CN102703552B - Preparation method and products of recombinant human insulin - Google Patents
Preparation method and products of recombinant human insulin Download PDFInfo
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Abstract
The invention discloses a method for preparing human insulin from a human insulin precursor. The method comprises the following steps: (1) conducting an enzyme digestion reaction on the human insulin precursor in an aqueous phase buffer solution to obtain a 30-position double-strand human insulin precursor desB30 lacking threonine; (2) enabling the double-strand human insulin precursor desB30 to be subjected to transpeptidation in an organic phase to obtain insulin ester; and (3) carrying out de-esterification on the insulin ester to obtain the human insulin. Compared with the existing method for transforming the human insulin precursor to the human insulin ester at one step, the method disclosed by the invention is remarkably improved in transpeptidation efficiency, greatly shortened in transpeptidation time, fewer in by-products, higher in overall yield and lower in cost. The invention provides a simple and efficient method for large scale and industrialized production of recombinant human insulin by pichia pastoris expression.
Description
Technical field
The present invention relates to a kind of recombinant human insulin's preparation method, relate in particular to recombinant human insulin's product that a kind of insulin precurosor by Pichia anomala expression is converted into recombinant human insulin's method and is obtained by this preparation method, belong to recombinant human insulin's preparation field.
Background technology
Along with the raising of people's living standard, the social aged increases, and the sickness rate of diabetes is rising year by year.Diabetes are a kind of clinical syndromes that caused by h and E factor interaction, and its basic pathology is characterized as definitely or the caused metabolism disorder of relativity hypoinsulinism.Diabetes have now become a kind of common disease, frequently-occurring disease, are a kind of diseases of serious harm human health.Preventing and treating diabetes has become one of important, urgent health care problem clinically, day by day by people are paid attention to.For islet cells pathology, can not excreting insulin, need to supplement foreign aid's Regular Insulin, also many insulin-dependent diabetes mellitus (IDDM) patient of relevant complication, Regular Insulin is unique medicine.In addition,, although also have 30%～40% the patients with NIDDM self can excreting insulin, because secretory volume is not enough or can not effectively utilize Regular Insulin, finally also need to use pancreas islet usually to treat.Insulin preparations has now accounted for more than 30% market share of whole diabetic.
China has replaced India at present becomes global diabetes the first big country, within 2010, diabetic subject has reached 9,200 ten thousand people, in addition three one-tenth of diabetes mellitus in China patient wholistic therapy rate less thaies and Regular Insulin usage quantity is on the low side per capita, be only U.S. diabetic subject per capita usage quantity 0.8%, so Chinese Regular Insulin market potential is huge.Therefore, Development and Production recombinant human insulin's new way, the means of production of optimizing existing Regular Insulin are important topics of medicine bioengineering chemical field always.
Industry is known, and the industrial requirement that genetically engineered is produced insulin human is quite high.One, Regular Insulin is the small molecules of a more complicated, production process complexity.The genetically engineered recombinant human insulin of listing mainly contains 2 kinds of production methods now, is respectively the escherichia coli expression of Li Lai company and the yeast expression of Novo Nordisk Co.,Ltd.The insulin precurosor that a kind of front method obtains need to be through the lower renaturation manipulation of yield and the more expensive trypsinase of price and the double digestion of carboxypeptidase; A kind of rear method need to be carried out the enzyme that cost is very high by the precursor of expression and be cut transpeptidation reaction.In addition, two kinds of methods all need high performance liquid chromatography to carry out polishing purification, and production cost and complexity are far above common gene engineering products such as picture Interferon, rabbit.Its two, this clinical drug using dosage is larger, is approximately 300 times of the clinical using dosage of Interferon, rabbit.Its three, low price.Come out more than 20 year as the gene engineering product of first listing, and the Regular Insulin listing history of existing 80 years of extracting from animal pancreas, in fact due to the competition of long-term technology and price, Regular Insulin has become in the world the most cheaply, the maximum gene engineering drug of consumption.This makes the suitability for industrialized production of this product will reach that scale is large, cost is low, technique is very ripe could realize profit.
