CN102532257B - Method for purifying recombinant human proinsulin - Google Patents
Method for purifying recombinant human proinsulin Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and particularly relates to a method for purifying recombinant human proinsulin, in particular to a method for purifying recombinant human proinsulin by using macroporous absorbent resin and cation exchange resin. In the method, a recombinant human proinsuli plural liquid is adsorbed and concentrated through the macroporous absorbent resin, and is loaded onto the cation exchange resin for further purifying, so that the collected proinsulin has high yield and high purity, and can be directly applied to enzyme digestion to obtain insulin. The method is easy and convenient to operate, has high process cohesion, and is suitable for industrial production.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of insulinogenic method of purification of recombinant human, particularly one utilizes macroporous adsorbent resin and the insulinogenic method of Zeo-karb purification of recombinant human.
Background technology
Regular Insulin is the still irreplaceable specifics for diabetes so far, the diabetic subject in the present whole world is more than 1.2 hundred million, and this numeral is all increasing progressively every year, expecting 2025 is 300,000,000, each diabetic subject needs to use 1.5 ~ 2.0 milligrams, Regular Insulin every day, and the whole world will consume about 5000 kilograms, Regular Insulin every year.Therefore researching and developing low cost, to prepare the method for Regular Insulin on a large scale imperative.
Regular Insulin is a kind of proteohormone secreted as the stimulation of glucose, lactose, ribose, arginine, hyperglycemic-glycogenolytic factor etc. by endogenous or exogenous material by beta Cell of islet, is uniquely fall hypoglycemic hormone in body.In current numerous patent, disclose a large amount of insulin derivates or analogue and corresponding method for making (CN1043719A, CN1068501A, CN1126761A, CN1126761A, CN1175106C, CN1247616C), these method for makings are divided into direct chemical synthesis and method of gene recombination, method of gene recombination comprises the secretion preparation method (CN100432104C utilizing colibacillary inclusion body preparation method (CN1059703C, ZL98812757.1, CN1663960A etc.) and utilize yeast cell, CN1873006A, CN101029077B etc.).
In the process preparing Regular Insulin, beta Cell of islet first synthesizes a macromolecular preproinsulin, is then processed into the proinsulin of 86 peptides, then becomes Regular Insulin through hydrolysis.Proinsulin comprise composition A, B chain of Regular Insulin and one be processed into C peptide cut in ripe insulin course, configuration is as follows: propetide-B-Arg Arg-C-Lys Arg-A.Chinese invention patent application CN101717442A discloses the technical scheme of polyethylene glycol recombination DesB 30 human insulin and its preparation method and application, in this technical scheme, the host cell of recombinant expression vector is pichia spp, and about insulinogenic purification process be after filtering fermentation liquor after absorption with macroporous adsorbent resin with the ethanol elution of 60%, then be further purified through strong cation exchange chromatography, precipitate through zinc chloride again after purifying, cut for enzyme after collection proinsulin precipitates and again dissolves and obtain Regular Insulin, it is more loaded down with trivial details that the program prepares insulinogenic production technique, proinsulin finally exists with the form of precipitation, be unfavorable for that enzyme cuts directly carrying out of step.
Chinese invention patent specification sheets CN88102311.6 discloses a kind of crystalline human proinsulin and production method thereof, first this technical scheme adds cationic salts in containing insulin human's original solution, pH is regulated to be 5.4 ~ 6.5 again, crystal is generated, then the proinsulin human of crystallization is reclaimed, although the method simplifies production technique, product yield is low, and proinsulin still exists with precipitation forms.Chinese invention patent specification sheets ZL98812757.1 discloses the preparation method of proinsulin human, this technical scheme is: inclusion body is first through dissolving and sulfonation, renaturation after cyanogen bromide process and cation exchange chromatography again, the correct proinsulin human of refolding is through adsorption chromatography chromatography, then the proinsulin human existed with precipitation forms is obtained through zinc chloride precipitation, can obtain the productive rate of 90%, the method complex manufacturing, product still exists with precipitation forms.
