CN103159849B - Recombinant human proinsulin preparation method - Google Patents
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Abstract
The invention belongs to the field of biomedicine, and particularly relates to a recombinant human proinsulin preparation method, specifically to a method for preparing recombinant human proinsulin by using an expanded bed absorption technology. According to the present invention, target protein is captured from a complex material solution containing recombinant human proinsulin in one step, and salt concentration gradient elution is performed so as to obtain high purity proinsulin protein, wherein a purity of the obtained recombinant human proinsulin is more than 90%; and the method has characteristics of simple process and convenient operation, wherein a production time is shortened with a high flow rate, production efficiency is increased, a yield of the purified recombinant human proinsulin is more than 85%, and the method is particularly suitable for industrial production of insulin.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of method preparing recombinant insulinum primary, particularly a kind of method utilizing ExPANDED BED ADSORPTION TECHNIQUE to prepare recombinant insulinum primary.
Background technology
The quantity of whole world diabetics increases just year by year, and expecting 2025 will reach 300,000,000, and Regular Insulin is the specific medicament of still widely used treatment diabetes so far, and therefore the consumption of Regular Insulin also can along with increasing progressively.The clinical application amount of Regular Insulin is large, and each diabetic subject need use about 1.5 ~ 2.0 milligrams, Regular Insulin every day, and lifelong medication, therefore develops a kind of processing method preparing Regular Insulin for large-scale low-cost and has good commercial value and social effect.Along with the fast development of biotechnology, genetically engineered recombinant human insulin emerge, and comparatively animal-origin Regular Insulin advantage is more obvious, completely the same with the sequence of Regular Insulin in human body, and purity is higher, few containing host protein, safe without toxic side effect.Recombinant human insulin used is clinically mainly expressed by E. coli system and Yeast system now.E. coli system is produced Regular Insulin and has been cooperated in nineteen eighty-two by Genetech company and Lilly company at first, and present multiplex fusion rotein makes it form inclusion body, is folded into proinsulin, after ferment treatment, forms Regular Insulin after solubilization of inclusion bodies.Yeast system is produced Regular Insulin and was studied successfully in 1986, and in yeast, the proinsulin of secreting, expressing can processing Regular Insulin further.Therefore no matter be with E. coli system or yeast expression system, all want first Expression product proinsulin, then be processed into insulin human further.A kind of simple process method preparing recombinant insulinum primary is provided to be the key of scale operation recombinant human insulin.
The filtration supernatant of renaturing inclusion bodies of colibacillus liquid or yeast expression nutrient solution all also exists such feature: the concentration of the recombinant insulinum primary contained is low; Containing a large amount of pigment and foreign protein; Material liquid volume is large, needs subsequent concentration process, therefore higher to processing requirement.Chinese invention patent CN101029077B discloses a kind of gene recombination proinsulin human purification process, and its technical scheme is yeast fermentation broth → add activated carbon → stirring → centrifugal → supernatant liquor → active carbon column → permeate → Cationic column chromatography → containing the former elutriant of gene-recombinant insulin.Due to a large amount of pigment and some foreign proteins can be produced in pichia spp fermentative production Recombulin process, the existence of pigment has a strong impact on the separation and purification in downstream, the method adopts the method for twice activated carbon treatment to remove pigment, purify target protein with cation seperation column again, but the activated carbon for decolouring easily brings the pollution of production environment.Chinese invention patent application prospectus CN1290299A discloses a kind of preparation method of recombinant insulinum primary, this technical scheme is by after feed liquid loading HP-2MG polarity methacrylate resin (ratio of every 1L resin 8g protein), wash post with 20mM acetate buffer, then use 30% acetone wash-out.Evaporation of eluate to remove acetone, thus obtains recombinant insulinum primary, but uses acetone to carry out wash-out, needs follow-up evaporation step to remove acetone, may affect product stability.
