CN103305581B - Preparation method of recombinant human insulin - Google Patents
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- CN103305581B CN103305581B CN201310279340.8A CN201310279340A CN103305581B CN 103305581 B CN103305581 B CN 103305581B CN 201310279340 A CN201310279340 A CN 201310279340A CN 103305581 B CN103305581 B CN 103305581B
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Abstract
The invention discloses a preparation method of recombinant human insulin. The preparation method comprises the following steps of: performing an enzyme digestion reaction on a recombinant human insulin precursor in a mixed solution of an organic solution and water to obtain a human insulin solution containing desB30; centrifuging or suction filtrating the human insulin solution containing desB30 to obtain water-containing powder of desB30 human insulin; performing a transpeptidation reaction on the water-containing powder of desB30 human insulin to obtain human insulin ester, wherein the final concentration of the desB30 human insulin in a transpeptidation reaction system is 12.1mM-33mM, the mol ratio of the desB30 human insulin to threonine ester or a derivative thereof is 1:(15-150), and the mass ratio of the desB30 human insulin to trypsin is (10-80):1; degreasing the recombinant human insulin ester to obtain the recombinant human insulin. The method is suitable for industrial production of converting yeast-expressed proinsulin into the human insulin, high in yield, simple to operation and low in cost.
Description
Technical field
The invention belongs to Regular Insulin preparation field, particularly the preparation method of a kind of recombinant human insulin.
Background technology
At present, according to IDF's statistics in 2011, global diabetes number of patients is 3.66 hundred million, estimates that the year two thousand thirty will reach 5.52 hundred million, is equivalent to increase a diabetic subject every ten seconds, or annual increase by 10,000,000 patients.In China, the morbidity of diabetes has turned over nearly twice at nearly 10 years.2010, the morbidity of China's maturity-onset diabetes was 9.7%, and patient populations, more than 9,000 ten thousand, becomes the first in the world diabetes big country; Meanwhile, the impaired glucose tolerance of pre-diabetes reaches 100,000,000 4 thousand 8 million peoples, and morbidity is 15.5%.So insulinize diabetes market is huge.
In current clinical application, Regular Insulin reduces the most effective medicine of blood sugar in body, the all necessary insulin injection of one diabetes mellitus type and serious type-II diabetes patient, supplementation with insulin, to control the level of blood sugar, reduces the generation of diabetic complication while improving the quality of life of patient.Being monopolized by foreign vendor of the market 95% of China's Regular Insulin, price comparison is expensive, badly influences the safety of China's diabetic; Simultaneously because offshore company holds China's Regular Insulin market, cause Regular Insulin lower in the rate of utilization of China diabetic subject.In order to break the monopolization of foreign vendor for China's Regular Insulin market, improving the competitive edge of domestic recombinant human insulin, certainly will need to reform, to reduce production cost existing recombinant human insulin's production technique.
The production of current recombinant human insulin and analogue thereof have employed two cover systems, first Yeast system (CN1414974A; CN1873006A; CN1125081C); It two is with E. coli system (CN1163600C; CN1011073006A).Yeast system have employed the mode of secretion proinsulin human analogue, and the human insulin precursor be secreted has been provided with natural disulfide linkage and correct N-holds.Shortcoming is that Yeast Growth is slow, cause the production cycle long, and expression level is very low.The mode that application escherichia coli expression recombinant human insulin produces mainly comprises three kinds of modes: a kind of A chain and B chain being the recombination bacillus coli adopted Lilly Co., Eli.'s nineteen eighty-two and distinguishing expression of insulin, expression product is through cyanogen bromide (brominecyanide, CNBr) cut after fusion leads peptide fragment, the A chain obtained and B chain purified after carry out renaturation again, finally obtain activated recombinant human insulin.Because the renaturation yield of which only has about 10%, adopt the Regular Insulin cost of this explained hereafter too high.The second Ye Shi Lilly Co., Eli. sets up, and remains the major way of many company Restruction insulin humans at present.Because the proinsulin human (B-C-A) of normal sequence is unstable and easily degrade in intestinal bacteria; the form of usual employing fusion rotein; proinsulin human being connected on a larger N-holds after fusion rotein; considerably increase insulinogenic expression amount (10%-30%) (Castellanos-Serra, et al.1996; Tikhonov, et al.2001), expression product discharges proinsulin human by cyanogen bromide.Due to the existence of C peptide, Regular Insulin proper energy forms good space conformation, and folding ratio reaches 70%, thus reduces the production cost of Regular Insulin to a certain extent.The third mode is the method that A, B chain of Novo Nordisk Co., Ltd of Denmark employing is expressed simultaneously, in the middle of A chain front end and A, B chain, all have methionine(Met).Expression product, through cyanogen bromide process, obtains A chain and B chain, then obtains activated recombinant human insulin through renaturation and purifying, and this mode and first kind of way also exist the same Cost Problems brought because of annealing efficiency.