In method disclosed above, operating procedure is complicated, and the precursor proinsulin of Regular Insulin finally exists with the form of precipitation, and be unfavorable for that follow-up enzyme cuts step direct control, technique connecting is poor, and insulinogenic yield and purity can not be taken into account simultaneously.Therefore, need to develop that a kind of purification efficiency is high, connecting good, and more simple and effective technique prepares proinsulin, is beneficial to prepare Regular Insulin on a large scale.
Summary of the invention
In order to solve problems of the prior art, simplify Escherichia coli fermentation recombinant insulinum primary purification step, improve insulinogenic yield and purity, connecting between enhanced process, contriver utilizes recombinant insulinum primary can be concentrated by absorption with macroporous adsorbent resin, and in acid condition can by cationic exchange resin adsorption, and the feature of desorption in the basic conditions.The invention provides a kind of insulinogenic method of purification of recombinant human, comprise macroporous adsorption resin chromatography and cation exchange resin layer is analysed.
First, by the colibacillus engineering containing recombinant insulinum primary gene through fermentation, centrifugal, broken bacterium, wash after collecting inclusion body, after dissolving, renaturation obtains recombinant insulinum primary renaturation solution.
In the present invention, macroporous adsorption resin chromatography concrete steps are:
With deionized water (acetic acid adjusts pH to be 3.0 ~ 5.0) balance macroporous adsorbent resin, by recombinant insulinum primary renaturation solution (hydrochloric acid adjusts pH to be 3.0 ~ 5.0) loading to macroporous adsorption resin chromatography post, loading flow velocity is 200 ~ 500cm/h, use deionized water (acetic acid adjusts pH to be 3.0 ~ 5.0) cleaning 5 ~ 10 column volumes again, cleaning flow velocity is 200 ~ 500cm/h, it is finally ethanol (acetic acid adjusts pH to be 3.0 ~ 5.0) the desorption wash-out of 65% ~ 95% by concentration, elution flow rate is 50 ~ 200cm/h, collects desorption elutriant.
The present invention carries out preferably macroporous adsorbent resin type, and preferably, above-mentioned macroporous adsorbent resin is D-101, D1300, X-5 type macroporous adsorbent resin, be more preferably D-101 type macroporous adsorbent resin.
The present invention carries out preferably the blade diameter length ratio of macroporous adsorption resin chromatography post, and preferably, the blade diameter length ratio of above-mentioned chromatography column is 1: 5 ~ 20, is more preferably 1: 10 ~ 15.
The pH of the present invention to balance macroporous absorption chromatography deionized water used carries out preferably, and preferably, the pH of above-mentioned deionized water is 3.5 ~ 4.0.
The present invention carries out preferably the pH of recombinant insulinum primary renaturation solution, and preferably, the pH of above-mentioned recombinant insulinum primary renaturation solution is 3.5 ~ 4.0.
The loading flow velocity of the present invention to macroporous adsorption resin chromatography carries out preferably, and preferably, above-mentioned loading flow velocity is 300 ~ 400cm/h.
The pH of the present invention to cleaning macroporous adsorption resin chromatography deionized water used carries out preferably, and preferably, the pH of above-mentioned deionized water is 3.5 ~ 4.0.
The cleaning flow velocity of the present invention to macroporous adsorption resin chromatography carries out preferably, and preferably, above-mentioned cleaning flow velocity is 300 ~ 400cm/h.
The present invention carries out preferably the elution flow rate of macroporous adsorption resin chromatography, and preferably, above-mentioned elution flow rate is 100cm/h.
The present invention carries out preferably the alcohol concn of desorption wash-out, and preferably, the concentration of above-mentioned ethanol is 85% ~ 95%.
The ethanol pH of the present invention to desorption wash-out carries out preferably, and preferably, the pH of above-mentioned ethanol is 3.5 ~ 4.0.
