CN103305581A - Preparation method of recombinant human insulin - Google Patents
Preparation method of recombinant human insulin Download PDFInfo
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- CN103305581A CN103305581A CN2013102793408A CN201310279340A CN103305581A CN 103305581 A CN103305581 A CN 103305581A CN 2013102793408 A CN2013102793408 A CN 2013102793408A CN 201310279340 A CN201310279340 A CN 201310279340A CN 103305581 A CN103305581 A CN 103305581A
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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 the Regular Insulin preparation field, particularly a kind of recombinant human insulin's preparation method.
Background technology
At present, according to IDF 2011 statistics, 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 per ten seconds a diabetic subject, perhaps annually increases by 10,000,000 patients.In China, the morbidity of diabetes was turned over nearly twice at nearly 10 years.2010, the morbidity of China's maturity-onset diabetes was 9.7%, and patient's sum surpasses 9,000 ten thousand, becomes the first in the world diabetes big country; Simultaneously, the impaired glucose tolerance of pre-diabetes has reached 100,000,000 4 thousand 8 million peoples, and morbidity is 15.5%.So insulinize diabetes market is huge.
In the at present clinical application, Regular Insulin is to reduce the most effective medicine of blood sugar in the body, one diabetes mellitus type and serious type-II diabetes patient must insulin injections, and supplementation with insulin reduces the generation of diabetic complication with the level of control blood sugar when improving Quality of Life.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; Because China Regular Insulin market is controlled by offshore company, cause Regular Insulin lower in China diabetic subject's rate of utilization simultaneously.In order to break foreign vendor for the monopolization in China Regular Insulin market, improve domestic recombinant human insulin's competitive edge, certainly will need to reform existing recombinant human insulin's production technique, to reduce production costs.
Recombinant human insulin and similar the production of material thereof have adopted two cover systems at present, first Yeast system (CN1414974A; CN1873006A; CN1125081C); It two is with intestinal bacteria (CN1163600C of system; CN1011073006A).Yeast system has adopted the mode of secretion proinsulin human analogue, and secretion human insulin precursor has out had natural disulfide linkage and correct N-end.Shortcoming is that Yeast Growth is slow, causes the production cycle long, and expression level is very low.The mode of using escherichia coli expression recombinant human insulin production mainly comprises three kinds of modes: a kind of is A chain and the B chain of the recombination bacillus coli difference expression of insulin of Lilly Co., Eli.'s nineteen eighty-two employing, expression product is through cyanogen bromide (bromine cyanide, CNBr) after peptide fragment is led in the cutting-out fusion, carry out again renaturation after the A chain that obtains and B chain are purified, obtain at last activated recombinant human insulin.Because the renaturation yield of this mode only has about 10%, adopts the Regular Insulin cost of this explained hereafter too high.The second also is that Lilly Co., Eli. sets up, and remains at present many Restruction insulin humans' of company major way.Because the proinsulin human (B-C-A) of normal sequence unstable and easy degraded in intestinal bacteria, usually adopt the form of fusion rotein, the proinsulin human is connected on a larger N-end fusion rotein back, insulinogenic expression amount (10%-30%) (Castellanos-Serra, et al.1996 have greatly been increased; Tikhonov, et al.2001), expression product discharges the proinsulin human by cyanogen bromide.Because the existence of C peptide, the Regular Insulin proper energy forms good space conformation, and folding ratio reaches 70%, thereby has reduced to a certain extent the production cost of Regular Insulin.The third mode is the A of Denmark Novo Nordisk Co.,Ltd employing, the method that the B chain is expressed simultaneously, at A chain front end and A, B chain centre methionine(Met) is arranged all.Expression product is processed through cyanogen bromide, obtains A chain and B chain, obtains activated recombinant human insulin through renaturation and purifying again, and this mode and first kind of way exist the same Cost Problems that brings because of annealing efficiency.
Pichia pastoris phaff (Pichia pastoris) is to carry out at present the focus that insulin precurosor expresses to carry out insulin production, also is the favor that is subject to numerous investigators over past ten years.Major cause is: one, this expression system has the advantage of prokaryotic expression system and eukaryotic expression system simultaneously concurrently; Two, cultivate processing ease, fast growth, expression amount is high, and product can excessive glycosylation, and protein antigenicity is low.
Although, high with Pichia anomala expression Restruction human insulin precursor amount, but most important step is to turn peptide, and this step is apparent that the most cost is high, yield is low, the time is long, want product competitive, reducing cost, improve yield will be that we turn the peptide emphasis.
