CN103694334B - A kind of method preparing hEGF raw product - Google Patents
A kind of method preparing hEGF raw product Download PDFInfo
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- CN103694334B CN103694334B CN201310715778.6A CN201310715778A CN103694334B CN 103694334 B CN103694334 B CN 103694334B CN 201310715778 A CN201310715778 A CN 201310715778A CN 103694334 B CN103694334 B CN 103694334B
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- hegf
- raw product
- urine
- metal chelate
- chelate chromatography
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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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF] (urogastrone)
Abstract
The invention discloses a kind of method preparing hEGF goods.The method is the sorbent material after collecting adsorbing urine protein, by the metal chelate chromatography post that the urine protein solution eluted has balanced, hEGF is attached on this metal chelate chromatography post, collects hEGF component elution peak, through ammonium sulfate precipitation, preparation hEGF raw product.The method is a kind of method preparing hEGF raw product from Male urine; this raw product can be used for the subsequent purification of hEGF; the hEGF be purified into is containing making human body produce anaphylactoid heterologous protein; the method is easy and simple to handle; be convenient to suitability for industrialized production, the hEGF preparing people source for mass-producing provides the foundation.
Description
Technical field
The present invention relates to a kind of extracting method of urine protein, particularly relate to a kind of preparation method of hEGF raw product.
Background technology
Containing 300 multiple proteins in people's urine, urokinase, human urine trypsin inhibitor, Human Urinary Kallidinogenase etc. are all separated from people's urine, and have played important effect in fields such as disease treatments.
Human epidermal growth factor's (Human Epidernal Growth Factor, is Urogastrone again, urogastrone, hereinafter referred to as hEGF), was separated first the seventies from people's urine.HEGF is the one in human growth factor, and the relative molecular weight be made up of 53 amino acid is 6045Da, and iso-electric point is the micromolecule polypeptide of about 4.6.HEGF contains three pairs of intramolecular disulfide bonds, therefore all more stable to chemical factors such as acid, alkali, heat.At present, hEGF has been developed to the treatment injection of gastric acid secretion inhibiting and burn and scald, ulcer, the externally applied medicine of all kinds of wound and corneal injury and cosmetics and skincare product.
It is very abundant that Chinese urinate resource, depends on this favourable condition, urine protein industry from last century late nineteen seventies just grow up, existing three more than ten years so far.Mainly contain two kinds in the large-scale production mode of urine protein raw product, method one: collect urine, be transported to processing stand, use adsorbent urine protein, due to the dispersion of urine source, compiling costs is high, adds that urban health is transformed, and collects more and more difficult; Method two: directly the resin of adsorbing urine protein is placed on lavatory carries out continuous on-line adsorption, the resin after then collecting absorption is transported processing stand back and is carried out separation and purification.This method environmentally safe, can collect in big and medium-sized cities, and urine source is concentrated, and transportation cost is lower, greatly expands the collecting zone of urine protein.
The main at present production process of urine protein product is: first, prepare urine protein raw product in various places through steps such as the absorption of urine protein, wash-out, precipitations; Secondly, the separation and purification of urine protein raw product through downstream, with refining, prepares urine protein bulk drug; Last filling finished product preparation, for clinical.
At present, hEGF source has two kinds.The first, hEGF is mainly through preparation, and title is restructuring hEGF(Recombind Human Epidermal Growth Factor, hereinafter referred to as rhEGF).Main approach of preparing has prokaryotic expression, mammalian cell expression, yeast expression etc., by collecting fermented liquid, carrying out separation and purification and obtaining rhEGF.The second, separation and Extraction hEGF from people's urine.At present, mainly from pregnant woman urine, hEGF is obtained through complicated purge process.
By the rhEGF of preparation, although easily purifying, output are high, and owing to wherein there is the heterologous protein of trace, rhEGF easily causes allergic reaction, and therefore, only uses as externally applied medicine at present.The hEGF prepared from urine, structures and characteristics is identical with human body, there is not heterologous protein, can be used as injection and enters clinical.There is the case of carrying out clinical study abroad at present.But hEGF at present most separation from urine of pregnant women obtains, and urine of pregnant women is difficult to collect and yield poorly, and limits the application of hEGF.
Summary of the invention
The object of the invention is the deficiency existed for prior art, a kind of method preparing hEGF raw product from Male urine is provided, this raw product can be used for the subsequent purification of hEGF, the hEGF be purified into is containing making human body produce anaphylactoid heterologous protein, easy and simple to handle being convenient to of the method industrially produces hEGF raw product, provides basis for extracting hEGF from natural source.