Pichia pastoris phaff (Pichia pastoris) is one of current most widely used exogenous protein expression system.This expression system has the advantage of prokaryotic expression system and eukaryotic expression system concurrently simultaneously, cultivate processing ease, growth fast, expression amount is high, processing and modification after external source eukaryotic gene correctly being translated and be translated again, and numerous protein product is carried out to secreting, expressing, make product be easy to purify.In addition, it is high that pichia spp has again secernment efficiency, and product can excessive glycosylation, and protein antigenicity is low, and expression strain is difficult for the advantages such as loss in thalline genome because recombinant plasmid is fused to.Therefore be subject to nearly ten years numerous investigators' favor.The expression that utilizes pichia spp to carry out insulin precurosor is focus of bio-pharmaceuticals in recent years always.
(the Preparative Biochemistry & Biotechnology.2008 such as, Liu Haifeng graceful according to thanking, 38:308-317) report, the expression amount of the insulin precurosor of Pichia anomala expression on the microorganism fermentation tank of 16L can reach 3.6g/L.Fermented liquid is after centrifugal, ion-exchange purification, desalination freeze-drying, insulin precurosor utilizes excessive tertiary butyl ether threonine tert-butyl ester under tryptic effect in organic phase, and direct enzyme cutting turns peptide and generated the insulin ester that has added Threonine on the 30th, B chain.This insulin ester, through trifluoroacetic acid deprotection and after high performance liquid chromatography, has obtained the recombinant human insulin consistent with insulin human's structure.
But with Pichia anomala expression Restruction insulin human, one of the most key step is that enzyme is cut and turned peptide.The most obvious feature of this step is that conversion is slow, yield is low, cost is high.Obtain competitive product, one of crucial index is that enzyme is cut the yield that turns peptide and wanted high, and cost is low.
Enzyme is cut and is turned that peptide process is actual comprises two reactions, and first reaction is with trypsinase, insulin precurosor enzyme to be cut, and need to cut altogether three times, could generate the double-stranded insulin precurosor desB30 that the 30th, B chain lacks Threonine.Second reaction be the desB30 that generates still under tryptic effect, turn a tertiary butyl ether threonine tert-butyl ester of peptide access at the C-terminal of B29 Methionin.In the document of having reported, it is all that a step is carried out in organic phase that enzyme is cut the two-step reaction process that turns peptide, as dimethylamine, BDO etc.For ensureing the higher peptide efficiency that turns, the mol ratio of expensive tertiary butyl ether threonine tert-butyl ester and desB30 will exceed or approach 100 times.But in the organic phase of higher concentration, tryptic activity obviously reduces, so the mass ratio of the trypsinase in transpeptidation reaction and precursor is approximately 1:5, even ratio is higher, finally cause the consumption of pancreatin also very large, cost is also higher.In addition, it is very slow that enzyme is cut transpeptidation reaction speed, and general 10 to 24 hours, more than at least 10 hours.After long reaction time, by product can increase, and can increase the difficulty of downstream purification, is also unfavorable for the raising of overall yield simultaneously.
Summary of the invention
Technical problem to be solved by this invention be overcome existingly in the existing method of being prepared human insulin ester by insulin precurosor turn that peptide efficiency is low, output yield is low, the many or high in cost of production defect of by product; a kind of new insulin human's preparation method is provided; the method to turn peptide efficiency high; by product is few; product yield is high; cost is low, the method suitability for scale production recombinant human insulin.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A method of being prepared recombinant human insulin by human insulin precursor, comprises the following steps: (1) is carried out human insulin precursor endonuclease reaction and obtained the double-stranded insulin precurosor desB30 of people that the 30th, B chain lacks Threonine in water damping fluid; (2) in organic phase, double-stranded human insulin precursor desB30 is carried out to transpeptidation reaction and obtain insulin-ester; (3) by de-insulin-ester ester, to obtain final product.
Water damping fluid described in step (1) is preferably tris buffer (Tris damping fluid) or ammonium bicarbonate buffers (NH
4hCO
3damping fluid); In order to reach better effect, it is the Tris damping fluid that 25-100mM, pH value are 7.0-8.5 that described Tris damping fluid is preferably concentration; More preferably concentration is the Tris damping fluid that 50mM, pH value are 8.0.Described NH
4hCO
3damping fluid is preferably concentration 50-200mM, the NH that pH value is 7.0-8.5
4hCO
3damping fluid.
In step (1), with trypsinase, insulin precurosor being carried out to enzyme cuts; The trypsinase adding and the mass ratio of insulin precurosor are preferably 1:100 ~ 400, more preferably 1:200.