In the document of having reported, transpeptidation reaction all be in the organic phase of few water a step finish.Organic solvent commonly used: DMF(N, dinethylformamide), DMA(N, N-N,N-DIMETHYLACETAMIDE), the DMSO(dimethyl sulfoxide (DMSO)), BDO, THF(tetrahydrofuran (THF)) or dioxane etc.Reduce the transpeptidation reaction time in order to improve yield, the mol ratio of uncle's butyl ether threonine tert-butyl ester and recombinant human insulin's precursor reaches nearly 100:1 even higher.And also 1:5 nearly of the mass ratio of trypsinase and recombinant human insulin's precursor, although the enzyme amount that adds is very high, but the transpeptidation reaction time is generally all wanted more than 10 hours, also be because the impurity that the long reaction time reaction is produced is also a lot, like this postorder purifying trouble and pressure have been brought, whole lower procedure cost is raise, and yield reduces.
In national inventing patent application CN102703552A, disclose the transformation efficiency with raising that adopts two-step approach to replace the insulin production technique of single stage method, reduced the cost of whole production technique.But in the process exploitation process, adopted the technique of twice freeze-drying, be not suitable for the amplification of industry and the reduction of cost, the concentration ratio of Regular Insulin is lower in the transpeptidation reaction in addition, is 2.5～10mM.In patent CN100424179C, also mentioned the thinking that turns peptide after adopting first enzyme to cut, product after its enzyme is cut does not need to separate, advantage on it possesses skills, but convert to the peptide system from the enzyme system of cutting, need to be added into a large amount of organic compound, also reduce the insulin concentration (approximately 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 a kind of recombinant human insulin's preparation method.Technical problem to be solved by this invention is multi-step in the present recombinant human insulin's preparation process of improvement, turns the shortcomings such as the peptide rate is low, expensive, unstable.Adopt the new preparation technology of improvement to produce, the upstream and downstream connection is smooth and easy, step is few, cost is low, it is high, stable to turn the peptide rate.This method has been rendered to from the experimental phase and has been verified and obtained expected effect the production.
Purpose of the present invention is achieved through the following technical solutions: a kind of recombinant human insulin's preparation method may further comprise the steps:
(1) in the mixing solutions of organic solvent and water, the restructuring human insulin precursor is carried out endonuclease reaction, obtain containing the 30th insulin human (desB that lacks Threonine of insulin human B chain
30The insulin human) solution;
(2) from containing desB
30Insulin human's solution, centrifugal or suction filtration obtains desB
30Insulin human's hydrous powdery;
(3) use desB
30Insulin human's hydrous powdery carries out transpeptidation reaction, obtains the human insulin ester; Wherein, desB
30The final concentration of insulin human in the transpeptidation reaction system is 12.1mM～33mM; This step is with desB
30Uncle's butyl ether threonine tert-butyl ester of B29 position Methionin C-terminal switching of insulin human;
(4) recombinant human insulin's ester is taken off ester, namely get the recombinant human insulin.
The concentration of organic solvent in described mixing solutions described in the step (1) 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 wash-out moving phase, is preferably Virahol or ethanol;
Step (1) is preferably: in the mixing solutions of organic solvent and water the restructuring human insulin precursor is carried out endonuclease reaction, the condition that enzyme is cut is for to be adjusted to 7～9 with the pH value, in 20～30 ℃ of reactions, obtain containing the 30th insulin human's solution that lacks Threonine of insulin human B chain; Wherein, trypsinase and recombinant human insulin's precursor 1:50～500 proportionings in mass ratio;
The time of described reaction is preferably 1～8 hour; More preferably 3～8 hours;
Described Recombulin precursor is preferably by adopting genetic engineering technique to be used for recombinant human insulin's precursor that Pichi strain carries out secreting, expressing, or the recombinant human insulin's precursor that obtains behind the inclusion body of expressing from prokaryotic cell prokaryocyte (intestinal bacteria), renaturation;
Described pH value is preferably regulated with ammoniacal liquor; More preferably use the ammoniacal liquor of 5mol/L to regulate;
Described enzyme tangent condition was preferably the pH value is adjusted to 8.5, in 25 ℃ of reactions 3 hours;
Described trypsinase and described recombinant human insulin's precursor be 1:300 proportioning in mass ratio preferably;
Before the centrifugal or suction filtration described in the step (2), first to containing desB
30Insulin human's solution precipitates;
Described to containing desB
30The mode that insulin human's solution precipitates staticly settles after being preferably and regulating the pH value, or adds Zn first
2+, re-adjustment pH value staticly settles afterwards;
Described pH value is 5.6～6, more preferably 5.8;
The reagent of described adjusting pH value can be a kind of in phosphoric acid, acetic acid and the ammoniacal liquor or at least two kinds; Be preferably the phosphoric acid solution of 3mol/L;
Described Zn
2+Preferred zinc chloride can be provided by zinc chloride or zinc sulfate;
