.8M salt concn.
In another preference, it is as follows to carry out the condition of purifying with sulfonic acid propyl group agarose ion exchange chromatography in the step (a): the balance liquid pH of sulfonic acid propyl group (SP) agarose ion exchange chromatography is preferably between 5.0～5.5; Electricity is led and is no more than 10ms.Last sample concentration is preferably at 80～110g albumen/L.Wash-out preferably carries out in the damping fluid of 0.3～0.4M salt concn
In another preference of purification process of the present invention, carry out purifying with diethyl quaternary amine agarose ion exchange chromatography in the step (b), comprising: (i ') diethyl quaternary amine agarose ion exchange resin equilibrium step; (ii ') last sample step; (iii ') elution step.Preferably, wherein said step (i ') and step (ii ') are carried out in the damping fluid of pH6～9.Described (iii ') elution step is to carry out in the damping fluid of 0.1～0.4M concentration.
In another preference, it is as follows to carry out the condition of purifying with diethyl quaternary amine agarose ion exchange chromatography in the step (b): balance and on sample preferably carry out in the damping fluid between 7～8; Electricity is led and is no more than 0.05ms.Last sample concentration is preferably at 20～30g albumen/L.Wash-out preferably carries out in the damping fluid of 0.2～0.3M salt concn.
In another preference of purification process of the present invention, the elution step of ion exchange chromatography described in step (a) or the step (b) adopt the linear gradient method or progressively terrace work carry out wash-out.
In another preference of purification process of the present invention, described in step (a) or the step (b) in the elution step of ion exchange chromatography, the wash-out that adopts comprises muriate, acetate, phosphoric acid salt, vitriol or its combination with the salt that contains salt buffer, and the positively charged ion of wherein said salt comprises Na
+, K
+, NH
4 +
Another aspect of the present invention provides human chorionic gonadotropin, and available following purification process obtains, and comprises the steps:
(a) solution that will contain human chorionic gonadotropin carries out purifying with sulfonic acid propyl group agarose ion exchange chromatography, obtains containing first purified product of human chorionic gonadotropin;
(b) first purified product that step (a) is obtained carries out purifying with diethyl quaternary amine agarose ion exchange chromatography, obtains the human chorionic gonadotropin of purifying.
The biological value of described human chorionic gonadotropin is not less than 6000IU/mg, and total recovery is not less than 90%.
Embodiment
The inventor, finds to use ion exchange chromatography can obtain highly purified HCG through uniting, and can guarantee very high yield by selecting appropriate resin and optimized Separation condition for use by improving preparation technology through extensive and deep research.By the HCG that purification process of the present invention is produced, its biological value is greater than 6000IU/mg, and total recovery is more than 90%.
The solution that contains human chorionic gonadotrophin (HCG)
The strength of solution of human chorionic gonadotropin that contains of the present invention is preferably at 50～120g albumen/L.Its preparation method makes by this area common technique.For example make: 1. the pregnant woman is urinated the extracting of enriched material process ethanol and make the HCG raw material by following method; 2.HCG raw material wiring solution-forming.The solution that contains HCG then carries out the subsequent purification reaction.
Described pregnant woman urinates enriched material and makes by this area routine techniques.For example, collect the healthy women urine in conceived 2～June, after the acceptance(check), add 2.2 kilograms of adding Sodium Benzoates, after the stirring and dissolving, regulate pH4～5, staticly settle and spend the night with rare HCl in the 100L urine.Siphon next day discards the useless urine in upper strata, filters collecting precipitation.Add 10 liters by the per kilogram precipitation and add 95% ethanol, stirred 1 hour, filter collecting precipitation, promptly get after the precipitation drying and urinate enriched material.Its biological value is about 7～12IU/mg.
Described HCG raw material makes by this area routine techniques.For example, above-mentioned pregnant woman urinates enriched material and extracts with extract, and the prescription of extract generally is: 0.01M CaCl
2+ 0.04M NaAc+0.25M NaCl+50% ethanol, pH4.5～5.5.Add 20L adding extract by per kilogram urine enriched material, 6～8 ℃ were stirred 2 hours, centrifugal collection supernatant, and supernatant adds 95% ethanol of 3 times of volumes, collecting precipitation, vacuum-drying gets the HCG raw material.
Human chorionic gonadotropin is a kind of hormone that is produced by placenta, and generally the urine from the pregnant woman extracts.HCG is a kind of polymer glycoprotein, is made up of α chain and two subunits of β chain, and the α chain has 89～92 amino acid, molecular weight 14500～18000D; The β chain has 145～147 amino acid, molecular weight 22200～39000D.Article two, polypeptide chain combines with carbohydrate chain with covalent linkage respectively, and carbohydrate chain accounts for 30% of the full molecular weight of HCG, is made up of seminose, semi-lactosi, Fucose and sialic acid etc.The iso-electric point of HCG is pH3.0, is amphoteric substance.HCG has identical α subunit with follicular stimulating hormone (FSH), lutropin (LH), the thyrotropin (TSH) in hypophysis source, and its β subunit has specificity, has determined biological activity and the immunocompetence of HCG.