Since 1993 come out, Expanded Bed Adsorption has been widely used in the research and development of gene engineering product, the advantage of Expanded Bed Adsorption is can directly by the direct loading of material without refiner impurity, eliminate step that is centrifugal or ultrafiltration, a step can play the effect of clarification, concentrated and preliminary purification.ExPANDED BED ADSORPTION TECHNIQUE is appeared in the newspapers in the application of genetically engineered association area, Chinese invention patent application prospectus CN1727361A discloses a kind of purifying process of recombinant human interferon alpha 1 b, the engineering strain that this technical scheme is used for from expressing human interferon alpha 1 b extracts recombinant human interferon alpha 1 b, comprise bacteria breaking, Expanded Bed Adsorption, affinity chromatography and gel filtration step, thus production efficiency is improved greatly, production cost reduces greatly simultaneously.Chinese invention patent application prospectus CN1800379A discloses a kind of method that ExPANDED BED ADSORPTION TECHNIQUE and affinity chromatography technology prepare pancreatic kallikrein, this technical scheme is first with ExPANDED BED ADSORPTION TECHNIQUE directly first Purification of Pig pancreas extracting solution, obtained high purity pancreatic kallikrein is further purified again by affinity chromatography technology, thus simplify production stage, improve production efficiency, improve product quality, be suitable for suitability for industrialized production.But whether ExPANDED BED ADSORPTION TECHNIQUE can be used for the purifying of the recombinant insulinum primary of Yeast system and E. coli system expression, yet there are no relevant report.
In the preparation technology of the recombinant insulinum primary of bibliographical information, ubiquitous problem is that production process is complicated, feed liquid containing recombinant insulinum primary need could obtain the higher proinsulin of purity by the processing step more than at least two steps or two steps, and the result that processing step causes more is exactly the decline of yield and the increase of production cost.
Summary of the invention
In order to solve problems of the prior art, the present inventor is according to insulinogenic physico-chemical property, through a large amount of tests, more suitable processing condition are screened, reduce processing step, improve the purity that ensure that product while product yield, provide a kind of novel method preparing recombinant insulinum primary, the method mainly make use of ExPANDED BED ADSORPTION TECHNIQUE one step from containing catching the method that also purified insulin is former the feed liquid of recombinant insulinum primary.
Initial feed liquid in the present invention is the filtration supernatant of the feed liquid containing recombinant insulinum primary, i.e. the renaturing inclusion bodies liquid of escherichia coli expression, or yeast expression nutrient solution.
Method of the present invention utilizes expanding bed resin column to prepare recombinant insulinum primary, comprises the steps:
A, column equilibration: expanding bed resin column first balances about 3 ~ 10 column volumes by moving phase I, and equilibrium velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, make resin in post be expanded to 1 ~ 5 times of fixed bed volume;
B, loading: be loaded to expanding bed resin column by containing the feed liquid of recombinant insulinum primary, loading flow velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, make resin in post be expanded to 1 ~ 5 times of fixed bed volume;
C, washing: loading is complete, wash about 3 ~ 10 column volumes by moving phase II, washing flow velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, makes resin in post be expanded to 1 ~ 5 times of fixed bed volume;
D, wash-out: make resin natural subsidence, capital sparger is dropped to resin surface and compresses resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 50 ~ 500cm/h, by moving phase II: moving phase III gradient elution about 5 ~ 20 column volumes, collect elution peak.
The present invention to carry out preferably to expanding bed resin, preferably, above-mentioned expanding bed resin is ion-exchange type resin or hydrophobic adsorbent type resin, more preferably, above-mentioned expanding bed resin is Streamline SP XL, or Streamline Q is XL, or Streamline phenyl is XL, most preferably, above-mentioned expanding bed resin is Streamline SP XL.