Described Zn
2+Consumption press desB
30Insulin human and Zn
2+Mol ratio be that interpolation is calculated in 1:0.5～1.5;
The described temperature that leaves standstill is preferably 4～8 ℃;
The condition optimization of the suction filtration described in the step (2) is decompress filter; The filter membrane that uses in the described suction filtration is preferably 300 orders;
Transpeptidation reaction described in the step (3) preferably includes following steps: with dmso solution Threonine or derivatives thereof, and desB
30Insulin human's hydrous powdery adds BDO and the trypsinase aqueous solution again or only adds the trypsinase aqueous solution, obtains to turn the reaction system of 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, such as o-tertbutyl ether-l-threonine tert-butyl ester acetate and o-tertbutyl ether-l-threonine tert-butyl ester hydrochloride;
The ratio of solvent is preferably dimethyl sulfoxide (DMSO) in the described reaction system: BDO: water is 15%～50%:0～65%:20%～50% by volume; Wherein comprised desB in the content of water
30The moisture that comprises 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 ℃.
The ester that takes off described in the step (4) takes off ester for employing trifluoroacetic acid reagent;
The concrete steps of taking off ester described in the step (4) are: behind the insulin-ester isoelectric precipitation, and lyophilize; After adopting acetone solution and detaching acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the recombinant human insulin.
The present invention has following advantage and effect with respect to prior art:
(1) guaranteeing under the high-quality prerequisite of recombinant human insulin, reducing recombinant human insulin's cost, raising yield.The present invention has adopted enzyme to cut the desB that obtains
30Insulin human's hydrous powdery turns the peptide degreasing again and obtains the recombinant human insulin.Overcome the complicated old technique that first freeze-drying in the prior art turns peptide again, improved simultaneously the concentration of recombinant human insulin's precursor in the process of reacting, reduced the consumption of trypsinase and Threonine ester, reduced the cost that turns peptide, improved the service efficiency of equipment.
(2) at the miscible fluid enzyme of organic solvent and the water desB that hits
30The insulin human, enzyme is cut rate and is surpassed 95%, and other impurity appears in nothing in 24 hours.Generate recombinant human insulin's ester by transpeptidation reaction, it turns peptide efficient and surpasses 85%.
Description of drawings
Fig. 1 is that HPLC detects the digested design sketch of recombinant human insulin's precursor among the embodiment 1; Wherein, figure a is that the HPLC of recombinant human insulin's precursor detects figure, and figure b is the HPLC detection figure after recombinant human insulin's pre-enzyme is cut.
Fig. 2 be HPLC detect embodiment 1 turn the peptide design sketch; Wherein, figure a, figure b, figure c, figure d and figure e be respectively 0,2,4,6 and 8 hour turn peptide sampling 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 is that recombinant human insulin's ester of embodiment 1 takes off behind the ester and recombinant human insulin's standard substance comparison diagram; Wherein, the HPLC of sample detected collection of illustrative plates after figure a and figure b were respectively recombinant human insulin's standard substance and take off ester.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) desB
30The preparation of insulin human's hydrous powdery: recombinant human insulin's precursor of pichia spp secreting, expressing, behind the purification with macroreticular resin (reference: Gao Jiankun, wealth continues fair-skinned, and Fan Kai etc. contain short C peptide proinsulin human analogue desB
30Expression and purification in pichia spp, Progress in Biochemistry and Biophysics, 2008,35(1): sample solution 63-68), carry out anti-phase purifying after diluting 2 times, adopting Virahol to carry out gradient elution, obtain containing recombinant human insulin's precursor aqueous solution (HPLC detects collection of illustrative plates as shown in Figure 1a) of Virahol (concentration is volume percent 24%), is 8.5 with 5M ammoniacal liquor adjust pH.Ratio in body mass ratio 1:300 before trypsinase and the recombinant human insulin adds trypsinase, cuts 3 hours in 25 ℃ of enzymes, obtains the solution that enzyme is cut, and detecting the recombinase rate of cutting through HPLC is that 97.55%(HPLC detects collection of illustrative plates shown in Fig. 1 b).Add zinc chloride by recombinant human insulin's precursor and zinc chloride mol ratio 1:1, then regulating the pH value with the phosphoric acid solution of 3M is that 5.8,4～8 ℃ of placements are spent the night, and uses 300 purpose 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 insulin human carries out transpeptidation reaction for the reaction density of 17mM, obtains recombinant human insulin's ester: the desB that obtains with dmso solution step (1)
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate adds BDO again, adds at last the trypsinase aqueous solution, obtains turning peptide liquid.DesB wherein
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 that turns is taken a sample respectively at 0,2,4,6,8 hour 25 ± 1 ℃ of lower mild stirring reactions, and 0,2,4,6,8 hour the peptide efficient that turns is respectively 0%, 76.03%, 88.02%, 91.82%, 90.66%.The collection of illustrative plates that HPLC detects is respectively shown in a～e among Fig. 2.