For achieving the above object, the present invention prepares the technical scheme of the method for hEGF raw product and is:
Prepare a method for hEGF raw product, collect the sorbent material after adsorbing urine protein, concentrate wash-out, by the metal chelate chromatography post that the urine protein solution loading eluted has balanced, collect the elution peak containing hEGF component, through ammonium sulfate precipitation, preparation hEGF raw product.
The described method preparing hEGF raw product, comprises the steps:
1), in urinal anionite-exchange resin being placed on Public toilets as sorbent material or urine funnel, the urine protein flowed through in urine or diluted urine is adsorbed;
2) collect the resin of adsorbing urine protein, carry out cleaning, desorb process;
3) ultrafiltration and concentration;
4) ultrafiltration and concentration liquid regulates pH and conductance, the metal chelate chromatography post that loading has balanced, then rinses with balance liquid;
5) use elute soln wash-out, collect the elution peak containing hEGF component;
6) in the elution peak collected, add ammonium sulfate powder to saturated, leave standstill after 2-10 hour, collecting precipitation, obtains hEGF raw product.
The metal ion of described metal chelate chromatography post chelating is Cu
2+, Zn
2+, Ni
2+, Fe
3+in any one.Preferred Cu
2+.
Metal chelate chromatography column equilibration liquid is 0.01-0.2M phosphate buffered saline buffer, and NaCl concentration is 0.2-2.0M, pH6.0-8.0.Preferred 0.02M phosphate buffered saline buffer, NaCl concentration is 0.5M.
Described metal chelate chromatography post elute soln is 0.05-0.2M phosphate buffered saline buffer, NH
4cl concentration is 0.5-1.0M, pH6.0-8.0.Preferred 0.1M phosphate buffered saline buffer, the NH of 0.8M
4cl, pH7.5.
In step 4, ultrafiltration and concentration liquid regulates pH6-8, conductance 0.5-10mS/cm.Preferred pH8.0, conductance 2.2mS/cm.
Between step 4 and step 5, increase a rinse step, use pH3-6, NaCl concentration≤0.2M to rinse metal chelate chromatography post, described washing fluid buffer medium is 0.01-0.2M phosphoric acid salt or acetate.The 0.1M acetate buffer of preferred pH5.0, sodium chloride concentration is 0.1M.
The present invention compared with prior art, has the following advantages: to achieve from Male urine enrichment first and be separated hEGF, and the method making to extract hEGF from people source can industrializing implementation; Introduce metal chelate chromatography technique first, be effectively separated hEGF, greatly reduce the difficulty of hEGF subsequent purification, the hEGF be purified into is containing making human body produce anaphylactoid heterologous protein; Adopt high-effect ionic exchange adsorbing substance, place in urine funnel or urinal, carry out the on-line preconcentration of urine protein, avoid the mass transit of urine, reduce cost.
Accompanying drawing explanation
Fig. 1 is the schema of the method preparing hEGF raw product.
Embodiment
Below in conjunction with embodiment, illustrate the present invention further, these embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention, after having read the present invention, the amendment of those skilled in the art to the various equivalent form of value of the present invention has all fallen within the application's claims limited range.
Prepare a method for hEGF raw product, comprise the steps:
1), in urinal ion exchange resin being placed on Public toilets as sorbent material or urine funnel, the urine protein flowed through in urine or diluted urine is adsorbed;
2) collect the resin of adsorbing urine protein, carry out cleaning, desorb process;
3) ultrafiltration and concentration;
4) ultrafiltration and concentration liquid regulates pH6-8, conductance 0.5-10mS/cm, preferred pH8.0, conductance 2.2mS/cm, on the metal chelate chromatography post that balanced, rinse with balance liquid, balance liquid is 0.01-0.2M phosphate buffered saline buffer, and NaCl concentration is 0.2-2.0M again, pH6.0-8.0, preferred 0.02M phosphate buffered saline buffer, NaCl concentration is 0.5M, and chelated metal ions is Cu
2+, Zn
2+, Ni
2+, Fe
3+in any one, preferred Cu
2+;
5) with elution, elution peak is collected.Described elute soln is 0.05-0.2M phosphate buffered saline buffer, NH
4cl concentration is 0.5-1.0M, pH6.0-8.0, preferred 0.1M phosphate buffered saline buffer, pH7.5,0.8MNH
4cl;
6) in the elution peak collected, add ammonium sulfate powder to saturated, leave standstill after 2-10 hour, collecting precipitation, obtains hEGF raw product.
