CN103159849A - Recombinant human proinsulin preparation method - Google Patents
Recombinant human proinsulin preparation method Download PDFInfo
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- CN103159849A CN103159849A CN2011104062821A CN201110406282A CN103159849A CN 103159849 A CN103159849 A CN 103159849A CN 2011104062821 A CN2011104062821 A CN 2011104062821A CN 201110406282 A CN201110406282 A CN 201110406282A CN 103159849 A CN103159849 A CN 103159849A
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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 for preparing recombinant insulinum primary, particularly a kind of method of utilizing ExPANDED BED ADSORPTION TECHNIQUE to prepare recombinant insulinum primary.
Background technology
Whole world diabetics's the positive increase year after year of quantity expects 2025 and will reach 300,000,000, and Regular Insulin is the specific medicament of widely used treatment diabetes still so far, so the consumption of Regular Insulin also can be along with increasing progressively.The clinical application amount of Regular Insulin is large, and each diabetic subject need use approximately 1.5～2.0 milligrams, Regular Insulin every day, and lifelong medication, therefore develops a kind of processing method for 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 emerges, and is more obvious than animal-origin Regular Insulin advantage, in full accord with the sequence of Regular Insulin in human body, and purity is higher, contains host protein few, safe without toxic side effect.Now clinically recombinant human insulin used be mainly the intestinal bacteria system and Yeast system expressed.The intestinal bacteria system produces Regular Insulin and cooperate to complete 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 solubilization of inclusion bodies, forms Regular Insulin after the enzyme processing.Yeast system is produced Regular Insulin and was studied successfully in 1986, the further processing Regular Insulin of the proinsulin of secreting, expressing in yeast.Therefore no matter be with intestinal bacteria system or yeast expression system, all want first Expression product proinsulin, more further be processed into the insulin human.It is scale operation recombinant human insulin's key that a kind of simple process method for preparing recombinant insulinum primary is provided.
The filtration supernatant of renaturing inclusion bodies of colibacillus liquid or yeast expression nutrient solution all exists such characteristics: the concentration of the recombinant insulinum primary that contains is low; Contain a large amount of pigments and foreign protein; Material liquid volume is large, needs follow-up concentration, and is 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 → see through liquid → cation seperation column chromatography → contain former elutriant of gene-recombinant insulin.Owing to can producing a large amount of pigments and some foreign proteins 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, use again cation seperation column purifying target protein, however the pollution that the activated carbon that is used for decolouring easily brings production environment.Chinese invention patent application prospectus CN1290299A discloses a kind of preparation method of recombinant insulinum primary, this technical scheme is with after feed liquid loading HP-2MG polarity methacrylate resin (ratio of every 1L resin 8g protein), wash post with the 20mM acetate buffer, then use 30% acetone wash-out.Evaporation of eluate to be removing acetone, thereby obtains recombinant insulinum primary, yet comes wash-out with acetone, needs follow-up evaporation step remove acetone, may affect product stability.
Since coming out in 1993, Expanded Bed Adsorption has been widely used in the research and development of gene engineering product, the advantage of Expanded Bed Adsorption be can be directly with without the direct loading of the material of refiner impurity, exempted the step of centrifugal or ultrafiltration, can one the step play clarification, concentrate and the effect of 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, this technical scheme is used for extracting recombinant human interferon alpha 1 b from the engineering strain of expressing human interferon alpha 1 b, comprise bacteria breaking, Expanded Bed Adsorption, affinity chromatography and gel-filtration step, thereby make production efficiency greatly improve, production cost reduces greatly simultaneously.Chinese invention patent application prospectus CN1800379A discloses a kind of method for preparing pancreatic kallikrein with ExPANDED BED ADSORPTION TECHNIQUE and affinity chromatography technology, this technical scheme is at first with the directly first Purification of Pig pancreas extracting solution of ExPANDED BED ADSORPTION TECHNIQUE, be further purified with the affinity chromatography technology again and make the high purity pancreatic kallikrein, thereby simplified production stage, improved 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 intestinal bacteria 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, the feed liquid that contains recombinant insulinum primary needs could obtain the higher proinsulin of purity through at least two steps or two above processing steps of step, 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 inventor is according to insulinogenic physico-chemical property, through a large amount of tests, more suitable processing condition have been screened, reduce processing step, guaranteed the purity of product when improving product yield, a kind of novel method for preparing recombinant insulinum primary is provided, the method is mainly to have utilized one step of ExPANDED BED ADSORPTION TECHNIQUE to catch from the feed liquid that contains recombinant insulinum primary and the former method of purified insulin.