The concrete steps of cationic exchange resin chromatography of the present invention are:
Adjust pH to be 5.0 ~ 7.0 with hydrochloric acid after the desorption elutriant that macroporous adsorption resin chromatography obtains mixes with deionized water equal-volume, room temperature leaves standstill and occurs flocks after 1 ~ 2 hour, centrifuging and taking supernatant liquor be 3.0 ~ 5.0 with hydrochloric acid polyphony pH.
First use deionized water (acetic acid adjusts pH to be 3.0 ~ 5.0) balance cation exchange resin, by pH be 3.0 ~ 5.0 supernatant liquor loading analyse post to cation exchange resin layer, loading flow velocity is 50 ~ 200cm/h, use deionized water (acetic acid adjusts pH to be 3.0 ~ 5.0) cleaning 5 ~ 10 column volumes again, cleaning flow velocity is 50 ~ 200cm/h, finally with pH be 8.5 ~ 10.5, the bicarbonate of ammonia wash-out of 0.1mol/L, elution flow rate is 50 ~ 200cm/h, collects elutriant.
The present invention carries out preferably the filler of Zeo-karb, and preferably, the filler of above-mentioned Zeo-karb is SP Sepharose Fast Flow.
The present invention carries out preferably to desorption elutriant and the mixed pH of deionized water, preferably, is 5.0 ~ 6.0 with hydrochloric acid tune pH after above-mentioned desorption elutriant mixes with deionized water.
The pH of the present invention to the supernatant liquor with hydrochloric acid polyphony gained carries out preferably, and preferably, the pH of above-mentioned supernatant liquor is 3.5 ~ 4.0.
The pH of the present invention to balance cation displacement chromatography deionized water used carries out preferably, and preferably, the pH of above-mentioned deionized water is 3.5 ~ 4.0.
PH that the present invention analyses deionized water used to cleaning cation exchange resin layer carries out preferably, and preferably, the pH of above-mentioned deionized water is 3.5 ~ 4.0.
The loading flow velocity that the present invention analyses cation exchange resin layer carries out preferably, and preferably, above-mentioned loading flow velocity is 100cm/h.
The cleaning flow velocity that the present invention analyses cation exchange resin layer carries out preferably, and preferably, above-mentioned cleaning flow velocity is 100cm/h.
The present invention carries out preferably the pH of bicarbonate of ammonia, and preferably, the pH of above-mentioned bicarbonate of ammonia is 9.0 ~ 10.0.
The present invention carries out preferably the elution flow rate that cation exchange resin layer is analysed, and preferably, above-mentioned elution flow rate is 100cm/h.
The present invention compared with prior art has following outstanding advantage:
Purification process provided by the invention utilizes macroporous adsorption resin chromatography to the efficient adsorption of recombinant insulinum primary and desorption, solve the problem that purification yield that renaturation solution volume brings greatly is low, use cation exchange resin layer to analyse simultaneously and effectively desorption buffer exchange is become time buffer system of step enzyme cutting process, and by optimizing cleaning and elution requirement, effectively removes impurity, the proinsulin yield obtained by technical scheme of the present invention reaches more than 90%, purity is more than 95%, and can directly carry out the operation of enzyme cutting process by the proinsulin that the present invention obtains, ensure that technique connecting, this technological operation is easy, reduce production cost, be conducive to the suitability for industrialized production of Regular Insulin.
Embodiment
Further describe the present invention below by way of specific embodiment, but the present invention is not limited only to following examples.
The preparation of embodiment 1, recombinant insulinum primary renaturation solution:
Colibacillus engineering containing recombinant insulinum primary gene, centrifugal that thalline is about 17.5kg after high-density cells fermentation, thalline is the centrifugal recombinant insulinum primary obtaining existing with inclusion bodies after homogeneous, inclusion body dissolves after washing under Denaturing, then renaturation, obtains about 500L renaturation solution.