The present invention carries out preferably to moving phase I component during balanced expansion bed resin column in step a, and preferably, moving phase I is salt concn is the damping fluid of 0 ~ 1.0M, the damping fluid of pH3.0 ~ 10.0; More preferably, moving phase I is 10mMNaAc-HAc, pH3.0 ~ 6.0,0 ~ 0.8M NaCl, or 10mM is Tris-HCl, pH8.0 ~ and 10.0,0 ~ 0.8MNaCl; Most preferably, moving phase I is 10mM NaAc-HAc, pH4.0, or 10mM Tris-HCl, pH9.0 or 10mM NaAc-HAc, pH4.0,0.5M be NaCl, or 10mM Tris-HCl, pH9.0,0.5M be NaCl.
The present invention carries out preferably the equilibrium velocity of expanding bed resin in step a, and preferably, the equilibrium velocity of above-mentioned expanding bed resin is 600 ~ 800cm/h.
The present invention carries out preferably the expansion multiple in step a during balanced expansion bed resin, and preferably, above-mentioned expanding bed resin should be expanded to 2 ~ 4 times of fixed bed volume.
The present invention carries out preferably the flow velocity in step b during expanding bed resin column loading, and preferably, the loading flow velocity of above-mentioned expanding bed resin is 600 ~ 800cm/h.
The present invention carries out preferably to expansion multiple during loading expanding bed resin in step b, and preferably, above-mentioned expanding bed resin should be expanded to 2 ~ 3 times of fixed bed volume.
The present invention carries out preferably the washing flow velocity of expanding bed resin column in step c, and preferably, the washing flow velocity of above-mentioned expanding bed resin is 600 ~ 800cm/h.
The present invention carries out preferably expansion multiple when washing expanding bed resin column in step c, and preferably, above-mentioned expanding bed resin should be expanded to 2 ~ 4 times of fixed bed volume.
The present invention carries out preferably to moving phase II component when washing expanding bed resin column in step c, preferably, moving phase II for salt concn be the damping fluid of 0 ~ 0.5M, the damping fluid of pH3.0 ~ 10.0; More preferably, moving phase II is 10mMNaAc-HAc, pH3.0 ~ 6.0,0.1 ~ 0.5M NaCl or 10mM is Tris-HCl, pH8.0 ~ 10.0,0.1 ~ 0.5M NaCl; Most preferably, moving phase II is 10mMNaAc-HAc, pH4.0,0.5M NaCl or 10mM Tris-HCl, pH9.0,0.5M NaCl, or 10mM NaAc-HAc, pH4.0,0.2M be NaCl, or 10mM Tris-HCl, pH9.0,0.2M be NaCl.
The present invention carries out preferably the elution flow rate of expanding bed resin column in steps d, and preferably, the elution flow rate of above-mentioned expanding bed resin is 100 ~ 200cm/h.
Moving phase III component when the present invention carries out gradient elution to expanding bed resin column in steps d is carried out preferably, preferably, moving phase III for salt concn be the damping fluid of 0 ~ 2.0M, the damping fluid of pH3.0 ~ 10.0; More preferably, moving phase III is 10mM NaAc-HAc, pH3.0 ~ 6.0,0 ~ 1.5M NaCl or 10mM is Tris-HCl, pH8.0 ~ 10.0,0 ~ 1.5M NaCl; Most preferably, moving phase III is 10mM NaAc-HAc, pH4.0,1.0M NaCl or 10mM Tris-HCl, pH9.0,1.0M NaCl, or 10mM is NaAc-HAc, pH4.0, or 10mM is Tris-HCl, pH9.0.
The present invention carries out gradient elution program to expanding bed resin column in steps d and carries out preferably, preferably, described gradient elution program is linear gradient, and the volume ratio that namely moving phase III accounts for eluent system rises to 100% gradually by 40%, and elution volume is 10 ~ 15 column volumes.
The present invention prepares the recombinant insulinum primary of gained, its aminoacid sequence and the former aminoacid sequence of natural human insulin identical or different.