Initial feed liquid in the present invention is the feed liquid that contains recombinant insulinum primary, i.e. the filtration supernatant of the renaturing inclusion bodies liquid of escherichia coli expression, or yeast expression nutrient solution.
Method of the present invention utilizes the expanding bed resin column to prepare recombinant insulinum primary, comprises the steps:
A, column equilibration: the expanding bed resin column is first used approximately 3～10 column volumes of moving phase I balance, and equilibrium velocity is 200～1000cm/h, and in pipeline, flow direction is upwards to flow from resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
B, loading: the feed liquid that will contain recombinant insulinum primary is loaded to the expanding bed resin column, and the loading flow velocity is 200～1000cm/h, and in pipeline, flow direction is upwards to flow from resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
C, washing: loading is complete, and with about 3～10 column volumes of moving phase II washing, washing flow velocity is 200～1000cm/h, and in pipeline, flow direction be to make progress mobilely from the resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
D, wash-out: make the resin natural subsidence, the capital sparger is dropped to resin surface and compresses resin, and from the downward feed liquor in expanding bed top, flow velocity is adjusted into 50～500cm/h, use the moving phase II: moving phase III gradient elution is 5～20 column volumes approximately, collect elution peak.
The present invention has carried out preferably the expanding bed resin, preferably, above-mentioned expanding bed resin is ion-exchange type resin or hydrophobic adsorptive 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 moving phase I component of the present invention during to the balanced expansion bed resin column in step a carried out preferably, and preferably, moving phase I is that salt concn is the damping fluid of 0～1.0M, the damping fluid of pH3.0～10.0; More preferably, the moving phase I is 10mM NaAc-HAc, pH3.0～6.0,0～0.8M NaCl, or 10mM is Tris-HCl, pH8.0～10.0,0～0.8M NaCl; Most preferably, the moving phase I is 10mM NaAc-HAc, pH4.0, or 10mM is Tris-HCl, pH9.0 or 10mM is NaAc-HAc, pH4.0,0.5M NaCl, or 10mM is Tris-HCl, pH9.0,0.5M NaCl.
The present invention has carried 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 has carried out preferably the expansion multiple during balanced expansion bed resin in step a, and preferably, above-mentioned expanding bed resin should be expanded to 2～4 times of fixed bed volume.
The present invention has carried out preferably the flow velocity during expanding bed resin column loading in step b, and preferably, the loading flow velocity of above-mentioned expanding bed resin is 600～800cm/h.
The expansion multiple of the present invention during to the loading expanding bed resin in step b carried out preferably, and preferably, above-mentioned expanding bed resin should be expanded to 2～3 times of fixed bed volume.
The present invention has carried 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 has carried out preferably the expansion multiple during 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 has carried out preferably the moving phase II component during washing expanding bed resin column in step c, and preferably, the moving phase II is that salt concn is the damping fluid of 0～0.5M, the damping fluid of pH3.0～10.0; More preferably, the moving phase II is 10mM NaAc-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, the moving phase II is 10mMNaAc-HAc, pH4.0,0.5M NaCl or 10mM is Tris-HCl, pH9.0,0.5M NaCl, or 10mM is NaAc-HAc, pH4.0,0.2M NaCl, or 10mM is Tris-HCl, pH9.0,0.2M NaCl.
The present invention has carried 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.
The moving phase III component of the present invention when in steps d, the expanding bed resin column carries out gradient elution carried out preferably, and preferably, the moving phase III is that salt concn is the damping fluid of 0～2.0M, the damping fluid of pH3.0～10.0; More preferably, the 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, the moving phase III is 10mM NaAc-HAc, pH4.0,1.0M NaCl or 10mM is 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 the gradient elution program to expanding bed resin column in steps d and has carried out preferably, preferably, described gradient elution program is linear gradient, and namely the moving phase III volume ratio that 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, and the former aminoacid sequence of its aminoacid sequence and natural human insulin is identical or different.
