CN113481268B - Method for continuously preparing aliphatic chain-GLP-1 receptor agonist polypeptide by using microreactor - Google Patents
Method for continuously preparing aliphatic chain-GLP-1 receptor agonist polypeptide by using microreactor Download PDFInfo
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- CN113481268B CN113481268B CN202110695290.6A CN202110695290A CN113481268B CN 113481268 B CN113481268 B CN 113481268B CN 202110695290 A CN202110695290 A CN 202110695290A CN 113481268 B CN113481268 B CN 113481268B
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention provides a method for continuously preparing a fat chain-GLP-1 receptor stimulant polypeptide by using a microreactor, belongs to the fields of biological engineering and pharmaceutical engineering, and relates to fat chain modification of medicinal protein polypeptides. The continuous preparation method comprises a separation process for continuously synthesizing the fatty chain-GLP-1 receptor stimulant polypeptide and the downstream modified product in the microreactor. The invention establishes a continuous preparation method which is suitable for the fat chain-GLP-1 receptor agonist polypeptide and has high efficiency, energy conservation and easy amplification.
Description
Technical Field
The invention belongs to the fields of biological engineering and pharmaceutical engineering, relates to fatty chain modification of medicinal protein polypeptides, and particularly relates to a continuous preparation method of a fatty chain modified GLP-1 receptor agonist polypeptide.
Background
Protein/polypeptide drugs are becoming the leading products of modern biomedical industries, including recombinant proteins, polypeptides, enzymes, antibodies and the like, have the advantages of high biological activity, strong specificity and the like, and are widely used for preventing and treating serious diseases such as cardiovascular and cerebrovascular diseases, cancers, diabetes and the like. However, the pharmaceutical protein polypeptide still has the defects of short in-vivo half-life, strong immunogenicity and the like in clinic, so that repeated administration or large-dose administration is needed in clinical treatment, adverse reactions such as drug tolerance, immune rejection and the like are easily generated, and economic burden is increased for patients, so that the development of long-acting protein polypeptide drugs is a main direction for future development.
The glucagon-like peptide-1 (GLP-1) receptor stimulant medicine not only has the function of reducing blood sugar similar to insulin, but also has other functions, including appetite control, weight reduction, cardiovascular benefit and the like, and has great market potential. The natural GLP-1 can be rapidly hydrolyzed by dipeptidyl peptidase IV (DPP-IV) in vivo to lose biological activity, the half life is only 1-2min, and the natural GLP-1 is not medicinal, so that the development of a long-acting dosage form of GLP-1 receptor agonist drugs is an important direction for the development of future hypoglycemic drugs. The loxeinai peptide is modified and reformed on the basis of the chemical structure of the exenatide, the 2 nd Gly, the 14 th Met and the 28 th Asn of the exenatide are respectively mutated into D-Ala, Nle and Gln, the enzyme medium stability and the chemical stability based on a polypeptide skeleton are improved, and meanwhile, the Ser at the C end of the peptide is changed into Cys.
The fatty chain modified protein polypeptide gradually becomes a new sustained-release drug, namely, the side chain of fatty acid, alcohol, aldehyde or amine is coupled to the medicinal protein polypeptide by means of chemical reaction. The drug connected with the fatty chain can be combined with the human serum albumin or self-assembled into a polymer after entering the human body, thereby improving the half life of the circulation in the human body. In addition, the modified protein polypeptide drug also has the advantages of good biological safety, high activity retention rate, no immunogenicity, improved fat solubility and the like. The research of the medicinal protein polypeptide modified by the fatty chain mainly relates to the research of novel medicine development, the safety and effectiveness evaluation of the medicine and the relevant research of process engineering. At present, the research on process engineering is relatively weak, and the problem is a bottleneck restricting the industrial application of the process engineering. The process of modifying protein polypeptide drugs by fatty chains mainly has the following problems: first, the mass transfer efficiency between the hydrophilic polypeptide and the hydrophobic fatty chain modifier is low, directly resulting in a low efficiency of the modification reaction; secondly, the preparation process of the fatty chain modified drug mainly adopts the traditional intermittent operation, has low production efficiency and large batch-to-batch instability, and is one of the obstacles of process expansion; thirdly, the separation and purification means is mainly chromatographic means, so that the efficiency is low and the cost is high.