The present invention compared with prior art has following outstanding advantage:
Expanding bed resin can directly by the material loading without refiner impurity, a step can play the effect of clarification/concentrated and preliminary purification.Target protein is caught from the step the feed liquid of recombinant insulinum primary that contains of complexity, simultaneous test is known, through processing method provided by the invention through salt concentration gradient wash-out, obtain highly purified proinsulin albumen, the purity of the recombinant insulinum primary of gained reaches more than 90%.And method technological process provided by the invention is simple, easy to operate, and preferred flow velocity shortens the production time, improve production efficiency, therefore greatly can reduce production cost, the insulinogenic yield of purification of recombinant human reaches more than 85%, is particularly suitable for the suitability for industrialized production of Regular Insulin.
Embodiment
Set forth the present invention further below by way of specific embodiment, these embodiments are only not used in restriction the present invention scope required for protection for illustration of the present invention.
The preparation of embodiment 1, recombinant insulinum primary
Streamline Q XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM Tris-HCl, pH9.0) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 200cm/h, makes expanding bed resin be expanded to 1 times of fixed bed volume, balances about 3 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 1000cm/h.Expanding bed resin is made to be expanded to 5 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 1000cm/h, makes expanding bed resin be expanded to 5 times of fixed bed volume, washs about 10 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 50cm/h, with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH9.0,1.0M NaCl) starts gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 5 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 89.2%, and purity is 91.3%.
The preparation of embodiment 2, recombinant insulinum primary
Streamline Q XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM Tris-HCl, pH9.0) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 700cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, balances about 10 column volumes;
Start loading and filter supernatant 50L containing the yeast expression nutrient solution of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 700cm/h.Expanding bed resin is made to be expanded to 1 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 600cm/h, makes expanding bed resin be expanded to 2 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 500cm/h, with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH9.0,1.0M NaCl) starts gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 20 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 90.5%, and purity is 94.5%.
The preparation of embodiment 3, recombinant insulinum primary
Streamline SP XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM sodium acetate-acetic acid, pH4.0) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 4 times of fixed bed volume, balances about 8 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 600cm/h.Expanding bed resin is made to be expanded to 2 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM sodium acetate-acetic acid, pH4.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, with moving phase II (10mM NaAc-HAc, pH4.0,0.5M NaCl): moving phase III (10mM NaAc-HAc, pH4.0,1.0M NaCl) starts gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 10 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 92.2%, and purity is 95.2%.
The preparation of embodiment 4, recombinant insulinum primary
Streamline SP XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM NaAc-HAc, pH4.0) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 600cm/h, makes expanding bed resin be expanded to 2 times of fixed bed volume, balances about 5 column volumes;
Start loading and filter supernatant 50L containing the yeast expression nutrient solution of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 800cm/h.Expanding bed resin is made to be expanded to 4 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM NaAc-HAc, pH4.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 700cm/h, makes expanding bed resin be expanded to 5 times of fixed bed volume, washs about 10 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 200cm/h, with moving phase II (10mM NaAc-HAc, pH4.0,0.5M NaCl): moving phase III (10mM NaAc-HAc, pH4.0,1.0M NaCl) starts gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 8 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 90.7%, and purity is 94.8%.
The preparation of embodiment 5, recombinant insulinum primary
Streamline phenyl XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM NaAc-HAc, pH4.0,0.5M NaCl) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, make expanding bed resin be expanded to 4 times of fixed bed volume, balance about 5 column volumes;
Start loading and filter supernatant 50L containing the yeast expression nutrient solution of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 200cm/h.Expanding bed resin is made to be expanded to 1 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM NaAc-HAc, pH4.0,0.2M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 200cm/h, makes expanding bed resin be expanded to 4 times of fixed bed volume, washs about 6 column volumes;
Wash complete, termination of pumping, makes resin natural subsidence, then capital is dropped to resin surface and compresses resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, with moving phase II (10mM NaAc-HAc, pH4.0,0.2M NaCl): moving phase III (10mM NaAc-HAc, pH4.0) start gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 5 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 91.6%, and purity is 94.1%.