The present invention compared with prior art has advantages of following outstanding:
The expanding bed resin can be directly with the material loading without refiner impurity, can play the effect of clarification/concentrated and preliminary purification a step.One step was caught target protein from the feed liquid that contains recombinant insulinum primary of complexity, simultaneous test as can be known, through the salt concn gradient elution, obtained highly purified proinsulin albumen through processing method provided by the invention, 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 has shortened the production time, improved production efficiency, therefore can greatly reduce production costs, 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
Below further set forth the present invention by specific embodiment, these embodiment only are used for explanation the present invention and are not used in restriction the present invention scope required for protection.
The preparation of embodiment 1, recombinant insulinum primary
With Streamline Q XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM Tris-HCl, pH9.0) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 200cm/h, makes the expanding bed resin be expanded to 1 times of fixed bed volume, and balance is 3 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 1000cm/h.Make the expanding bed resin be expanded to 5 times of fixed bed volume;
Loading is complete, with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 1000cm/h, makes the expanding bed resin be expanded to 5 times of fixed bed volume, and washing is 10 column volumes approximately;
Wash completely, termination of pumping makes the 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 into 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) beginning gradient elution, the gradient elution program is 5 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 89.2%, and purity is 91.3%.
The preparation of embodiment 2, recombinant insulinum primary
With Streamline Q XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM Tris-HCl, pH9.0) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 700cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and balance is 10 column volumes approximately;
The yeast expression nutrient solution that the beginning loading contains recombinant insulinum primary filters supernatant 50L, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 700cm/h.Make the expanding bed resin be expanded to 1 times of fixed bed volume;
Loading is complete, with moving phase II (10mM Tris-HCl, pH9.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 600cm/h, makes the expanding bed resin be expanded to 2 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash completely, termination of pumping makes the 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 into 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) beginning gradient elution, the gradient elution program is 20 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 90.5%, and purity is 94.5%.
The preparation of embodiment 3, recombinant insulinum primary
With Streamline SP XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM sodium acetate-acetic acid, pH4.0) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 4 times of fixed bed volume, and balance is 8 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 600cm/h.Make the expanding bed resin be expanded to 2 times of fixed bed volume;
Loading is complete, with moving phase II (10mM sodium acetate-acetic acid, pH4.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash completely, termination of pumping makes the 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 into 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) beginning gradient elution, the gradient elution program is 10 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 92.2%, and purity is 95.2%.
The preparation of embodiment 4, recombinant insulinum primary
With Streamline SP XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM NaAc-HAc, pH4.0) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 600cm/h, makes the expanding bed resin be expanded to 2 times of fixed bed volume, and balance is 5 column volumes approximately;
The yeast expression nutrient solution that the beginning loading contains recombinant insulinum primary filters supernatant 50L, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 800cm/h.Make the expanding bed resin be expanded to 4 times of fixed bed volume;
Loading is complete, with moving phase II (10mM NaAc-HAc, pH4.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 700cm/h, makes the expanding bed resin be expanded to 5 times of fixed bed volume, and washing is 10 column volumes approximately;
Wash completely, termination of pumping makes the 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 into 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) beginning gradient elution, the gradient elution program is 8 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 90.7%, and purity is 94.8%.
The preparation of embodiment 5, recombinant insulinum primary
With Streamline phenyl XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM NaAc-HAc, pH4.0,0.5M NaCl) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, make the expanding bed resin be expanded to 4 times of fixed bed volume, balance is 5 column volumes approximately;
The yeast expression nutrient solution that the beginning loading contains recombinant insulinum primary filters supernatant 50L, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 200cm/h.Make the expanding bed resin be expanded to 1 times of fixed bed volume;
Loading is complete, with moving phase II (10mM NaAc-HAc, pH4.0,0.2M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 200cm/h, makes the expanding bed resin be expanded to 4 times of fixed bed volume, and washing is 6 column volumes approximately;
Wash complete, termination of pumping makes the 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 into 100cm/h, with moving phase II (10mM NaAc-HAc, pH4.0,0.2M NaCl): moving phase III (10mM NaAc-HAc, pH4.0) beginning gradient elution, the gradient elution program is 5 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 91.6%, and purity is 94.1%.