Method of the present invention also can be used for from hypophysis purifying human chorionic gonadotropin, or purification of Recombinant human chorionic gonadotropin the cell culture medium that obtains after reorganization.Purification process of the present invention can also other mammiferous chorionic gona dotropin of purifying, for example ox, horse, pig, sheep and monkey.
Purification process
The purification process of human chorionic gonadotropin of the present invention comprises the steps:
(a) solution that will contain human chorionic gonadotropin carries out purifying with sulfonic acid propyl group agarose ion exchange chromatography, obtains containing first purified product of human chorionic gonadotropin;
(b) first purified product that step (a) is obtained carries out purifying with diethyl quaternary amine agarose ion exchange chromatography, obtains the human chorionic gonadotropin of purifying.
In the above-mentioned steps, can carry out step (a) earlier and then carry out step (b), also can carry out step (b) earlier, carry out step (a) again.
Sulfonic acid propyl group (SP) agarose ion exchange chromatography purification step
Sulfonic acid propyl group of the present invention (SP) agarose ion exchange chromatography purification step can be removed most of foreign protein.
In preferred embodiment of the present invention, during with sulfonic acid propyl group (SP) agarose ion exchange chromatography purifying, comprising: (i) sulfonic acid propyl group agarose ion exchange resin equilibrium step; (ii) go up the sample step; (iii) elution step.
The balance liquid pH of sulfonic acid propyl group (SP) agarose ion exchange chromatography is preferably 4～6, more preferably between 5.0～5.5; Electricity is led and is no more than 10ms.
The pH of sample solution, electricity are led and are adjusted to consistently with balance liquid, preferably carry out in the damping fluid of pH4～6, and last sample concentration is preferably at 50～120g albumen/L.With the balance liquid washing, wash volumes can make a large amount of foreign proteins flow out preferably at 5～15 column volumes behind the end of the sample.
Carry out in the damping fluid of the preferred 0.1～0.8M salt concn of wash-out.Described wash-out comprises muriate, acetate, phosphoric acid salt, vitriol or its combination with the salt that contains salt buffer, and the positively charged ion of wherein said salt is Na
+, K
+, NH
4 +Elution step can be used linear gradient method wash-out, also can use progressively terrace work wash-out.With the salt concn of terrace work wash-out progressively more preferably at 0.2～0.5M, particularly preferably in 0.3～0.4M.
Collection contains the cut of HCG, adds ethanol sedimentation.Collecting precipitation, vacuum-drying.
In another preference, it is as follows to carry out the condition of purifying with sulfonic acid propyl group agarose ion exchange chromatography in the step (a): the balance liquid pH of sulfonic acid propyl group (SP) agarose ion exchange chromatography is preferably between 5.0～5.5; Electricity is led and is no more than 10ms.Last sample concentration is preferably at 80～110g albumen/L.Wash-out preferably carries out in the damping fluid of 0.3～0.4M salt concn.
Diethyl quaternary amine (Q) agarose ion exchange chromatography purification step
In preferred embodiment of the present invention, during with diethyl quaternary amine (Q) agarose ion exchange chromatography purifying, comprising: (i) diethyl quaternary amine (Q) agarose ion exchange resin equilibrium step; (ii) go up the sample step; (iii) elution step.
Balance is to carry out in the damping fluid of pH6～9, more preferably between 7～8; Electricity is led and is no more than 0.05ms.The pH of sample solution, electricity are led and are adjusted to consistently with balance liquid, and last sample concentration is preferably at 20～50g/L.With the balance liquid washing, wash volumes is preferably at 5～15 column volumes behind the end of the sample.
Wash-out is to carry out in the damping fluid that contains 0.1～0.4M salt concn.Described wash-out comprises muriate, acetate, phosphoric acid salt, vitriol or its combination with the salt that contains salt buffer, and the positively charged ion of wherein said salt is Na
+, K
+, NH
4 +Elution step can be used linear gradient method wash-out, also can use progressively terrace work wash-out.With the salt concn of terrace work wash-out progressively preferably at 0.1～0.4M, more preferably at 0.2～0.3M.
Collection contains the cut of HCG, adds ethanol sedimentation, collecting precipitation, vacuum-drying; Also can be by carrying out lyophilize after the ultrafiltration desalination.Promptly obtain the HCG purified product through above step.