The endonuclease reaction time described in step (1) is preferably 2.0 ~ 4.0h, more preferably 3.0h.
Endonuclease reaction temperature described in step (1) is preferably 20-30 DEG C, more preferably 25 DEG C.
" human insulin precursor " described in the present invention can be that any one adopts the insulin precurosor that genetically engineered is expressed or prepare, for example, can be to adopt pichia spp or Wine brewing yeast strain to carry out the insulin precurosor of secreting, expressing.Adopting Pichi strain secreting, expressing insulin precurosor is the ordinary skill in the art, for various kinds of document reported (Li Jun etc., the structure of the Pichi strain of expression human insulin precursor. the journal .2004 of Beijing University of Chemical Technology, the 31st volume the 2nd phase .21-23; Hao Helong etc., the optimization of insulin human B27K-DTr I precursor expression condition in pichia spp. food and pharmaceutical .2011 the 13rd volume the 09th phase .305-308).
Transpeptidation reaction described in step (2) is under tryptic effect, turns a tertiary butyl ether threonine tert-butyl ester of peptide access at the C-terminal of the B29 of double-stranded insulin precurosor desB30 Methionin.
Concrete, described transpeptidation reaction can carry out with reference to following methods: after dimethyl sulfoxide (DMSO) (DMSO) is dissolved for threonine tert-butyl ester by desB30 and tertiary butyl ether, then add successively BDO, Tris damping fluid, finally adds trypsinase to react; Described Tris damping fluid preferably concentration is the Tris damping fluid that 0.5M, pH value are 6.5;
The volume ratio of DMSO, Tris and BDO is preferably 15:15:70;
The final concentration of the double-stranded insulin precurosor desB30 of step (2) in reaction soln is preferably 2.5 ~ 10mM, more preferably 5mM; The mol ratio of desB30 protein freeze-dried powder and tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) is 1:25 ~ 200, more preferably 1:50;
Trypsinase and desB30 mass ratio 1:10 ~ 40, more preferably 1:20;
Wherein, described temperature of reaction is preferably 20-30 DEG C, more preferably 25 DEG C; The described reaction times is preferably 1 ~ 3h, more preferably 2.0h.
De-ester described in step (3) can be for adopting trifluoroacetic acid to take off ester.
For the insulin precurosor that improves Pichia anomala expression changes into insulin human more efficiently, the insulin precurosor that the present invention has adopted two-step approach to replace single stage method to carry out Pichia anomala expression changes into human insulin ester's enzyme and cuts transpeptidation reaction.The first step is first cut insulin precurosor enzyme to obtain desB30 in water, and pre-enzyme is cut efficiency and exceeded 95%; Second step carries out desB30 and turns peptide generation insulin-ester in organic phase, turns peptide efficiency and exceedes 85%.By this two-step reaction; make to utilize the peptide efficiency that turns of the insulin-ester that insulin precurosor that Pichia anomala expression obtains generates to significantly improve; the reaction times that turns peptide significantly shortens; by product is few; entirety yield is higher; cost is lower, for mass-producing, industrialized utilization Pichia anomala expression Restruction insulin human, provides a kind of simple and direct efficient method.
Brief description of the drawings
Fig. 1 HPLC detection two-step approach of the present invention is prepared recombinant human insulin's enzyme and is cut efficiencies.
What Fig. 2 HPLC detected that two-step approach of the present invention prepares recombinant human insulin turns peptide efficiencies.
The ESI mass spectroscopy result of the prepared insulin-ester of Fig. 3 the inventive method.
The prepared recombinant human insulin's of Fig. 4 the inventive method ESI mass spectroscopy result.
The HPLC measurement result of the prepared recombinant human insulin of Fig. 5 the inventive method and recombinant human insulin's standard substance.
What Fig. 6 HPLC detected existing One-step production recombinant human insulin turns peptide efficiencies.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 recombinant human insulin's preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the Tris damping fluid of pH 8.0 of 50mM, the ratio that is 1:200 in trypsinase and human insulin precursor mass ratio, 25 DEG C of enzymes are cut 3.0h, obtain enzyme and cut the aqueous solution completely.After testing, pre-enzyme to cut efficiency be 98.5%(Fig. 1).Precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:50, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 5mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:20; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein 15min, 1h, 2h and 3h turn peptide efficiency be respectively 45.2%, 80.3%, 84.2% and 75.9%(Fig. 2).