React the solution that obtains containing recombinant human insulin's ester after 8 hours, use the phosphate aqueous solution of pH3.0 with 5 times of its dilutions.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying, is 5925 through the mass spectroscopy molecular weight, consistent with theoretical molecular (mass spectrometric detection the results are shown in Figure 3).
(3) take off ester: behind the insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the 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 and fit like a glove (as shown in Figure 5), illustrate that the recombinant human insulin who obtains in the present embodiment has identical structure with recombinant human insulin's standard substance.
(1) desB
30The preparation of insulin human's hydrous powdery: the concentration that the recombinant human insulin's precursor aqueous solution among the embodiment 1 is diluted to Virahol is volume percent 10%, is 9 with 5M ammoniacal liquor adjust pH.The mass ratio of pressing trypsinase and recombinant human insulin's precursor 1:400 adds trypsinase, cuts 5 hours in 25 ℃ of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 95.31% after testing.The mol ratio of pressing human insulin precursor and zinc chloride 1:1.5 adds zinc chloride, and regulating the pH value with the 3M phosphoric acid solution is that 5.8,4～8 ℃ of placements are spent the night, and uses 300 purpose filter membrane suction filtrations to obtain desB
30Insulin human's hydrous powdery, and survey moisture and content.
(2) with desB
30The insulin human obtains recombinant human insulin's ester: use dmso solution desB for the reaction density of 13.4mM carries out transpeptidation reaction
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate adds BDO again, adds at last the trypsinase aqueous solution.DesB wherein
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 that turns is taken a sample respectively at 2,4,6 hours 25 ± 1 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6 hours the peptide efficient that turns is respectively 87.12%, 89.51%, 87.68%.
(3) take off ester: react the solution that obtains containing recombinant human insulin's ester after 6 hours, use the phosphate aqueous solution of pH3.0 with 5 times of its dilutions.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying, behind the insulin-ester isoelectric precipitation, and lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the recombinant human insulin.
Embodiment 3
(1) desB
30Carry out wash-out with ethanol during the anti-phase purifying of recombinant human insulin's precursor among the preparation of insulin human's hydrous powdery: the embodiment 1, it is volume percent 30% that elutriant is diluted to alcohol concn, regulating the pH value with 5M ammoniacal liquor is 8.5, the mass ratio of pressing trypsinase and recombinant human insulin's precursor 1:300 adds trypsinase, cuts 6 hours in 25 ℃ of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 96.07% after testing.The mol ratio of pressing human insulin precursor and zinc chloride 1:0.5 adds zinc chloride, regulates pH5.80 with 3M phosphoric acid, and 4～8 ℃ of placements are spent the night, the centrifugal desB that obtains of 8000g
30Insulin human's hydrous powdery.Measure its moisture and content.
(2) with desB
30The insulin human obtains recombinant human insulin's ester: use dmso solution desB for the reaction density of 12.1mM carries out transpeptidation reaction
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolving adds the trypsinase aqueous solution again.DesB wherein
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 that turns is taken a sample respectively at 2,4,6,8 hours 25 ± 1 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6,8 hours the peptide efficient that turns is respectively 83.58%, 84.11%, 87.76%, 86.60%.