In another preference, it is as follows to carry out the condition of purifying with diethyl quaternary amine agarose ion exchange chromatography in the step (b): balance and on sample preferably carry out in the damping fluid between 7～8; Electricity is led and is no more than 0.05ms.Last sample concentration is preferably at 20～30g/L.Wash-out preferably carries out in the damping fluid of 0.2～0.3M salt concn.
The chorionic gona dotropin that the present invention makes, biological value is not less than 6000IU/mg, and total recovery is not less than 90%.
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Ratio and per-cent are based on weight, unless stated otherwise.
Embodiment 1
Get HCG urine enriched material (the survey biological value is 8.1IU/mg) 2500 grams, total titer is 0.202 hundred million IU.It is joined 8 ℃ 50L extract (0.01M CaCl
2+ 0.04M NaAc+0.25M NaCl+50% ethanol, pH5.0) in, stirred 2 hours, centrifugal collection supernatant, supernatant add 150L 95% ethanol, precipitation is spent the night.Next day, centrifugal collecting precipitation dewatered with dehydrated alcohol, and vacuum-drying gets 77.3 gram HCG raw materials.
With above-mentioned 77.3 gram HCG raw materials add to the 700mL balance liquid (the 0.03M SODIUM PHOSPHATE, MONOBASIC, pH5.2) in, stirred the centrifuging and taking supernatant 40 minutes.Supernatant liquor is 8cm, highly is SP-Sepharose FF (Amersham provides) chromatography column of 12cm that by diameter this post has used identical balance liquid balance good in advance with the flow velocity of 12cm/h.Behind the end of the sample, with 6 liters of balance liquid washings, flow velocity 12cm/h.Use balance liquid and elutriant (0.03M SODIUM PHOSPHATE, MONOBASIC+1M NaCl then, pH5.2) carry out linear gradient elution, flow velocity 12cm/h, with UV-detector monitoring 280nm place, distributing to collect respectively distillates the peak, detect its HCG immunizing potency, be associated with the about 0.4L of effective constituent, the dehydrated alcohol precipitation that adds 1.6 liters-10 ℃ is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 4.30 gram dry products.
With above-mentioned 4.30 gram dry products, be dissolved in 150mL balance liquid (0.01M SODIUM PHOSPHATE, MONOBASIC, pH7.0) in, be 8cm, highly be Q-Sepharose FF (Amersham provides) chromatography column of 8cm that by diameter this post has used identical balance liquid balance good in advance with the flow velocity of 8cm/h.Behind the end of the sample, with 4 liters of balance liquid washings, flow velocity 8cm/h.Use elutriant (0.01M SODIUM PHOSPHATE, MONOBASIC+0.2M NaCl then, pH7.0) carry out wash-out, flow velocity 8cm/h, with UV-detector monitoring 280nm place, the about 0.4L of collected volume, the dehydrated alcohol precipitation that adds 2.0 liters-10 ℃ is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 2.61 gram HCG purified products.The survey biological value is 7316IU/mg, and total titer is 0.191 hundred million IU, and total recovery is 94.6%.The measuring method of biological value is pressed 2005 editions appendix XIIE checks of Chinese Pharmacopoeia.
Embodiment 2
Get HCG urine enriched material (biological value be 8.1IU/mg) 25.0 kilogram identical with embodiment 1, total titer is 2.02 hundred million IU.It is joined 8 ℃ 500L extract (0.01M CaCl
2+ 0.04MNaAc+0.25M NaCl+50% ethanol, pH5.0) in, stirred 2 hours, centrifugal collection supernatant, supernatant add 1500L 95% ethanol, precipitation is spent the night.Next day, centrifugal collecting precipitation dewatered with dehydrated alcohol, and vacuum-drying gets 826 gram HCG raw materials.
With above-mentioned 826 gram HCG raw materials add to the 9L balance liquid (the 0.03M SODIUM PHOSPHATE, MONOBASIC, pH5.2) in, stirred the centrifuging and taking supernatant 40 minutes.Supernatant liquor is 18cm, highly is SP-Sepharose FF (Amersham provides) chromatography column of 24cm that by diameter this post uses identical balance liquid balance good earlier with the flow velocity of 24cm/h.Behind the end of the sample, with 50 liters of balance liquid washings, flow velocity 24cm/h.Use elutriant (0.03M SODIUM PHOSPHATE, MONOBASIC+0.4M NaCl then, pH5.2) carry out wash-out, flow velocity 24cm/h, with UV-detector monitoring 280nm place, the about 4.3L of collected volume, the dehydrated alcohol precipitation that adds 18 liters-10 ℃ is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 45.5 gram dry products.Survey biological value 4351IU/mg.