Pichia pastoris phaff (Pichia pastoris) carries out insulin precurosor at present to express the focus carrying out insulin production, is also the favor being subject to numerous investigators nearly ten years.Major cause is: one, this expression system has the advantage of prokaryotic expression system and eukaryotic expression system concurrently simultaneously; Two, cultivate processing ease, fast growth, expression amount is high, and product can not excessive glycosylation, and protein antigenicity is low.
Although, high by Pichia anomala expression Restruction human insulin precursor amount, but most important step turns peptide, and this step is apparent that cost is high, yield is low, the time is long the most, wanting product competitive, reduce costs, improve yield will be that we turn peptide emphasis.
In the document reported, transpeptidation reaction is all that a step has been carried out in the organic phase of few water.Conventional organic solvent: DMF(N, dinethylformamide), DMA(N, N-N,N-DIMETHYLACETAMIDE), DMSO(dimethyl sulfoxide (DMSO)), BDO, THF(tetrahydrofuran (THF)) or dioxane etc.Reduce the transpeptidation reaction time to improve yield, it is even higher that the mol ratio of tertiary butyl ether threonine tert-butyl ester and recombinant human insulin's precursor reaches nearly 100:1.And the mass ratio of trypsinase and recombinant human insulin's precursor also nearly 1:5, although the enzyme amount added is very high, but the transpeptidation reaction time generally all wants more than 10 hours, also be because long reaction time to react the impurity produced also a lot, like this trouble and pressure are brought to postorder purifying, make whole lower procedure cost raise, yield reduces simultaneously.
In national inventing patent application CN102703552A, disclose the transformation efficiency with raising adopting two-step approach to replace the insulin production technique of single stage method, reduce the cost of whole production technique.But in process exploitation process, have employed the technique of twice freeze-drying, be not suitable for the amplification of industry and the reduction of cost, in transpeptidation reaction, the concentration of Regular Insulin is lower in addition, is 2.5 ~ 10mM.Also the thinking turning peptide after adopting first enzyme to cut is refer in patent CN100424179C, its enzyme cut after product do not need be separated, its possess skills on advantage, but cut system transform from enzyme to become to turn peptide system, need to be added into a large amount of organic compound, also reduce the insulin concentration (about 5mM) of transpeptidation reaction simultaneously.
Summary of the invention
For overcoming the shortcoming and defect of prior art, the object of the present invention is to provide the preparation method of a kind of recombinant human insulin.Technical problem to be solved by this invention is multi-step in the current recombinant human insulin's preparation process of improvement, turns the shortcomings such as peptide rate is low, high cost, instability.Adopt the new preparation technology that improves to produce, upstream and downstream connection is smooth and easy, step is few, cost is low, it is high, stable to turn peptide rate.This method has been rendered to production from the experimental phase and has been verified and obtain expected effect.