The preparation of embodiment 6, recombinant insulinum primary
Streamline phenyl XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10Mm Tris-Cl, pH9.0,0.5M NaCl) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 1000cm/h, make expanding bed resin be expanded to 5 times of fixed bed volume, balance about 5 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 1000cm/h.Expanding bed resin is made to be expanded to 5 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM Tris-Cl, pH9.0,0.2M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 4 times of fixed bed volume, washs about 10 column volumes;
Wash complete, termination of pumping, makes resin natural subsidence, then capital is dropped to resin surface and compresses resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 200cm/h, with moving phase II (10mM Tris-Cl, pH9.0,0.2M NaCl): moving phase III (10mM Tris-Cl, pH9.0) start gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 10 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 89.8%, and purity is 90.4%.
The preparation of embodiment 7, recombinant insulinum primary
Streamline SP XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM sodium acetate-acetic acid, pH3.0) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 4 times of fixed bed volume, balances about 8 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 600cm/h.Expanding bed resin is made to be expanded to 2 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM sodium acetate-acetic acid, pH3.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, with moving phase II (10mMNaAc-HAc, pH3.0,0.5M NaCl): moving phase III (10mMNaAc-HAc, pH3.0,1.0M NaCl) start gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 5 column volumes.From the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 87.7%, and purity is 90.0%.
The preparation of embodiment 8, recombinant insulinum primary
Streamline SP XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), with moving phase I (10mM sodium acetate-acetic acid, pH6.0,0.8M NaCl) balance, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, make expanding bed resin be expanded to 4 times of fixed bed volume, balance about 8 column volumes;
Start loading and filter supernatant 50L containing the yeast expression nutrient solution of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 600cm/h.Expanding bed resin is made to be expanded to 2 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM sodium acetate-acetic acid, pH6.0,0.1M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, with moving phase II (10mM NaAc-HAc, pH6.0,0.1M NaCl): moving phase III (10mM NaAc-HAc, pH6.0,1.5M NaCl) starts gradient elution, gradient elution program is 40% ~ 100% moving phase III gradient elution, 20 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 88.1%, and purity is 90.6%.
The preparation of embodiment 9, recombinant insulinum primary
Streamline phenyl XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), balance by moving phase I (10mM Tris-HCl, pH8.0,0.8M NaCl), upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, make expanding bed resin be expanded to 4 times of fixed bed volume, balance about 8 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 600cm/h.Expanding bed resin is made to be expanded to 2 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM Tris-HCl, pH8.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, with moving phase II (10mM Tris-HCl, pH8.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH8.0) starts gradient elution, and gradient elution program is 40% ~ 100% moving phase III gradient elution, 6 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 87.1%, and purity is 90.3%.
The preparation of embodiment 10, recombinant insulinum primary
Streamline phenyl XL is poured into expanding bed chromatography column (Streamline 100 chromatography column, GE), balance by moving phase I (10mM Tris-HCl, pH10.0,0.5M NaCl), upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, make expanding bed resin be expanded to 4 times of fixed bed volume, balance about 8 column volumes;
Start loading containing the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, upwards feed liquor bottom expanding bed, adjustment loading flow velocity is 600cm/h.Expanding bed resin is made to be expanded to 2 times of fixed bed volume;
Loading is complete, and with moving phase II (10mM Tris-HCl, pH10.0,0.5M NaCl) washing, upwards feed liquor bottom expanding bed, flow velocity is adjusted to 800cm/h, makes expanding bed resin be expanded to 3 times of fixed bed volume, washs about 5 column volumes;
Wash complete, termination of pumping, make resin natural subsidence, then capital dropped to resin surface and compress resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted to 100cm/h, with moving phase II (10mM Tris-HCl, pH10.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH10.0) starts gradient elution, and gradient elution program is 40% ~ 100% moving phase III gradient elution, 6 column volumes, collects elution peak.
After testing and calculate, the yield of recombinant insulinum primary is 87.6%, and purity is 90.6%.