With above-mentioned 45.5 gram dry products, be dissolved in 2L balance liquid (0.01M SODIUM PHOSPHATE, MONOBASIC, pH7.0) in, be 18cm, highly be Q-Sepharose FF (Amersham provides) chromatography column of 16cm that by diameter this post has used identical balance liquid balance good in advance with the flow velocity of 16cm/h.Behind the end of the sample, with 40 liters of balance liquid washings, flow velocity 16cm/h.Use elutriant (0.01M SODIUM PHOSPHATE, MONOBASIC+0.2M NaCl then, pH7.0) carry out wash-out, flow velocity 16cm/h, with UV-detector monitoring 280nm place, the about 4.5L of collected volume, the dehydrated alcohol precipitation that adds 23 liters-10 ℃ is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 29.8 gram HCG purified products.The survey biological value is 6539IU/mg, and total titer is 1.95 hundred million IU, and total recovery is 96.5%.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (9)
1. the purification process of a human chorionic gonadotropin is characterized in that, comprises the steps:
(a) solution that will contain human chorionic gonadotropin carries out purifying with sulfonic acid propyl group agarose ion exchange chromatography, obtains containing first purified product of human chorionic gonadotropin;
(b) first purified product that step (a) is obtained carries out purifying with diethyl quaternary amine agarose ion exchange chromatography, obtains the human chorionic gonadotropin of purifying.
2. the method for claim 1 is characterized in that: carry out purifying with sulfonic acid propyl group agarose ion exchange chromatography in the step (a), comprising: (i) sulfonic acid propyl group agarose ion exchange resin equilibrium step; (ii) go up the sample step; (iii) elution step.
3. method as claimed in claim 2 is characterized in that: wherein said step (i) and step are (ii) carried out in the damping fluid of pH4～6, and/or described (iii) elution step is to carry out in the damping fluid of 0.1～0.8M salt concn.
4. the method for claim 1 is characterized in that: carries out purifying with diethyl quaternary amine agarose ion exchange chromatography in the step (b), comprising: (i ') diethyl quaternary amine agarose ion exchange resin equilibrium step; (ii ') last sample step; (iii ') elution step.
5. method as claimed in claim 4 is characterized in that: wherein said step (i ') and step (ii ') are carried out in the damping fluid of pH6～9, and/or described (iii ') elution step is to carry out in the damping fluid of 0.1～0.4M concentration.
6. as claim 2 or 4 described methods, it is characterized in that: the elution step of described ion exchange chromatography adopt the linear gradient method or progressively terrace work carry out wash-out.
7. as claim 2 or 4 described methods, it is characterized in that: described in step (a) or the step (b) in the elution step of ion exchange chromatography, the wash-out that adopts comprises muriate, acetate, phosphoric acid salt, vitriol or its combination with the salt that contains salt buffer, and the positively charged ion of wherein said salt is Na
+, K
+, NH
4 +
8. human chorionic gonadotropin with the preparation of the described purification process of claim 1.
9. human chorionic gonadotropin as claimed in claim 8 is characterized in that the biological value of described human chorionic gonadotropin is not less than 6000IU/mg, and total recovery is not less than 90%.
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| CN1958603B CN1958603B (en) | 2010-05-05 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010034198A1 (en) * | 2008-09-24 | 2010-04-01 | 上海天伟生物制药有限公司 | A method for removing/inactivating virus in glycoprotein |
| CN103288950A (en) * | 2012-12-28 | 2013-09-11 | 青岛九龙生物医药有限公司 | Method for improving ratio of washing liquid to eluent in column separation process to remove impurities |
| CN103739701A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Technical solution for purification of menotrophin |
| CN105968185A (en) * | 2016-07-20 | 2016-09-28 | 宁波人健药业集团股份有限公司 | Chorionic gonadotrophin purification method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RS50741B (en) * | 2000-02-22 | 2010-08-31 | Laboratoires Serono Sa. | Process for purification of recombinant hcg |
| CN1302818A (en) * | 2000-12-12 | 2001-07-11 | 上海惠海生化制品厂 | Human chorionic gonadotropin and its preparing process |
-
2005
- 2005-11-04 CN CN 200510110035 patent/CN1958603B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010034198A1 (en) * | 2008-09-24 | 2010-04-01 | 上海天伟生物制药有限公司 | A method for removing/inactivating virus in glycoprotein |
| CN103288950A (en) * | 2012-12-28 | 2013-09-11 | 青岛九龙生物医药有限公司 | Method for improving ratio of washing liquid to eluent in column separation process to remove impurities |
| CN103739701A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Technical solution for purification of menotrophin |
| CN105968185A (en) * | 2016-07-20 | 2016-09-28 | 宁波人健药业集团股份有限公司 | Chorionic gonadotrophin purification method |
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| CN1958603B (en) | 2010-05-05 |
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