React after 2 hours acetic acid (HAc) termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5924.6, consistent with theoretical molecular (Fig. 3).
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.3, consistent with theoretical molecular (Fig. 4).To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure (Fig. 5) with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 28.2U/mg, suitable with recombinant human insulin's standard substance.
Embodiment 2 recombinant human insulins' preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the Tris damping fluid of pH 8.5 of 25mM, the ratio that is 1:400 in trypsinase and human insulin precursor mass ratio, 30 DEG C of enzymes are cut 2.0h, obtain enzyme and cut the aqueous solution completely.After testing, to cut efficiency be 96.5% to pre-enzyme; Precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:25, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 2.5mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:10; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein the peptide efficiency that turns of 15min, 1h, 2h and 3h is respectively 37.8%, 76.5%, 81.3% and 72.8%.
React after 3 hours the HAc termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5923.9, consistent with theoretical molecular.
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.1, consistent with theoretical molecular.To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 27.2U/mg, suitable with insulin human's standard substance.
Embodiment 3 recombinant human insulins' preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the Tris damping fluid of pH 7.5 of 100mM, the ratio that is 1:100 in trypsinase and human insulin precursor mass ratio, 20 DEG C of enzymes are cut 3.0h, obtain enzyme and cut the aqueous solution completely, after testing, to cut efficiency be 95.7% to pre-enzyme; Precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:200, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 5mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:40; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein the peptide efficiency that turns of 15min, 1h, 2h and 3h is respectively 41.8%, 77.9%, 82.1% and 72.3%.
React after 3 hours the HAc termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5924.4, consistent with theoretical molecular.
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.5, consistent with theoretical molecular.To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 27.6U/mg, suitable with insulin human's standard substance.
Embodiment 4 recombinant human insulins' preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the Tris damping fluid of pH 7.0 of 80mM, the ratio that is 1:300 in trypsinase and human insulin precursor mass ratio, 20 DEG C of enzymes are cut 4.0h, obtain enzyme and cut the aqueous solution completely, after testing, to cut efficiency be 95.1% to pre-enzyme; Precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:100, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 8mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:30; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein the peptide efficiency that turns of 15min, 1h, 2h and 3h is respectively 41.2%, 76.8%, 81.9% and 72.1%.
React after 3 hours the HAc termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5924.3, consistent with theoretical molecular.
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.2, consistent with theoretical molecular.To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 27.8U/mg, suitable with insulin human's standard substance.
Embodiment 5 recombinant human insulins' preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the ammonium bicarbonate buffers of pH 7.0 of 50mM, the ratio that is 1:400 in trypsinase and human insulin precursor mass ratio, 25 DEG C of enzymes are cut 4.0h, obtain enzyme and cut the aqueous solution completely.After testing, to cut efficiency be 94.6% to pre-enzyme; Precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:25, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 2.5mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:10; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein the peptide efficiency that turns of 15min, 1h, 2h and 3h is respectively 37.8%, 76.5%, 81.3% and 72.8%.
React after 3 hours the HAc termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5923.9, consistent with theoretical molecular.
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.1, consistent with theoretical molecular.To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 27.2U/mg, suitable with insulin human's standard substance.
Embodiment 6 recombinant human insulins' preparation and qualification
Through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography), freeze-drying obtains protein powder to the human insulin precursor supernatant liquor of Pichi strain secreting, expressing, be dissolved in the ammonium bicarbonate buffers of pH 8.5 of 200mM, the ratio that is 1:100 in trypsinase and human insulin precursor mass ratio, 30 DEG C of enzymes are cut 2.0h, obtain enzyme and cut the aqueous solution completely.After testing, it is 93.9% that pre-enzyme is cut efficiency, and precipitation freeze-drying obtains the protein powder of desB30;
Taking mol ratio is desB30 protein freeze-dried powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:200, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add successively again BDO, the Tris damping fluid of the 0.5M that pH is 6.5; Wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and controlling the final concentration of desB30 in solution is 5mM; Finally add trypsinase, trypsinase and desB30 mass ratio are 1:40; Stirring reaction at 25 DEG C, HPLC detect respectively turn peptide 0min, 15min, 1h, 2h and 3h turn peptide efficiency, wherein the peptide efficiency that turns of 15min, 1h, 2h and 3h is respectively 41.8%, 77.9%, 82.1% and 72.3%.