(3) take off ester: reaction obtained containing the solution of recombinant human insulin's ester in 8 hours, used the phosphate aqueous solution of pH3.0 with 5 times of its dilutions.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.Behind the insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the 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 among the embodiment 1 is diluted to isopropyl alcohol concentration, regulating the pH value with 5M ammoniacal liquor is 8.0, press trypsinase and recombinant human insulin's precursor 1:500 mass ratio add trypsinase, cut 8 hours in 20 ℃ of enzymes, recombinant human insulin's pre-enzyme rate of cutting is 94.75% after testing.Regulating the pH value with the 3M phosphoric acid solution is that 5.6,4～8 ℃ of placements are spent the night, and uses 300 purpose filter membrane suction filtrations to obtain desB
30Insulin human's hydrous powdery is measured moisture and content.
(2) with desB
30The insulin human obtains recombinant human insulin's ester: use dmso solution desB for the reaction density of 33mM carries out transpeptidation reaction
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolving adds BDO again, adds at last the trypsinase aqueous solution.DesB wherein
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 that turns is taken a sample respectively at 2,4,6,8 hours 25 ± 1 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6,8 hours the peptide efficient that turns is respectively 75.04%, 84.05%, 89.42%, 87.46%.
(3) take off ester: reaction obtained containing the solution of recombinant human insulin's ester in 8 hours, used the phosphate aqueous solution of pH3.0 with 5 times of its dilutions.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.Behind the insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the 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 among the embodiment 1 is diluted to isopropyl alcohol concentration, and regulating the pH value with 5M ammoniacal liquor is 7.5.The mass ratio of pressing trypsinase and recombinant human insulin's precursor 1:200 adds trypsinase, cuts 6 hours in 20 ℃ of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 96.49% after testing.The mol ratio of pressing human insulin precursor and zinc chloride 1:1.2 adds zinc chloride, and regulating the pH value with the 3M phosphoric acid solution is that 5.8,4～8 ℃ 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 insulin human obtains recombinant human insulin's ester: use dmso solution desB for the reaction density of 25mM carries out transpeptidation reaction
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolving adds BDO again, adds at last the trypsinase aqueous solution.DesB wherein
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 that turns is taken a sample respectively at 2,4,6,8 hours 20 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6,8 hours the peptide efficient that turns is respectively 81.39%, 82.56%, 87.22%, 85.97%.
(3) take off ester: reaction obtained containing the solution of recombinant human insulin's ester in 8 hours, used the phosphate aqueous solution of pH3.0 with 8 times of its dilutions.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.Behind the insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the 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 among the embodiment 1 is added Virahol to Virahol final concentration, and regulating the pH value with 5M ammoniacal liquor is 7.The mass ratio of pressing trypsinase and recombinant human insulin's precursor 1:50 adds trypsinase, cuts 4.0 hours in 30 ℃ of enzymes.Recombinant human insulin's pre-enzyme rate of cutting is 97.22% after testing.The mol ratio of pressing human insulin precursor and zinc chloride 1:1.5 adds zinc chloride, regulates pH5.8 with the 3M phosphoric acid solution, and 4～8 ℃ 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 insulin human obtains recombinant human insulin's ester: use dmso solution desB for the reaction density of 12.1mM carries out transpeptidation reaction
30Insulin human's hydrous powdery and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolving adds BDO again, adds at last the trypsinase aqueous solution.DesB wherein
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 that turns is taken a sample respectively at 2,4,6,8 hours 25 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6,8 hours the peptide efficient that turns is respectively 79.60%, 82.97%, 85.70%, 85.69%.
(3) take off ester: reaction obtained containing the solution of recombinant human insulin's ester in 8 hours,, with the phosphate aqueous solution of pH3.0 it is diluted 5 times.Recombinant human insulin's ester obtains the concentrated sample of high purity through anti-phase purifying.Behind the insulin-ester isoelectric precipitation, lyophilize.After adopting acetone solution and vacuum to detach acetone, press the 1g insulin-ester: after the amount of 5ml trifluoroacetic acid was dissolved insulin-ester, vacuum detached unnecessary trifluoroacetic acid, namely gets the recombinant human insulin.
1 one steps of Comparative Examples turn the peptide method
Recombinant human insulin's precursor aqueous solution solution among the embodiment 1, the lyophilized powder of acquisition recombinant human insulin precursor is measured its content after the lyophilize.