Embodiment 2, macroporous adsorption resin chromatography purifying:
Filling D-101 macroporous adsorbent resin is to glass chromatography column (blade diameter length ratio is 1: 15), deionized water (acetic acid adjusts pH to be 3.5) balance chromatography media, the renaturation solution (hydrochloric acid adjusts pH to be 3.5) obtained according to embodiment 1 is total to about 500L through peristaltic pump loading, loading flow velocity is kept to be 300cm/h, with deionized water (acetic acid adjusts pH to be 3.5) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 300cm/h, then the ethanol of 95% (acetic acid adjusts pH to be 3.5) is used to carry out wash-out, elution flow rate remains 100cm/h, collection obtains about 11.8L elutriant.
Embodiment 3, macroporous adsorption resin chromatography purifying:
Filling AB-8 macroporous adsorbent resin is to glass chromatography column (blade diameter length ratio is 1: 20), deionized water (acetic acid adjusts pH to be 5.0) balance chromatography media, the renaturation solution (hydrochloric acid adjusts pH to be 5.0) obtained according to embodiment 1 is total to about 500L through peristaltic pump loading, loading flow velocity is kept to be 200cm/h, with deionized water (acetic acid adjusts pH to be 5.0) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 200cm/h, then the ethanol of 65% (acetic acid adjusts pH to be 5.0) is used to carry out wash-out, elution flow rate remains 50cm/h, collects and obtains about 12.5L elutriant.
Embodiment 4, macroporous adsorption resin chromatography purifying:
Filling D1300 macroporous adsorbent resin is to glass chromatography column (blade diameter length ratio is 1: 5), deionized water (acetic acid adjusts pH to be 3.0) balance chromatography media, the renaturation solution (hydrochloric acid adjusts pH to be 3.0) obtained according to embodiment 1 is total to about 500L through peristaltic pump loading, loading flow velocity is kept to be 400cm/h, with deionized water (acetic acid adjusts pH to be 3.0) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 400cm/h, then the ethanol of 75% (acetic acid adjusts pH to be 3.0) is used to carry out wash-out, elution flow rate remains 200cm/h, collects and obtains about 13.1L elutriant.
Embodiment 5, macroporous adsorption resin chromatography purifying:
Filling X-5 macroporous adsorbent resin is to glass chromatography column (blade diameter length ratio is 1: 10), deionized water (acetic acid adjusts pH to be 4.0) balance chromatography media, the renaturation solution (hydrochloric acid adjusts pH to be 4.0) obtained according to embodiment 1 is total to about 500L through peristaltic pump loading, loading flow velocity is kept to be 500cm/h, with deionized water (acetic acid adjusts pH to be 4.0) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 500cm/h, then the ethanol of 85% (acetic acid adjusts pH to be 4.0) is used to carry out wash-out, elution flow rate remains 100cm/h, collects and obtains about 12.1L elutriant.
Embodiment 6, cation exchange resin layer are analysed:
It is 5.0 with deionized water equal-volume with salt acid for adjusting pH that the desorption elutriant that macroporous adsorption resin chromatography obtains mixes rear, room temperature leaves standstill and occurs flocks after 1 ~ 2 hour, with 8000rpm, after room temperature is centrifugal, get supernatant liquor, and be 3.5 with the pH of hydrochloric acid polyphony supernatant liquor.
Be 10cm with diameter, it is highly the axial compression glass chromatography column filling SP sepharose Fast Flow resin of 30cm, with deionized water (acetic acid adjusts pH to be 3.5) balance chromatography media, pH is that the supernatant liquor of 3.5 is through peristaltic pump loading, loading flow velocity is 100cm/h, with deionized water (acetic acid adjusts pH to be 3.5) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 100cm/h, then 0.1mol/L bicarbonate of ammonia (sodium hydroxide adjusts pH to be 9.0) is used to carry out wash-out, elution flow rate remains 100cm/h, collection obtains about 13.2L elutriant, detect recombinant insulinum primary yield is 91.2%, purity is 96.9%.