React after 3 hours the HAc termination reaction with the 1.0M of 2 times of volumes, obtain insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction, after anti-phase purifying, obtain insulin-ester, through ESI mass spectroscopy, molecular weight is 5924.4, consistent with theoretical molecular.
Insulin-ester, through the de-ester of trifluoroacetic acid, generates recombinant human insulin.Through ESI mass spectroscopy, molecular weight is 5808.5, consistent with theoretical molecular.To under recombinant human insulin prepared the present embodiment and recombinant human insulin's standard substance identical conditions, carry out HPLC detection.The appearance time of the recombinant human insulin that the present embodiment is prepared and recombinant human insulin's standard substance is in full accord.Above description of test is produced the recombinant human insulin who obtains and is had identical structure with recombinant human insulin's standard substance according to two-step approach of the present invention.
According to " Chinese Pharmacopoeia " 2010 editions annex XII Regular Insulin bioassay methods (hypoglycemic method), the recombinant human insulin who selects adult mice to prepare the present embodiment carries out activity in vivo mensuration.Institute's test sample product specific activity is 27.6U/mg, suitable with insulin human's standard substance.
The enzyme of the insulin precurosor that comparative example's 1 single stage method is carried out is cut transpeptidation reaction
The freeze-drying after preliminary purification (purifying mode is ion-exchange and reversed phase chromatography) of the insulin precurosor supernatant liquor of Pichi strain secreting, expressing obtains protein powder.Taking mol ratio is insulin precursor protein lyophilized powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:50, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add again 1 successively, 4-butyleneglycol, pH is the Tris damping fluid of 6.5 0.5M, wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and the final concentration of insulin precurosor is 5mM.Finally add trypsinase, the mass ratio 1:5 of trypsinase and insulin precurosor, stirring reaction 24h at 25 DEG C, by the HAc termination reaction of the 1.0M of 2 times of volumes, obtains insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction.What HPLC detected respectively 0h, 10h, 20h and 24h turns peptide efficiency, wherein 10h, 20h and 24h turn peptide efficiency be respectively 28.1%, 43.9% and 42.1%(Fig. 6).
The enzyme of the insulin precurosor that comparative example's 2 single stage method are carried out is cut transpeptidation reaction
The insulin precurosor supernatant liquor of Pichi strain secreting, expressing obtains protein powder through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography) freeze-drying.Taking mol ratio is insulin precursor protein lyophilized powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:25, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add again 1 successively, 4-butyleneglycol, pH is the Tris damping fluid of 6.5 0.5M, wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and the final concentration of insulin precurosor is 2.5mM.Finally add trypsinase, the mass ratio 1:10 of trypsinase and insulin precurosor, stirring reaction 24h at 25 DEG C, by the HAc termination reaction of the 1.0M of 2 times of volumes, obtains insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction.What HPLC detected respectively 0h, 10h, 20h and 24h turns peptide efficiency, and wherein the peptide efficiency that turns of 10h, 20h and 24h is respectively 27.3%, 42.4% and 41.5%.
The enzyme of the insulin precurosor that comparative example's 3 single stage method are carried out is cut transpeptidation reaction
The insulin precurosor supernatant liquor of Pichi strain secreting, expressing obtains protein powder through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography) freeze-drying.Taking mol ratio is insulin precursor protein lyophilized powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:200, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add again 1 successively, 4-butyleneglycol, pH is the Tris damping fluid of 6.5 0.5M, wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and the final concentration of insulin precurosor is 5mM.Finally add trypsinase, trypsinase and insulin precurosor mass ratio 1:40, stirring reaction 24h at 25 DEG C, by the HAc termination reaction of the 1.0M of 2 times of volumes, obtains insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction.What HPLC detected respectively 0h, 10h, 20h and 24h turns peptide efficiency, and wherein the peptide efficiency that turns of 10h, 20h and 24h is respectively 26.7%, 42.7% and 41.8%.