Reaction density take recombinant human insulin's precursor as 6.7mM turns peptide.With dmso solution recombinant human insulin precursor lyophilized powder and o-tertbutyl ether-l-threonine tert-butyl ester acetate dissolving, add again BDO, add at last 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 that turns is taken a sample respectively at 2,4,6 hours 25 ℃ of lower mild stirring reactions, and HPLC detects analytic sample.2,4,6 hours the peptide efficient that turns is respectively 25.00%, 43.13%, 68.60%.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a recombinant human insulin preparation method is characterized in that may further comprise the steps:
(1) in the mixing solutions of organic solvent and water, the restructuring human insulin precursor is carried out endonuclease reaction, obtain containing desB
30Insulin human's solution;
(2) from containing desB
30Insulin human's solution, centrifugal or suction filtration obtains desB
30Insulin human's hydrous powdery;
(3) use desB
30Insulin human's hydrous powdery carries out transpeptidation reaction, obtains the human insulin ester; Wherein, desB
30The final concentration of insulin human in the transpeptidation reaction system is 12.1mM～33mM;
(4) recombinant human insulin's ester is taken off ester, namely get the recombinant human insulin.
2. recombinant human insulin's according to claim 1 preparation method, it is characterized in that: the concentration of the organic solvent described in the step (1) is volume percent 0.1～30%;
Described organic solvent is Virahol or ethanol.
3. recombinant human insulin's according to claim 1 preparation method, it is characterized in that: step (1) is: in the mixing solutions of organic solvent and water the restructuring human insulin precursor is carried out endonuclease reaction, the condition that enzyme is cut is for to be adjusted to 7～9 with the pH value, in 20～30 ℃ of reactions, obtain containing the 30th insulin human's solution that lacks Threonine of insulin human B chain; Wherein, trypsinase and recombinant human insulin's precursor 1:50～500 proportionings in mass ratio.
4. recombinant human insulin's according to claim 3 preparation method, it is characterized in that: the time of the reaction described in the step (1) is 1～8 hour;
PH value described in the step (1) is regulated with ammoniacal liquor;
Described trypsinase and described recombinant human insulin's precursor be the 1:300 proportioning in mass ratio.
5. recombinant human insulin's according to claim 1 preparation method is characterized in that: before the centrifugal or suction filtration described in the step (2), first to containing desB
30Insulin human's solution precipitates.
6. recombinant human insulin's according to claim 5 preparation method is characterized in that: described to containing desB
30The mode that insulin human's solution precipitates staticly settles after regulating the pH value, or adds Zn first
2+, re-adjustment pH value staticly settles afterwards.
7. recombinant human insulin's according to claim 6 preparation method, it is characterized in that: described pH value is 5.6～6;
The reagent of described adjusting pH value can be for a kind of in phosphoric acid, acetic acid and the ammoniacal liquor or at least two kinds;
Described Zn
2+Consumption press desB
30Insulin human and Zn
2+Mol ratio be that interpolation is calculated in 1:0.5～1.5;
The described temperature that leaves standstill is 4～8 ℃.
8. recombinant human insulin's according to claim 1 preparation method is characterized in that:
Transpeptidation reaction described in the step (3) comprises the steps: with dmso solution Threonine or derivatives thereof, and desB
30Insulin human's hydrous powdery adds BDO and the trypsinase aqueous solution again or only adds the trypsinase aqueous solution, obtains to turn the reaction system of 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.
9. recombinant human insulin's according to claim 8 preparation method 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;
The ratio of solvent is dimethyl sulfoxide (DMSO) in the described reaction system: BDO: water is 15%～50%:0～65%:20%～50% by volume; Wherein comprised desB in the content of water
30The moisture that comprises in insulin human's hydrous powdery.
10. recombinant human insulin's according to claim 1 preparation method is characterized in that: take off ester for adopting trifluoroacetic acid reagent to take off ester described in the step (4).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164594A (en) * | 2017-12-08 | 2018-06-15 | 珠海冀百康生物科技有限公司 | The recovery method that a kind of insulin precurosor for removing 30 amino acids residue of B chains precipitates |
Citations (2)
| 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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Patent Citations (2)
| 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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| XIE, T.等: "Secretory Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin", 《PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164594A (en) * | 2017-12-08 | 2018-06-15 | 珠海冀百康生物科技有限公司 | The recovery method that a kind of insulin precurosor for removing 30 amino acids residue of B chains precipitates |
| CN108164594B (en) * | 2017-12-08 | 2020-03-31 | 珠海冀百康生物科技有限公司 | Recovery method of insulin precursor precipitate without 30-bit amino acid residue in B chain |
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