Object of the present invention is achieved through the following technical solutions: the preparation method of a kind of recombinant human insulin, comprises the following steps:
(1) in the mixing solutions of organic solvent and water, endonuclease reaction is carried out to human insulin precursor of recombinating, obtain the insulin human (desB lacking Threonine containing the 30th, insulin human B chain
30insulin human) solution;
(2) from containing desB
30the solution of insulin human, centrifugal or suction filtration obtains desB
30insulin human's hydrous powdery;
(3) desB is used
30insulin human's hydrous powdery carries out transpeptidation reaction, obtains human insulin ester; Wherein, desB
30the final concentration of insulin human in transpeptidation reaction system is 12.1mM ~ 33mM; This step is by desB
30the B29 position Methionin C-terminal of insulin human is transferred a tertiary butyl ether threonine tert-butyl ester;
(4) recombinant human insulin's ester is taken off ester, obtain recombinant human insulin.
The concentration of organic solvent described in step (1) in described mixing solutions is volume percent 0.1 ~ 30%; Be preferably volume percent 5 ~ 30%;
Described organic solvent is the organic solvent that can be used as reverse-phase chromatography elution flow phase, is preferably Virahol or ethanol;
Step (1) is preferably: in the mixing solutions of organic solvent and water, carry out endonuclease reaction to human insulin precursor of recombinating, the condition that enzyme is cut is for be adjusted to 7 ~ 9 by pH value, in 20 ~ 30 DEG C of reactions, obtain the insulin human's solution lacking Threonine containing the 30th, insulin human B chain; Wherein, trypsinase and recombinant human insulin's precursor 1:50 ~ 500 proportioning in mass ratio;
The time of described reaction is preferably 1 ~ 8 hour; Be more preferably 3 ~ 8 hours;
Described Recombulin precursor carries out recombinant human insulin's precursor of secreting, expressing preferably by adopting genetic engineering technique to be used for Pichi strain, or the inclusion body of expressing from prokaryotic cell prokaryocyte (intestinal bacteria), recombinant human insulin's precursor of obtaining after renaturation;
Described pH value preferably regulates with ammoniacal liquor; More preferably the ammoniacal liquor of 5mol/L is used to regulate;
PH value was preferably adjusted to 8.5 by described enzyme tangent condition, in 25 DEG C of reactions 3 hours;
Described trypsinase and the preferred 1:300 proportioning in mass ratio of described recombinant human insulin's precursor;
Before centrifugal or suction filtration described in step (2), first to containing desB
30the solution of insulin human precipitates;
Described to containing desB
30the mode that the solution of insulin human carries out precipitating staticly settles after being preferably adjust ph, or first adds Zn
2+, then adjust ph, staticly settle afterwards;
Described pH value is 5.6 ~ 6, is more preferably 5.8;
The reagent of described adjust ph can be one in phosphoric acid, acetic acid and ammoniacal liquor or at least two kinds; Be preferably the phosphoric acid solution of 3mol/L;
Described Zn
2+can be provided by zinc chloride or zinc sulfate, preferred zinc chloride;
Described Zn
2+consumption press desB
30insulin human and Zn
2+mol ratio be 1:0.5 ~ 1.5 carry out calculating add;
The described temperature left standstill is preferably 4 ~ 8 DEG C;
The condition optimization of the suction filtration described in step (2) is decompress filter; The filter membrane used in described suction filtration is preferably 300 orders;
Transpeptidation reaction described in step (3) preferably includes following steps: with dmso solution Threonine or derivatives thereof, and desB
30insulin human's hydrous powdery, then add BDO and the trypsinase aqueous solution or only add the trypsinase aqueous solution, obtain the reaction system turning peptide; Then mild stirring is reacted; DesB
30the mol ratio of insulin human and Threonine ester or derivatives thereof is 1:15 ~ 150, desB
30insulin human and trypsinase are 10 ~ 80:1 in mass ratio;
Described Threonine ester is preferably o-tertbutyl ether-l-threonine tert-butyl ester;
Described derivative refers to o-tertbutyl ether-l-threonine tert-butyl ester salt derivative, as o-tertbutyl ether-l-threonine tert-butyl ester acetate and o-tertbutyl ether-l-threonine tert-butyl ester hydrochloride;
In described reaction system, the ratio of solvent is preferably dimethyl sulfoxide (DMSO): BDO: water 15% ~ 50%:0 by volume ~ 65%:20% ~ 50%; DesB is contained in the content of wherein water
30the moisture comprised in insulin human's hydrous powdery, is measured by the mode of moisture content tester or weight loss on drying;
The time of described stirring reaction is preferably 1 ~ 8 hour, and temperature is preferably 25 ± 1 DEG C.