Embodiment 7. cation exchange resin layer is analysed:
PH is adjusted to be 6.0 with hydrochloric acid after the desorption elutriant that macroporous adsorption resin chromatography obtains mixes with deionized water equal-volume, room temperature leaves standstill and occurs flocks after 1 ~ 2 hour, with 8000rpm, get supernatant liquor after room temperature is centrifugal and be 4.0 with the pH of hydrochloric acid polyphony supernatant liquor;
Be 10cm with diameter, it is highly the axial compression glass chromatography column filling SP sepharose Fast Flow resin of 30cm, with the deionized water balance Zeo-karb that acetic acid tune pH is 4.0, by pH be 4.0 supernatant liquor loading analyse post to cation exchange resin layer, loading flow velocity is 50cm/h, with deionized water (acetic acid adjusts pH to be 4.0) cleaning 5 ~ 10 column volumes after loading, cleaning flow velocity is 50cm/h, use the bicarbonate of ammonia of 0.1mol/L (sodium hydroxide adjusts pH to be 8.5) wash-out again, elution flow rate is 50cm/h, collect elutriant, detect recombinant insulinum primary yield is 90.3%, purity is 95.8%.
Embodiment 8. cation exchange resin layer is analysed:
PH is adjusted to be 7.0 with hydrochloric acid after the desorption elutriant that macroporous adsorption resin chromatography obtains mixes with deionized water equal-volume, room temperature leaves standstill and occurs flocks after 1 ~ 2 hour, with 8000rpm, get supernatant liquor after room temperature is centrifugal and be 5.0 with the pH of hydrochloric acid polyphony supernatant liquor;
Be 10cm with diameter, it is highly the axial compression glass chromatography column filling SP Sepharose Fast Flow resin of 30cm, with the deionized water balance Zeo-karb that acetic acid tune pH is 5.0, by pH be 5.0 supernatant liquor loading analyse post to cation exchange resin layer, loading flow velocity is 200cm/h, be washed with de-ionized water 5 ~ 10 column volumes of 5.0 again with acetic acid tune pH, cleaning flow velocity is 200cm/h, finally use the bicarbonate of ammonia of 0.1mol/L (sodium hydroxide adjusts pH to be 10.5) wash-out, elution flow rate is 200cm/h, collect elutriant, detect recombinant insulinum primary yield is 90.5%, purity is 97.0%.
Embodiment 9. cation exchange resin layer is analysed:
PH is adjusted to be 5.0 with hydrochloric acid after the desorption elutriant that macroporous adsorption resin chromatography obtains mixes with deionized water equal-volume, room temperature leaves standstill and occurs flocks after 1 ~ 2 hour, with 8000rpm, get supernatant liquor after room temperature is centrifugal and be 3.0 with the pH of hydrochloric acid polyphony supernatant liquor;
Be 10cm with diameter, it is highly the axial compression glass chromatography column filling SP Sepharose Fast Flow resin of 30cm, with the deionized water balance Zeo-karb that acetic acid tune pH is 3.0, by pH be 3.0 supernatant liquor loading analyse post to cation exchange resin layer, loading flow velocity is 100cm/h, be washed with de-ionized water 5 ~ 10 column volumes of 3.0 again with acetic acid tune pH, cleaning flow velocity is 100cm/h, finally use the bicarbonate of ammonia of 0.1mol/L (sodium hydroxide adjusts pH to be 10) wash-out, elution flow rate is 100cm/h, collect elutriant, detect recombinant insulinum primary yield is 91.5%, purity is 96.3%.