Disclosure of Invention
The invention aims to provide a continuous preparation method which is high-efficiency, energy-saving and easy to amplify and is used for the GLP-1 receptor stimulant polypeptide modified by the fatty chain.
The continuous preparation method comprises a separation process for continuously synthesizing the fat chain-GLP-1 receptor stimulant polypeptide and the downstream modification product in the microreactor. Aiming at sulfhydryl or amino of GLP-1 receptor agonist polypeptide, a specific modifier, such as fatty chain maleimide, fatty chain succinimide or fatty aldehyde, is adopted to realize continuous synthesis of the fatty chain-GLP-1 receptor agonist polypeptide through a microreactor system, and then a buffer solution is added into a crude product solution to realize separation of the modified product, namely the fatty chain-GLP-1 receptor agonist polypeptide by a precipitation method.
In order to achieve the purpose of the invention, the technical scheme of the invention is implemented by the following scheme:
a method for continuously preparing aliphatic chain-GLP-1 receptor stimulant polypeptides by using a microreactor mainly comprises the following steps:
(i) determining the technological parameters of the GLP-1 receptor stimulant polypeptide modified by the aliphatic chain modifier in a traditional batch reactor;
(ii) designing and constructing a micro-reactor system consisting of a pump, a micro-mixer and a micro-channel, and continuously synthesizing the fat chain-GLP-1 receptor agonist polypeptide in the micro-reactor under the process parameters determined in the step (i) to obtain a crude product solution;
(iii) and (3) adding a buffer solution into the crude product solution prepared in the step (ii) by utilizing the solubility difference between the aliphatic chain modifier and the aliphatic chain-GLP-1 receptor agonist polypeptide in the buffer solution, precipitating the aliphatic chain modifier, carrying out solid-liquid separation, taking a supernatant, and concentrating to obtain the aliphatic chain-GLP-1 receptor agonist polypeptide.
Further, based on the above technical scheme, the fatty chain modifier is a medium-long chain fatty acid, a fatty alcohol or a fatty amine with activated carboxyl, hydroxyl or amino, the number of carbon atoms of the fatty acid, alcohol or amine is 8-36, and the activated group comprises a maleimide group, a succinimide group and an aldehyde group.
Further, based on the technical scheme, the GLP-1 receptor agonist polypeptide comprises natural GLP-1, exenatide, lisinopeptide, lixisenatide, abilutide and loxapide.
Further, based on the above technical scheme, the specific steps of synthesizing the fatty chain-GLP-1 receptor agonist polypeptide in step (ii) are as follows: dissolving a fatty chain modifier in a reaction solvent to prepare a homogeneous solution A; dissolving GLP-1 receptor agonist polypeptide in a reaction solvent to prepare a homogeneous solution B, simultaneously pumping the homogeneous solution A and the homogeneous solution B into a micro mixer of a micro reactor, mixing uniformly, and introducing into a micro channel for reaction.
Further, based on the above technical scheme, the reaction solvent is a mixed solution of a buffer solution and an organic solvent, wherein the buffer solution comprises a phosphate buffer solution, a citric acid-sodium citrate buffer solution, and a glycine-hydrochloric acid buffer solution, and the organic solvent comprises methanol, ethanol, acetonitrile, and dimethyl sulfoxide (DMSO); the volume percentage of the buffer solution in the reaction solvent is 10-60%.
Further, based on the technical scheme, the concentration of the aliphatic chain modifier in the homogeneous solution A is 0.1-5 mg/mL; the concentration of GLP-1 receptor agonist polypeptide in the homogeneous solution B is 0.5-10 mg/mL; the reaction molar ratio of the fatty chain modifier to the GLP-1 receptor agonist polypeptide is 20: 1-1: 1.
Further, based on the technical scheme, the flow rate of pumping the homogeneous phase solution A into the microreactor is 0.05-1.5 mL/h; the flow rate of pumping the homogeneous phase solution B into the microreactor is 0.05-1.5 mL/h.
Further, based on the above technical scheme, the pump comprises an injection pump and a peristaltic pump; the micro mixer is a Y-shaped micro mixer or a T-shaped micro mixer, and the inner diameter size range of the micro mixer is 0.01-0.2 cm; the inner diameter of the micro-channel ranges from 0.01 cm to 0.2 cm.