Between step 4 and step 5, increase a rinse step, use pH3-6, NaCl concentration≤0.2M rinses metal chelate chromatography post, described washing fluid buffer medium is 0.01-0.2M phosphoric acid salt or acetate, the 0.1M acetate buffer of preferred pH5.0, and sodium chloride concentration is 0.1M.
example one
1. macroreticular ion exchange resin 100 kilograms is as sorbent material, after manipulation of regeneration, water punching is to neutral, loading aperture is in 50 object filter cloth bags, in the just bucket filter cloth bag installing resin being placed on the more lavatory of flow of the people or urinal, make urine or flow through resin, after 24 hours by the urine after water dilutes, collect the resin of adsorbing urine protein, resin tap water after absorption is rinsed well, filled post; Carry out wash-out with the NaCl of 1M, collect elution peak.
2. carry out ultrafiltration and concentration with the ultra-filtration membrane of molecular weight cut-off 10K, regulate pH7.0, conductance 0.5mS/cm, on the metal ion that balanced be Cu
2+metal chelate chromatography post (Chelating sepharose FF); Balance liquid (0.2M phosphate buffered, 0.2MNaCl, pH7.0) is used to rinse metal chelate chromatography post again; Metal chelate chromatography post is rinsed with the acetate buffer (pH5.0) of 0.1M; Use 0.05M phosphate buffered saline buffer, the NH of 1.0M
4cl, pH8.0, elution chromatography post, collects elution peak (E liquid).
3. the E liquid will collected, add ammonium sulfate powder to saturated, limit edged stirs, and leaves standstill 10 hours, and E liquid adds diatomite 20g collected by centrifugation and dries up precipitation, obtains hEGF raw product 32g.
By detecting the yield about 93% of hEGF, activity concentrates in hEGF raw product.HEGF content is as shown in table 1:
Table 1
| Sample ID | Weight (g) | HEGF content (U) |
| HEGF raw product | 32 | 1.236×10 6 |
example two
1. macroreticular ion exchange resin 100 kilograms is as sorbent material, after manipulation of regeneration, water punching is to neutral, loading aperture is in 50 object filter cloth bags, in the just bucket filter cloth bag installing resin being placed on the more lavatory of flow of the people or urinal, makes urine or is flow through resin by the urine after water dilutes, after 24 hours, collect the resin of adsorbing urine protein, resin tap water after absorption is rinsed well, finally fills post; Carry out wash-out with the NaCl of 1M, collect elution peak.
2. carry out ultrafiltration and concentration with the ultra-filtration membrane of molecular weight cut-off 10K, regulate pH to 6.0 and conductance to 10mS/cm ultrafiltration and concentration liquid, on the metal ion that balanced be Ni
2+metal chelate chromatography post (Chelating sepharoseFF); Balance liquid (balance liquid formula: 0.01M phosphate buffered, NaCl, the pH6.0 of 2M) is used to rinse metal chelate chromatography post again; Metal chelate chromatography post is rinsed with the acetate buffer (pH6.0) of the 0.01M containing 0.2M sodium-chlor; Use 0.2M phosphate buffered saline buffer, the NH of 0.5M
4cl, pH6.0, elution chromatography post, collects elution peak (E liquid).
3. will collect E liquid, add ammonium sulfate powder to saturated, limit edged stirs, and leaves standstill 2 hours, and E liquid adds diatomite 20g collected by centrifugation and dries up precipitation, hEGF raw product 31g.
By detecting the yield about 91% of hEGF, activity concentrates in hEGF raw product.HEGF content is as shown in table 2:
Table 2
example three
1. 100 kilograms, macroporous type anionite-exchange resin is as sorbent material, after manipulation of regeneration, water punching is to neutral, loading aperture is in 50 object filter cloth bags, in the just bucket filter cloth bag installing resin being placed on the more lavatory of flow of the people or urinal, makes urine or is flow through resin by the urine after water dilutes, after 24 hours, collect the resin of adsorbing urine protein, resin tap water after absorption is rinsed well, finally fills post; Carry out wash-out with the NaCl of 1M, collect elution peak.
2. carry out ultrafiltration and concentration with the ultra-filtration membrane of molecular weight cut-off 10K, regulate pH to 8.0 and conductance to 2.2mS/cm ultrafiltration and concentration liquid, on the metal ion that balanced be Zn
2+metal chelate chromatography post (Chelating sepharoseFF); Balance liquid (balance liquid formula: 0.02M phosphate buffered, 0.5MNaCl, pH8.0) is used to rinse metal chelate chromatography post again; Metal chelate chromatography post is rinsed with the 0.2M acetate buffer (pH3.0) containing 0.1M sodium-chlor; Use 0.1M phosphate buffered saline buffer, the NH of 0.8M
4cl, pH7.5, elution chromatography post, collects elution peak (E liquid).