De-ester described in step (4) carries out de-ester for adopting trifluoroacetic acid reagent;
The concrete steps of the de-ester described in step (4) are: after insulin-ester isoelectric precipitation, lyophilize; After adopting acetone solution and detaching acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
The present invention has following advantage and effect relative to prior art:
(1) under the high-quality prerequisite of guarantee recombinant human insulin, reduce the cost of recombinant human insulin, improve yield.Present invention employs enzyme and cut the desB obtained
30insulin human's hydrous powdery, then turn peptide degreasing and obtain recombinant human insulin.Overcome the old technique of complexity that first freeze-drying in prior art turns peptide again, improve the concentration of recombinant human insulin's precursor in the process of reaction simultaneously, reduce the consumption of trypsinase and Threonine ester, decrease the cost turning peptide, improve the service efficiency of equipment.
(2) to hit desB at the miscible fluid enzyme of organic solvent and water
30insulin human, enzyme cuts rate more than 95%, and without occurring other impurity in 24 hours.Generate recombinant human insulin's ester by transpeptidation reaction, it turns peptide efficiency more than 85%.
Accompanying drawing explanation
Fig. 1 is that HPLC detects the digested design sketch of recombinant human insulin's precursor in embodiment 1; Wherein, scheme the HPLC detection figure that a is the precursor of recombinant human insulin, figure b is the HPLC detection figure after the pre-enzyme of recombinant human insulin is cut.
Fig. 2 be HPLC detect embodiment 1 turn peptide design sketch; Wherein, the peptide that turns that figure a, figure b, figure c, figure d and figure e are respectively 0,2,4,6 and 8 hour samples HPLC detection figure spectrogram.
Fig. 3 is recombinant human insulin's ester mass spectrum of embodiment 1.
Fig. 4 is recombinant human insulin's mass spectrum of embodiment 1.
Fig. 5 be recombinant human insulin's ester of embodiment 1 take off after ester with recombinant human insulin's standard substance comparison diagram; Wherein, after figure a and figure b is respectively recombinant human insulin's standard substance and de-ester, the HPLC of sample detects collection of illustrative plates.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) desB
30the preparation of insulin human's hydrous powdery: recombinant human insulin's precursor of pichia spp secreting, expressing, after purification with macroreticular resin, (reference: Gao Jiankun, wealth continues fair-skinned, Fan Kai etc., containing short C peptide proinsulin human analogue desB
30expression and purification in pichia spp, Progress in Biochemistry and Biophysics, 2008, sample solution 35(1): 63-68), anti-phase purifying is carried out after diluting 2 times, adopt Virahol to carry out gradient elution, obtaining the recombinant human insulin's precursor aqueous solution (HPLC detects collection of illustrative plates as shown in Figure 1a) containing Virahol (concentration is volume percent 24%), is 8.5 with 5M ammoniacal liquor adjust pH.Add trypsinase in trypsinase and recombinant human insulin's precursor mass than the ratio of 1:300, cut 3 hours, obtain the solution that enzyme is cut in 25 DEG C of enzymes, detecting the recombinase rate of cutting through HPLC is that 97.55%(HPLC detects collection of illustrative plates as shown in Figure 1 b).Add zinc chloride by recombinant human insulin's precursor and zinc chloride mol ratio 1:1, be then that 5.8,4 ~ 8 DEG C of placements are spent the night by the phosphoric acid solution adjust ph of 3M, use 300 object filter membrane suction filtrations to obtain desB
30insulin human's hydrous powdery, adopts moisture content tester to survey moisture and HPLC detects its content.