The preparation of embodiment 6, recombinant insulinum primary
With Streamline phenyl XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10Mm Tris-Cl, pH9.0,0.5M NaCl) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 1000cm/h, make the expanding bed resin be expanded to 5 times of fixed bed volume, balance is 5 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 1000cm/h.Make the expanding bed resin be expanded to 5 times of fixed bed volume;
Loading is complete, with moving phase II (10mM Tris-Cl, pH9.0,0.2M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 4 times of fixed bed volume, and washing is 10 column volumes approximately;
Wash complete, termination of pumping makes the 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 into 200cm/h, with moving phase II (10mM Tris-Cl, pH9.0,0.2M NaCl): moving phase III (10mM Tris-Cl, pH9.0) beginning gradient elution, the gradient elution program is 10 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 89.8%, and purity is 90.4%.
The preparation of embodiment 7, recombinant insulinum primary
With Streamline SP XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM sodium acetate-acetic acid, pH3.0) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 4 times of fixed bed volume, and balance is 8 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 600cm/h.Make the expanding bed resin be expanded to 2 times of fixed bed volume;
Loading is complete, with moving phase II (10mM sodium acetate-acetic acid, pH3.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash complete, termination of pumping, make the 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) the beginning gradient elution, the gradient elution program is 5 column volumes of 40%～100% moving phase III gradient elution.From the downward feed liquor in expanding bed top, flow velocity is adjusted into 100cm/h, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 87.7%, and purity is 90.0%.
The preparation of embodiment 8, recombinant insulinum primary
With Streamline SP XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM sodium acetate-acetic acid, pH6.0,0.8M NaCl) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, make the expanding bed resin be expanded to 4 times of fixed bed volume, balance is 8 column volumes approximately;
The yeast expression nutrient solution that the beginning loading contains recombinant insulinum primary filters supernatant 50L, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 600cm/h.Make the expanding bed resin be expanded to 2 times of fixed bed volume;
Loading is complete, with moving phase II (10mM sodium acetate-acetic acid, pH6.0,0.1M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash completely, termination of pumping makes the 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 into 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) beginning gradient elution, the gradient elution program is 20 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 88.1%, and purity is 90.6%.
The preparation of embodiment 9, recombinant insulinum primary
With Streamline phenyl XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM Tris-HCl, pH8.0,0.8M NaCl) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, make the expanding bed resin be expanded to 4 times of fixed bed volume, balance is 8 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 600cm/h.Make the expanding bed resin be expanded to 2 times of fixed bed volume;
Loading is complete, with moving phase II (10mM Tris-HCl, pH8.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash complete, termination of pumping, make the 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 into 100cm/h, with moving phase II (10mM Tris-HCl, pH8.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH8.0) beginning gradient elution, the gradient elution program is 6 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 87.1%, and purity is 90.3%.
The preparation of embodiment 10, recombinant insulinum primary
With Streamline phenyl XL perfusion expanding bed chromatography column (Streamline 100 chromatography columns, GE), with moving phase I (10mM Tris-HCl, pH10.0,0.5M NaCl) balance, the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, make the expanding bed resin be expanded to 4 times of fixed bed volume, balance is 8 column volumes approximately;
The beginning loading contains the escherichia coli expression renaturing inclusion bodies liquid 50L of recombinant insulinum primary, the feed liquor upwards from the expanding bed bottom, and adjusting the loading flow velocity is 600cm/h.Make the expanding bed resin be expanded to 2 times of fixed bed volume;
Loading is complete, with moving phase II (10mM Tris-HCl, pH10.0,0.5M NaCl) washing, and the feed liquor upwards from expanding bed bottom, flow velocity is adjusted into 800cm/h, makes the expanding bed resin be expanded to 3 times of fixed bed volume, and washing is 5 column volumes approximately;
Wash complete, termination of pumping, make the 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 into 100cm/h, with moving phase II (10mM Tris-HCl, pH10.0,0.5M NaCl): moving phase III (10mM Tris-HCl, pH10.0) beginning gradient elution, the gradient elution program is 6 column volumes of 40%～100% moving phase III gradient elution, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 87.6%, and purity is 90.6%.
The preparation of embodiment 11, recombinant insulinum primary
Other step changes the gradient elution program into the direct moving phase III (10mM NaAc-HAc, pH4.0,1.0M NaCl) of using and carries out wash-out with embodiment 3, collects elution peak.