Further, based on the technical scheme, the reaction conditions are that the reaction temperature is 0-50 ℃ and the reaction residence time is 2-10 h.
Further, based on the above technical scheme, the volume percentage of the buffer solution added in step (iii) in the total system is 70-95%.
Further, based on the above technical scheme, the solid-liquid separation in step (iii) can adopt centrifugation, membrane filtration, filter pressing or suction filtration.
Further, based on the above technical scheme, the concentration in step (iii) includes, but is not limited to, distillation and dialysis.
In another aspect, the invention provides the polypeptide of the aliphatic chain-GLP-1 receptor agonist class prepared by the method.
Compared with the prior art, the continuous preparation process has the advantages that:
(1) according to the invention, the reaction of modifying GLP-1 receptor stimulant polypeptides by the fatty chain modifier is carried out by using the microreactor, so that the continuous preparation of the fatty chain-GLP-1 receptor stimulant polypeptides can be realized, the mass and heat transfer efficiency of the modification reaction is improved, and the reaction efficiency is improved; compared with the traditional reactor, the micro-reactor has great advantages in the aspects of mass and heat transfer, process control and process amplification, the micron-sized channel in the micro-reactor enables the size of the formed liquid drop to be equal to or smaller than the characteristic size in the channel, and the specific surface area between two phases is improved, so that the mass and heat transfer efficiency is enhanced; the system composed of the microreactor can realize high integration, can control the reaction conditions on line and ensure that the process of the production process is highly controllable; the process amplification of the traditional reactor needs the process from small test, pilot test to amplification, all process parameters are changed along with the process, the amplification related to the micro-reactors is parallel amplification, the process amplification process is only the increase of the number of the micro-reactors, and other conditions are basically unchanged. In addition, the microreactor is safer and more environment-friendly, and can realize continuous operation and high controllability of the production process; these advantages effectively solve the problem of low efficiency of the fatty chain modifier for modifying GLP-1 receptor agonist polypeptide.
(2) The invention utilizes the solubility difference of the hydrophobic fatty chain modifier, the hydrophilic protein polypeptide drug and the fatty chain modified product in the buffer solution, adopts a precipitation method to separate and purify the fatty chain-GLP-1 receptor agonist polypeptide, replaces the prior chromatographic separation technology, reduces the separation cost in the drug production process and improves the production efficiency of the separation process.
(3) The invention has universality for fatty chain modification of other protein polypeptide drugs.
Drawings
FIG. 1 is C 14 Of MAL 1 H-NMR analysis results.
FIG. 2 is C 14 RP-HPLC analysis of the MAL modified LOX reaction mixture.
FIG. 3 is a pictorial representation of a microreactor. Wherein A is a two-channel syringe pump, B and C are syringes containing reaction solution, D is a micromixer, and E is a microchannel.
FIG. 4 is a comparison of the reaction efficiency of microreactors of varying internal diameter dimensions with conventional batch reactors.
FIG. 5 is a graph of the change in capacity of a 0.02 inch microreactor for 40 h.
FIG. 6 is a diagram of precipitation separation of C 14 -a physical map of the LOX.
FIG. 7 shows RP-HPLC analysis of supernatant (A) and precipitate (B).
Detailed Description
In order to more clearly show the technical solutions of the present invention and the outstanding technical effects obtained thereby, the following will describe in detail the specific embodiments of the present invention by examples, but the specific embodiments should not be construed as limiting the present invention. Unless otherwise stated, the experimental methods used are conventional methods, the reagents used are all available from chemical reagent companies, and the chemical reagents used are all analytical reagents. The Loxapine (LOX) starting material described in this example (greater than 95% purity) was obtained from the pharmaceutical group, hounsfield, inc.
The analysis method of the reactants and the modified products in the invention is as follows:
the contents of the synthetic crude fatty chain loxapine and the fatty chain loxapine in the synthetic crude fatty chain loxapine and the purities of the fatty chain maleimide and the fatty chain loxapine are detected by using a reversed phase high performance liquid chromatography (RP-HPLC). The chromatographic column used was LiChrospher 100RP-18(250 mm. times.4.0 mm, 5 μm) from Merck, Germany, mobile phase A was pure water containing 0.05% (v/v) trifluoroacetic acid, mobile phase B was acetonitrile containing 0.05% (v/v) trifluoroacetic acid, and the UV detection wavelength was 210 nm. Sample introduction is carried out under the condition of 40% B, the sample introduction amount is 40 mu L, and the flow rate is 1 mL/min. The elution gradient was: eluting with 40-100% B gradient for 0-60 min. The trifluoroacetic acid and acetonitrile reagents used were chromatographically pure.