(2) with desB
30the reaction density that insulin human is 17mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: the desB obtained by dmso solution step (1)
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate, then add BDO, finally add the trypsinase aqueous solution, obtain turning peptide liquid.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:15, desB
30insulin human and tryptic mass ratio are 20:1, water (desB
30the summation of the water in the moisture in insulin human's hydrous powdery and the trypsinase aqueous solution), the volume ratio of dimethyl sulfoxide (DMSO) and BDO is 20%:40%:40%.The whole peptide solution mild stirring at 25 ± 1 DEG C that turns is reacted, and sampled respectively at 0,2,4,6,8 hour, the peptide efficiency that turns of 0,2,4,6,8 hour is 0%, 76.03%, 88.02%, 91.82%, 90.66% respectively.The collection of illustrative plates that HPLC detects is respectively as shown in a ~ e in Fig. 2.
React the solution obtaining containing recombinant human insulin's ester after 8 hours, diluted 5 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying, is 5925 through mass spectroscopy molecular weight, consistent with theoretical molecular (mass spectrometric detection the results are shown in Figure 3).
(3) de-ester: after insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.Be 5708 consistent with theoretical molecular (mass spectrometric detection the results are shown in Figure 4) through its molecular weight of mass spectroscopy, HPLC under the recombinant human insulin of embodiment 1 manufacture and recombinant human insulin's standard substance (National Institute for Food and Drugs Control's purchase) the same terms is detected, compare peak time to fit like a glove (as shown in Figure 5), illustrate that the recombinant human insulin obtained in the present embodiment has identical structure with recombinant human insulin's standard substance.
Embodiment 2
(1) desB
30the preparation of insulin human's hydrous powdery: the concentration recombinant human insulin's precursor aqueous solution in embodiment 1 being diluted to Virahol is volume percent 10%, is 9 with 5M ammoniacal liquor adjust pH.Add trypsinase by the mass ratio of trypsinase and recombinant human insulin's precursor 1:400, cut 5 hours in 25 DEG C of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 95.31% after testing.Add zinc chloride by the mol ratio of human insulin precursor and zinc chloride 1:1.5, be that 5.8,4 ~ 8 DEG C of placements are spent the night by 3M phosphoric acid solution adjust ph, use 300 object filter membrane suction filtrations to obtain desB
30insulin human's hydrous powdery, and survey moisture and content.
(2) with desB
30the reaction density that insulin human is 13.4mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: use dmso solution desB
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate, then add BDO, finally add the trypsinase aqueous solution.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:100, desB
30insulin human and tryptic mass ratio are 10:1, water (desB
30water summation in moisture in insulin human's hydrous powdery and the trypsinase aqueous solution), the volume ratio 50%:40%:10% of dimethyl sulfoxide (DMSO) and BDO.The whole peptide solution mild stirring at 25 ± 1 DEG C that turns is reacted, and sampled respectively at 2,4,6 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6 hours is 87.12%, 89.51%, 87.68% respectively.
(3) de-ester: react the solution obtaining containing recombinant human insulin's ester after 6 hours, diluted 5 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying, after insulin-ester isoelectric precipitation, and lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
Embodiment 3
(1) desB
30the preparation of insulin human's hydrous powdery: carry out wash-out with ethanol during the anti-phase purifying of the recombinant human insulin's precursor in embodiment 1, elutriant being diluted to alcohol concn is volume percent 30%, be 8.5 by 5M ammoniacal liquor adjust ph, add trypsinase by the mass ratio of trypsinase and recombinant human insulin's precursor 1:300, cut 6 hours in 25 DEG C of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 96.07% after testing.Add zinc chloride by the mol ratio of human insulin precursor and zinc chloride 1:0.5, regulate pH5.80 with 3M phosphoric acid, 4 ~ 8 DEG C of placements are spent the night, and 8000g is centrifugal obtains desB
30insulin human's hydrous powdery.Measure its moisture and content.