Also calculate after testing, the yield of recombinant insulinum primary is 75.6%, and purity is 84.3%.
The preparation of embodiment 12, recombinant insulinum primary
Other step is with embodiment 3, and the flow velocity of column equilibration, loading, washing step is adjusted into 1200cm/h, and the elution flow rate of elution step is adjusted into 600cm/h.
Also calculate after testing, the yield of recombinant insulinum primary is 70.1%, and purity is 80.3%.
Claims (10)
1. a method for preparing recombinant insulinum primary, is characterized in that, utilizes ExPANDED BED ADSORPTION TECHNIQUE purified insulin from the feed liquid that contains recombinant insulinum primary former.
2. the method for preparing recombinant insulinum primary according to claim 1, is characterized in that, the described feed liquid that contains recombinant insulinum primary is the renaturing inclusion bodies liquid of escherichia coli expression, or the filtration supernatant of yeast expression nutrient solution.
3. the method for preparing recombinant insulinum primary according to claim 1, is characterized in that, described recombinant insulinum primary, and the former aminoacid sequence of its aminoacid sequence and natural human insulin is identical or different.
4. the method for preparing recombinant insulinum primary according to claim 1, is characterized in that, comprises following steps:
A, column equilibration: the expanding bed resin column is first used approximately 3～10 column volumes of moving phase I balance, and equilibrium velocity is 200～1000cm/h, and in pipeline, flow direction is upwards to flow from resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
B, loading: the feed liquid that will contain recombinant insulinum primary is loaded to the expanding bed resin column, and the loading flow velocity is 200～1000cm/h, and in pipeline, flow direction is upwards to flow from resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
C, washing: loading is complete, and with about 3～10 column volumes of moving phase II washing, washing flow velocity is 200～1000cm/h, and in pipeline, flow direction be to make progress mobilely from the resin column bottom, makes resin in post be expanded to 1～5 times of fixed bed volume;
D, wash-out: make the resin natural subsidence, the capital sparger is dropped to resin surface and compresses resin, from the downward feed liquor in expanding bed top, flow velocity is adjusted into 50～500cm/h, uses the moving phase II: moving phase III gradient elution is 5～20 column volumes approximately, collect elution peak,
Wherein:
The component of moving phase I is that salt concn is the damping fluid of 0～1.0M, the damping fluid of pH3.0～10.0; The component of moving phase II is that salt concn is the damping fluid of 0～0.5M, the damping fluid of pH3.0～10.0; The component of moving phase III is that salt concn is the damping fluid of 0～2.0M, the damping fluid of pH3.0～10.0.
5. the method for preparing recombinant insulinum primary according to claim 4, is characterized in that, described expanding bed resin is ion-exchange type or hydrophobic adsorptive type expanding bed resin.
6. the method for preparing recombinant insulinum primary according to claim 4, it is characterized in that, described step a moving phase I is 10mM NaAc-HAc, pH3.0～6.0,0～0.8M NaCl, or 10mM is Tris-HCl, pH8.0～10.0,0～0.8M NaCl.
7. the method for preparing recombinant insulinum primary according to claim 4, it is characterized in that, described step c moving phase II is 10mM NaAc-HAc, pH3.0～6.0,0.1～0.5M NaCl or 10mM is Tris-HCl, pH8.0～10.0,0.1～0.5M NaCl.
8. the method for preparing recombinant insulinum primary according to claim 4, is characterized in that, the flow velocity in steps d wash-out expanding bed resin column process is 50～500cm/h.
9. the method for preparing recombinant insulinum primary according to claim 4, it is characterized in that, described steps d 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.
10. the method for preparing recombinant insulinum primary according to claim 4, is characterized in that, described steps d gradient elution program is linear gradient, and namely the moving phase III volume ratio that 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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| 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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| PETER BRIXIUS等: "Expanded Bed Adsorption as a Primary Recovery Step for the Isolation of the Insulin Precursor MI3 Process Development and Scale Up", 《BIOTECHNOLOGY AND BIOENGINEERING》, vol. 93, no. 1, 5 January 2006 (2006-01-05), pages 14 - 20 * |
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| CN114276432A (en) * | 2020-09-27 | 2022-04-05 | 鲁南制药集团股份有限公司 | Purification method of recombinant human insulin C peptide |
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