3. the E liquid will collected, add ammonium sulfate powder to saturated, limit edged stirs, and leaves standstill 8 hours, and E liquid adds the centrifugal filter of diatomite 20g and collects and dry up precipitation, obtains hEGF raw product 31g.
By detecting the yield about 94% of hEGF, activity concentrates in hEGF raw product.HEGF content is as shown in table 3:
Table 3
| Sample ID | Weight (g) | HEGF content (U) |
| HEGF raw product | 31 | 1.66×10 6 |
Claims (6)
1. prepare the method for hEGF raw product for one kind, it is characterized in that: collect the sorbent material after adsorbing urine protein, concentrate wash-out, by the metal chelate chromatography post that the urine protein solution loading eluted has balanced, collect the elution peak containing hEGF component, through ammonium sulfate precipitation, preparation hEGF raw product, comprises the steps:
1), in urinal anionite-exchange resin being placed on Public toilets as sorbent material or urine funnel, the urine protein flowed through in urine or diluted urine is adsorbed;
2) collect the resin of adsorbing urine protein, carry out cleaning, desorb process;
3) ultrafiltration and concentration;
4) ultrafiltration and concentration liquid regulates pH and conductance, the metal chelate chromatography post that loading has balanced, then rinses with balance liquid;
5) use elute soln wash-out, collect the elution peak containing hEGF component;
6) in the elution peak collected, add ammonium sulfate powder to saturated, leave standstill after 2-10 hour, collecting precipitation, obtains hEGF raw product.
2. the method preparing hEGF raw product according to claim 1, is characterized in that: the metal ion of described metal chelate chromatography post chelating is Cu
2+, Zn
2+, Ni
2+, Fe
3+in any one.
3. the method preparing hEGF raw product according to claim 1, is characterized in that, metal chelate chromatography column equilibration liquid is 0.01-0.2M phosphate buffered saline buffer, and NaCl concentration is 0.2-2.0M, pH6.0-8.0.
4. the method preparing hEGF raw product according to claim 1, is characterized in that: described metal chelate chromatography post elute soln is 0.05-0.2M phosphate buffered saline buffer, and NH4Cl concentration is 0.5-1.0M, pH6.0-8.0.
5. the method preparing hEGF raw product according to claim 1, is characterized in that, in step 4, ultrafiltration and concentration liquid regulates pH6-8, conductance 0.5-10mS/cm.
6. the method preparing hEGF raw product according to claim 1, it is characterized in that, between step 4 and step 5, increase a rinse step, use pH3-6, NaCl concentration≤0.2M rinses metal chelate chromatography post, and described washing fluid buffer medium is 0.01-0.2M phosphoric acid salt or acetate.
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| CN104497127A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Preparation method of human epidermal growth factor |
| CN107987150A (en) * | 2017-12-19 | 2018-05-04 | 江苏艾迪药业有限公司 | A kind of method that hEGF can be prepared in the slave urine of industrialized production |
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| KR960009051B1 (en) * | 1992-05-26 | 1996-07-10 | 재단법인 목암생명공학연구소 | Simultaneous preparation process of useful proteins from human urine |
| SG171764A1 (en) * | 2008-12-16 | 2011-07-28 | Millipore Corp | Purification of proteins |
| CN202155072U (en) * | 2011-06-27 | 2012-03-07 | 扬州艾迪生物科技有限公司 | Device applicable to adsorbing urine protein in urinal trough effectively |
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Effective date of registration: 20170809 Address after: 225008 Jiangsu city of Yangzhou province Liu Zhuang Weiyang Hanjiang Road Economic Development Zone No. 2 Patentee after: Jiangsu Addie Pharmaceutical Co., Ltd. Address before: 225008 Jiangsu city of Yangzhou province from the Hanjiang District of Weiyang Road Economic Development Zone No. 2 Patentee before: Yangzhou Aidea Biotech Co., Ltd. |
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Address after: 225008 Liu Zhuang Road, Hanjiang District, Yangzhou, Jiangsu Province, No. 2 Patentee after: Jiangsu Aidi Pharmaceutical Co., Ltd. Address before: 225008 No. 2, Liuzhuang Road, Weiyang Economic Development Zone, Hangjiang District, Yangzhou City, Jiangsu Province Patentee before: Jiangsu Addie Pharmaceutical Co., Ltd. |