Claims (1)
1. the insulinogenic method of purification of recombinant human, is characterized in that comprising following steps,
A. macroporous adsorption resin chromatography:
With the deionized water balance macroporous adsorbent resin that acetic acid tune pH is 3.0-5.0, pH is adjusted by hydrochloric acid to be that the recombinant insulinum primary renaturation solution loading of 3.0-5.0 is to macroporous adsorption resin chromatography post, loading flow velocity is 200-500cm/h, be washed with de-ionized water 5-10 the column volume of 3.0-5.0 again with acetic acid tune pH, cleaning flow velocity is 200-500cm/h, finally adjust with acetic acid the ethanolysis absorb-elute that pH is 3.0-5.0, concentration is 65%-95%, elution flow rate is 50-200cm/h, collects desorption elutriant;
B. cation exchange resin layer is analysed:
Adjusting pH to be 5.0-7.0 with hydrochloric acid after the desorption elutriant that macroporous adsorption resin chromatography obtains mixes with deionized water equal-volume, there is flocks after leaving standstill 1-2 hour in room temperature, centrifuging and taking supernatant liquor be 3.0-5.0 with hydrochloric acid polyphony pH;
It is first the deionized water balance Zeo-karb of 3.0-5.0 with acetic acid tune pH, by pH be 3.0-5.0 supernatant liquor loading analyse post to cation exchange resin layer, loading flow velocity is 50-200cm/h, be washed with de-ionized water 5-10 the column volume of 3.0-5.0 again with acetic acid tune pH, cleaning flow velocity is 50-200cm/h, be finally the bicarbonate of ammonia wash-out of 8.5-10.5,0.lmol/L with pH, elution flow rate is 50-200cm/h, collects elutriant; Chromatography column filler is SP sepharose FastFlow.
2., according to the method described in claim 1, it is characterized in that macroporous adsorbent resin described in step a is D-101, AB-8, D1300, X-5 type macroporous adsorbent resin.
3., according to the method described in claim 1, it is characterized in that the blade diameter length ratio of the post of macroporous adsorption resin chromatography described in step a is 1:5-20.
4., according to the method described in claim 1, it is characterized in that the flow velocity in step a in macroporous adsorption resin chromatography loading, cleaning process is 300-400cm/h.
5., according to the method described in claim 1, it is characterized in that the flow velocity in step a in macroporous adsorption resin chromatography elution process is 1OOcm/h.
6. according to the method described in claim 1, it is characterized in that in step a from the alcohol concn of desorption wash-out macroporous adsorbent resin be 85%-95%.
7., according to the method described in claim 1, it is characterized in that the flow velocity in the loading in step b, cleaning, elution process is 1OOcm/h.
8., according to the method described in claim 1, it is characterized in that the pH of bicarbonate of ammonia in elution process in step b is 9.0-10.0.
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| CN103833828B (en) * | 2012-11-21 | 2017-01-11 | 广东东阳光药业有限公司 | Extraction method of insulin glargine precursor protein |
| CN103145829B (en) * | 2013-03-29 | 2015-06-03 | 江苏诺泰制药有限公司 | Purification method of insulin detemir |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029077A (en) * | 2007-02-02 | 2007-09-05 | 广东东阳光药业有限公司 | Method for purifying gene-recombinant insulin precursor |
| CN101173006A (en) * | 2006-10-30 | 2008-05-07 | 江苏万邦生化医药股份有限公司 | Method for producing recombined insulin human |
| CN101717442A (en) * | 2008-10-09 | 2010-06-02 | 重庆富进生物医药有限公司 | Polyethylene glycol recombination DesB30 human insulin, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173006A (en) * | 2006-10-30 | 2008-05-07 | 江苏万邦生化医药股份有限公司 | Method for producing recombined insulin human |
| CN101029077A (en) * | 2007-02-02 | 2007-09-05 | 广东东阳光药业有限公司 | Method for purifying gene-recombinant insulin precursor |
| CN101717442A (en) * | 2008-10-09 | 2010-06-02 | 重庆富进生物医药有限公司 | Polyethylene glycol recombination DesB30 human insulin, preparation method and application thereof |
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