(2) with desB
30the reaction density that insulin human is 12.1mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: use dmso solution desB
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolve, then add the trypsinase aqueous solution.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:120, desB
30insulin human and tryptic mass ratio are 20:1, water (desB
30the summation of the water in the moisture in insulin human's hydrous powdery and the trypsinase aqueous solution) and the volume ratio 50%, 50% of dimethyl sulfoxide (DMSO).The whole peptide solution mild stirring at 25 ± 1 DEG C that turns is reacted, and sampled respectively at 2,4,6,8 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6,8 hours is 83.58%, 84.11%, 87.76%, 86.60% respectively.
(3) de-ester: react the solution obtained for 8 hours containing recombinant human insulin's ester, diluted 5 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.After insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
Embodiment 4
(1) desB
30the preparation of insulin human's hydrous powdery: it is 20% that recombinant human insulin's precursor aqueous solution in embodiment 1 is diluted to isopropyl alcohol concentration, be 8.0 by 5M ammoniacal liquor adjust ph, by trypsinase and recombinant human insulin's precursor 1:500 mass ratio add trypsinase, cut 8 hours in 20 DEG C of enzymes, recombinant human insulin's pre-enzyme rate of cutting is 94.75% after testing.Be that 5.6,4 ~ 8 DEG C of placements are spent the night by 3M phosphoric acid solution adjust ph, use 300 object filter membrane suction filtrations to obtain desB
30insulin human's hydrous powdery, measures moisture and content.
(2) with desB
30the reaction density that insulin human is 33mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: use dmso solution desB
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolve, then add BDO, finally add the trypsinase aqueous solution.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:150, desB
30insulin human and tryptic mass ratio are 80:1, water (desB
30the summation of the water in the moisture in insulin human's hydrous powdery and the trypsinase aqueous solution), the volume ratio of dimethyl sulfoxide (DMSO) and BDO is 30%:15%:55%.The whole peptide solution mild stirring at 25 ± 1 DEG C that turns is reacted, and sampled respectively at 2,4,6,8 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6,8 hours is 75.04%, 84.05%, 89.42%, 87.46% respectively.
(3) de-ester: react the solution obtained for 8 hours containing recombinant human insulin's ester, diluted 5 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.After insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
Embodiment 5
(1) desB
30the preparation of insulin human's hydrous powdery: it is volume percent 5% that the recombinant human insulin's precursor aqueous solution in embodiment 1 is diluted to isopropyl alcohol concentration, is 7.5 by 5M ammoniacal liquor adjust ph.Add trypsinase by the mass ratio of trypsinase and recombinant human insulin's precursor 1:200, cut 6 hours in 20 DEG C of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 96.49% after testing.Add zinc chloride by the mol ratio of human insulin precursor and zinc chloride 1:1.2, be that 5.8,4 ~ 8 DEG C of placements are spent the night by 3M phosphoric acid solution adjust ph, suction filtration obtains desB
30insulin human's hydrous powdery, and survey moisture and content.
(2) with desB
30the reaction density that insulin human is 25mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: use dmso solution desB
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolve, then add BDO, finally add the trypsinase aqueous solution.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:150, desB
30insulin human and tryptic mass ratio are 40:1, water (desB
30the summation of the water in the moisture in insulin human's hydrous powdery and the trypsinase aqueous solution), the volume ratio of dimethyl sulfoxide (DMSO) and BDO is 40%:30%:30%.The whole peptide solution mild stirring at 20 DEG C that turns is reacted, and sampled respectively at 2,4,6,8 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6,8 hours is 81.39%, 82.56%, 87.22%, 85.97% respectively.