The enzyme of the insulin precurosor that comparative example's 4 single stage method are carried out is cut transpeptidation reaction
The insulin precurosor supernatant liquor of Pichi strain secreting, expressing obtains protein powder through preliminary purification (purifying mode is ion-exchange and reversed phase chromatography) freeze-drying.Taking mol ratio is insulin precursor protein lyophilized powder and the tertiary butyl ether threonine tert-butyl ester (Thr (But)-OBut) of 1:100, complete to dissolving by dimethyl sulfoxide (DMSO) (DMSO), add again 1 successively, 4-butyleneglycol, pH is the Tris damping fluid of 6.5 0.5M, wherein the volume ratio of DMSO, 0.5M Tris (pH6.5), BDO is 15:15:70, and the final concentration of insulin precurosor is 8mM.Finally add trypsinase, trypsinase and insulin precurosor mass ratio 1:30, stirring reaction 24h at 25 DEG C, by the HAc termination reaction of the 1.0M of 2 times of volumes, obtains insulin-ester (desB30-Thr (But)-OBut) solution after transpeptidation reaction.What HPLC detected respectively 0h, 10h, 20h and 24h turns peptide efficiency, and wherein the peptide efficiency that turns of 10h, 20h and 24h is respectively 27.8%, 43.4% and 41.7%.
Claims (6)
1. a method of being prepared recombinant human insulin by human insulin precursor, comprises the following steps: (1) is carried out endonuclease reaction with trypsinase to human insulin precursor and obtained the double-stranded insulin precurosor desB30 of people that the 30th, B chain lacks Threonine in water damping fluid; The trypsinase adding and the mass ratio of insulin precurosor are 1:100～400; Described water damping fluid is that concentration is the tris buffer that 25～100mM, pH value are 7.0～8.5; (2) in organic phase, double-stranded human insulin precursor desB30 is carried out to transpeptidation reaction and obtain insulin-ester; Described transpeptidation reaction is: double-stranded insulin precurosor desB30 and tertiary butyl ether threonine tert-butyl ester, with after dmso solution, then are added to BDO, tris buffer successively, finally add trypsinase to react; The concentration of described tris buffer is that 0.5M, pH value are 6.5; The volume ratio of DMSO, Tutofusin tris and BDO is 15:15:70; The final concentration of double-stranded insulin precurosor desB30 in reaction soln is 2.5～10mM; The mol ratio of desB30 protein freeze-dried powder and tertiary butyl ether threonine tert-butyl ester is 1:25～200; Mass ratio 1:10～40 of described trypsinase and desB30; Described temperature of reaction is 20～30 DEG C; The described reaction times is 1～3h; (3) by de-insulin-ester ester, to obtain final product.
2. it is characterized in that in accordance with the method for claim 1: the water damping fluid described in step (1) is that concentration is the tris buffer that 50mM, pH value are 8.0.
3. it is characterized in that in accordance with the method for claim 1: the trypsinase adding in step (1) and the mass ratio of insulin precurosor are 1:200.
4. it is characterized in that in accordance with the method for claim 1: the enzyme time of cutting described in step (1) is 2.0～4.0h; Described endonuclease reaction temperature is 20～30 DEG C.
5. it is characterized in that in accordance with the method for claim 4: the enzyme time of cutting described in step (1) is 3.0h; Described endonuclease reaction temperature is 25 DEG C.
6. it is characterized in that in accordance with the method for claim 1: the final concentration of step (2) double center chain insulin precurosor desB30 in reaction soln is 5mM; The mol ratio of desB30 protein freeze-dried powder and tertiary butyl ether threonine tert-butyl ester is 1:50; The mass ratio of described trypsinase and desB30 is 1:20; Described temperature of reaction is 25 DEG C; The described reaction times is 2h.
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| CN116355101A (en) * | 2023-03-09 | 2023-06-30 | 北京惠之衡生物科技有限公司 | Preparation method of insulin aspart |
| CN116284452A (en) * | 2023-03-09 | 2023-06-23 | 北京惠之衡生物科技有限公司 | Preparation method of recombinant human insulin |
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| CN1040222A (en) * | 1988-08-10 | 1990-03-07 | 赫彻斯特股份公司 | Preparation goes-method of B30-Regular Insulin and going-B30-insulin derivates |
| CN1287124A (en) * | 1999-09-08 | 2001-03-14 | 中国科学院上海生物化学研究所 | Recombined natural and new-type human insulin and its preparation |
| CN1589328A (en) * | 2001-11-19 | 2005-03-02 | 诺沃挪第克公司 | Process for preparing insulin compounds |
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| CN1287124A (en) * | 1999-09-08 | 2001-03-14 | 中国科学院上海生物化学研究所 | Recombined natural and new-type human insulin and its preparation |
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