The preparation of embodiment 11, recombinant insulinum primary
Gradient elution program, with embodiment 3, changes into and directly carries out wash-out by moving phase III (10mM NaAc-HAc, pH4.0,1.0M NaCl), collect elution peak by other step.
After testing and calculate, the yield of recombinant insulinum primary is 75.6%, and purity is 84.3%.
The preparation of embodiment 12, recombinant insulinum primary
The flow velocity of column equilibration, loading, washing step, with embodiment 3, is adjusted to 1200cm/h by other step, and the elution flow rate of elution step is adjusted to 600cm/h.
After testing and calculate, the yield of recombinant insulinum primary is 70.1%, and purity is 80.3%.
Claims (1)
1. prepare a method for recombinant insulinum primary, it is characterized in that, utilize ExPANDED BED ADSORPTION TECHNIQUE from the renaturing inclusion bodies liquid of escherichia coli expression, or in the filtration supernatant of yeast expression nutrient solution, purified insulin is former, operation steps is:
A, column equilibration: expanding bed resin column first balances 3 ~ 10 column volumes by moving phase I, and equilibrium velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, make resin in post be expanded to 1 ~ 5 times of fixed bed volume;
B, loading: be loaded to expanding bed resin column by containing the feed liquid of recombinant insulinum primary, loading flow velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, make resin in post be expanded to 1 ~ 5 times of fixed bed volume;
C, washing: loading is complete, wash 3 ~ 10 column volumes by moving phase II, and washing flow velocity is 200 ~ 1000cm/h, and in pipeline, flow direction for upwards to flow bottom resin column, makes resin in post be expanded to 1 ~ 5 times of fixed bed volume;
D, wash-out: make resin natural subsidence, drop to resin surface by capital sparger and compress resin, and from the downward feed liquor in expanding bed top, flow velocity is adjusted to 50 ~ 500cm/h, by moving phase II: moving phase III gradient elution 5 ~ 20 column volumes, collects elution peak;
Wherein:
Described expanding bed resin is ion-exchange type or hydrophobic adsorbent type expanding bed resin; The component of described moving phase I is 10mM NaAc-HAc, pH3.0 ~ 6.0,0 ~ 0.8M NaCl, or 10mM is Tris-HCl, pH8.0 ~ and 10.0, the damping fluid of 0 ~ 0.8M NaCl; The component of described moving phase II is 10mM NaAc-HAc, pH3.0 ~ 6.0,0.1 ~ 0.5M NaCl, or 10mM is Tris-HCl, pH8.0 ~ and 10.0,0.1 ~ 0.5M NaCl damping fluid; Described moving phase III is the damping fluid of 10mM NaAc-HAc, pH3.0 ~ 6.0,0 ~ 1.5M NaCl or 10mM is Tris-HCl, pH8.0 ~ 10.0,0 ~ 1.5M NaCl; Gradient elution in described steps d is linear gradient, and the volume ratio that namely moving phase III accounts for eluent system rises to 100% gradually by 40%.
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|---|---|---|---|---|
| CN1680550A (en) * | 2004-04-05 | 2005-10-12 | 天津天士力制药股份有限公司 | Purification of recommbined human urokinase zymogen |
| CN1727361A (en) * | 2004-07-28 | 2006-02-01 | 深圳科兴生物工程股份有限公司 | Technique for purifying recombined human interferon alpha 1b |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680550A (en) * | 2004-04-05 | 2005-10-12 | 天津天士力制药股份有限公司 | Purification of recommbined human urokinase zymogen |
| CN1727361A (en) * | 2004-07-28 | 2006-02-01 | 深圳科兴生物工程股份有限公司 | Technique for purifying recombined human interferon alpha 1b |
Non-Patent Citations (1)
| Title |
|---|
| Expanded Bed Adsorption as a Primary Recovery Step for the Isolation of the Insulin Precursor MI3 Process Development and Scale Up;Peter Brixius等;《BIOTECHNOLOGY AND BIOENGINEERING》;20060105;第93卷(第1期);14-20 * |
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