(3) de-ester: react the solution obtained for 8 hours containing recombinant human insulin's ester, diluted 8 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.After insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
Embodiment 6
(1) desB
30the preparation of insulin human's hydrous powdery: it is volume percent 30% that the recombinant human insulin's precursor aqueous solution in embodiment 1 is added Virahol to Virahol final concentration, is 7 by 5M ammoniacal liquor adjust ph.Add trypsinase by the mass ratio of trypsinase and recombinant human insulin's precursor 1:50, cut 4.0 hours in 30 DEG C of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 97.22% after testing.Add zinc chloride by the mol ratio of human insulin precursor and zinc chloride 1:1.5, regulate pH5.8 with 3M phosphoric acid solution, 4 ~ 8 DEG C of placements are spent the night, and suction filtration obtains desB
30insulin human's hydrous powdery, and survey moisture and content.
(2) with desB
30the reaction density that insulin human is 12.1mM carries out transpeptidation reaction, obtains recombinant human insulin's ester: use dmso solution desB
30insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolve, then add BDO, finally add the trypsinase aqueous solution.Wherein desB
30the mol ratio of insulin human and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:30, desB
30insulin human and tryptic mass ratio are 30:1, water (desB
30water in moisture in insulin human's hydrous powdery and the trypsinase aqueous solution), the volume ratio of dimethyl sulfoxide (DMSO) and BDO is 20%:15%:65%.The whole peptide solution mild stirring at 25 DEG C that turns is reacted, and sampled respectively at 2,4,6,8 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6,8 hours is 79.60%, 82.97%, 85.70%, 85.69% respectively.
(3) de-ester: react the solution obtained for 8 hours containing recombinant human insulin's ester, diluted 5 times with the phosphate aqueous solution of pH3.0.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.After insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, by 1g insulin-ester: the amount of 5ml trifluoroacetic acid is by after insulin-ester dissolving, and vacuum detaches unnecessary trifluoroacetic acid, obtains recombinant human insulin.
Comparative example 1 one step turns peptide method
Recombinant human insulin's precursor aqueous solution solution in embodiment 1, obtains the lyophilized powder of recombinant human insulin's precursor, measures its content after lyophilize.
The reaction density being 6.7mM with recombinant human insulin's precursor carries out turning peptide.Dissolve with dmso solution recombinant human insulin precursor lyophilized powder and o-tertbutyl ether-l-threonine tert-butyl ester acetate, then add BDO, finally add the trypsinase aqueous solution.Wherein the mol ratio of recombinant human insulin's precursor and o-tertbutyl ether-l-threonine tert-butyl ester acetate is 1:35, recombinant human insulin's precursor and tryptic mass ratio are 10:1, the volume ratio 15%, 15%, 70% of water (water in the trypsinase aqueous solution), dimethyl sulfoxide (DMSO) and BDO.The whole peptide solution mild stirring at 25 DEG C that turns is reacted, and sampled respectively at 2,4,6 hours, HPLC detects analytic sample.2, the peptide efficiency that turns of 4,6 hours is 25.00%, 43.13%, 68.60% respectively.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. a recombinant human insulin's preparation method, is characterized in that comprising the following steps:
(1) in the mixing solutions of organic solvent and water, endonuclease reaction is carried out to human insulin precursor of recombinating, obtain containing desB
30insulin human's solution;
(2) from containing desB
30the solution of insulin human, centrifugal or suction filtration obtains desB
30insulin human's hydrous powdery;
(3) desB is used
30insulin human's hydrous powdery carries out transpeptidation reaction, obtains human insulin ester; Wherein, desB
30the final concentration of insulin human in transpeptidation reaction system is 12.1mM ~ 33mM;
(4) recombinant human insulin's ester is taken off ester, obtain recombinant human insulin;
The concentration of the organic solvent described in step (1) is volume percent 0.1 ~ 30%;
Described organic solvent is Virahol or ethanol.
2. the preparation method of recombinant human insulin according to claim 1, it is characterized in that: step (1) is: in the mixing solutions of organic solvent and water, endonuclease reaction is carried out to human insulin precursor of recombinating, the condition that enzyme is cut is for be adjusted to 7 ~ 9 by pH value, in 20 ~ 30 DEG C of reactions, obtain the insulin human's solution lacking Threonine containing the 30th, insulin human B chain; Wherein, trypsinase and recombinant human insulin's precursor 1:50 ~ 500 proportioning in mass ratio.
3. the preparation method of recombinant human insulin according to claim 2, is characterized in that: the time of the reaction described in step (1) is 1 ~ 8 hour;
PH value ammoniacal liquor described in step (1) regulates;
Described trypsinase and described recombinant human insulin's precursor 1:300 proportioning in mass ratio.
4. the preparation method of recombinant human insulin according to claim 1, is characterized in that: before the centrifugal or suction filtration described in step (2), first to containing desB
30the solution of insulin human precipitates.
5. the preparation method of recombinant human insulin according to claim 4, is characterized in that: described to containing desB
30the mode that the solution of insulin human carries out precipitating staticly settles after being adjust ph, or first adds Zn
2+, then adjust ph, staticly settle afterwards.
6. the preparation method of recombinant human insulin according to claim 5, is characterized in that: described pH value is 5.6 ~ 6;
The reagent of described adjust ph can be a kind of in phosphoric acid, acetic acid and ammoniacal liquor or at least two kinds;
Described Zn
2+consumption press desB
30insulin human and Zn
2+mol ratio be 1:0.5 ~ 1.5 carry out calculating add;
The described temperature left standstill is 4 ~ 8 DEG C.
7. the preparation method of recombinant human insulin according to claim 1, is characterized in that:
Transpeptidation reaction described in step (3) comprises the steps: with dmso solution Threonine ester or derivatives thereof, and desB
30insulin human's hydrous powdery, then add BDO and the trypsinase aqueous solution or only add the trypsinase aqueous solution, obtain the reaction system turning peptide; Then stir and react;
DesB
30the mol ratio of insulin human and Threonine ester or derivatives thereof is 1:15 ~ 150, desB
30insulin human and trypsinase are 10 ~ 80:1 in mass ratio.
8. the preparation method of recombinant human insulin according to claim 7, is characterized in that:
Described Threonine ester is o-tertbutyl ether-l-threonine tert-butyl ester;
Described derivative refers to o-tertbutyl ether-l-threonine tert-butyl ester salt derivative;
In described reaction system, the ratio of solvent is dimethyl sulfoxide (DMSO): BDO: water 15% ~ 50%:0 by volume ~ 65%:20% ~ 50%; DesB is contained in the content of wherein water
30the moisture comprised in insulin human's hydrous powdery.
9. the preparation method of recombinant human insulin according to claim 1, is characterized in that: the de-ester described in step (4) carries out de-ester for adopting trifluoroacetic acid reagent.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1589328A (en) * | 2001-11-19 | 2005-03-02 | 诺沃挪第克公司 | Process for preparing insulin compounds |
| CN102703552A (en) * | 2012-05-15 | 2012-10-03 | 山东阿华生物药业有限公司 | Preparation method and products of recombinant human insulin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1589328A (en) * | 2001-11-19 | 2005-03-02 | 诺沃挪第克公司 | Process for preparing insulin compounds |
| CN102703552A (en) * | 2012-05-15 | 2012-10-03 | 山东阿华生物药业有限公司 | Preparation method and products of recombinant human insulin |
Non-Patent Citations (2)
| Title |
|---|
| Biotechnology》.2008,第38卷(第3期), * |
| Xie, T.等.Secretory Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin.《Preparative Biochemistry & * |
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