The reaction product yield calculation formula is:
example 1 Synthesis and preparation of a Tetradecarbyl fatty chain modifier
Fourteen-carbon fatty chain-specific modifiers were synthesized against the thiol group of LOX. The synthesis process comprises the following steps: 2.295g (23.45mmol) of maleic anhydride was dissolved in 5mL of anhydrous dichloromethane and charged into a 50mL three-necked flask. 5g (23.45mmol) of tetradecylamine was dissolved in 10mL of anhydrous dichloromethane and added dropwise to a three-necked flask and refluxed at 40 ℃ for 30 minutes (partial curing). After cooling, the mixture was filtered under suction and washed twice with cold anhydrous dichloromethane, and dried to obtain 4.81g of a white solid (N-tetradecylmaleic acid). 4g (12.8mmol) of N-tetradecylmaleic acid and 0.72g (8.8mmol) of sodium acetate are dissolved in 15mL of acetic anhydride, added to a 100mL round-bottomed flask, heated at 100 ℃ for two hours, cooled, added with 50mL of water, allowed to stand in a separatory funnel, washed twice with diethyl ether (2X 30mL), twice with 2% KOH solution (2X 30mL), once with 30mL of water, the organic phase is taken off, spun dry, dissolved in 20mL of dichloromethane, MgSO 4 Drying, filtering, and spin-drying to obtain 3.197g of crude tetradecanemaleimide, and separating and purifying with YMC preparative liquid chromatography column (YMC-Pack ODS-A, 20 mm. times.250 mm, 10 μm) under the RP-HPLC analysis method. Dissolving the purified sample in deuterated methanol, detecting chemical shift of each hydrogen atom of the purified sample by using 500MHz nuclear magnetic resonance apparatus, determining chemical structure, and showing that the synthetic product is tetradecyl aliphatic chain maleimide (1-tetradecyl-1H-pyrrole-2, 5-diketone, C, and shown in figure 1 14 -MAL)。
Tetradecyl aliphatic chain maleimide (C) 14 MAL) is represented by formula (1):
example 2 conventional batch reactor C 14 MAL-modified LOX
Using purified C 14 MAL screening different reaction solvent systems in a conventional batch reactor (in a test tube) to effect reaction with LOX, such as a water-methanol/ethanol system. Using water-methanol (1: 9, v/v) as an example, a 1mL reaction system was constructed, using a PBS aqueous solution-methanol (1: 9, v/v) mixed solution as a reaction solvent, pH 6.07, C 14 The molar ratio MAL/LOX is 5: 1, and the reaction is carried out for 2h at 20 ℃. The specific process is as follows: a2 mL tube was charged with 0.1mL of LOX in PBS (pH 6.07) at 10mg/mL, and 1mg/mL of C was added 14 0.35mL of the MAL methanol solution, 0.55mL of the methanol solvent, and 50. mu.L of 1M DTT solution after reacting in a shaker at 200rpm and 20 ℃ for 4 hours to terminate the reaction. After completion of the reaction, the results of detection by liquid chromatography are shown in FIG. 2. From this, it was found that the modified product tetradecapeptide fatty chain loseatide (C) 14 LOX), which shows that C is achieved in a mixed solution of PBS aqueous solution and methanol (1: 9, v/v) 14 Reaction of MAL to modify LOX.
C 14 The reaction equation for MAL modified LOX is shown in formula (2):
example 3 construction of microreactors and preparation of microreactors by Using microreactors C 14 -LOX
FIG. 3 shows a constructed microreactor in which A is a two-channel syringe pump, B and C are each provided with C 14 -syringes of reaction solutions of MAL and LOX, D is a micromixer with an inner diameter of 1/16 inches (0.158cm), E is a microchannel with an inner diameter of 0.04 inches (0.1cm), wherein the volume from syringe to micromixer is 0.22mL, and the volume of the microchannel is 1.5 mL. Continuous synthesis of C in microreactors 14 LOX, using a mixed solution of PBS aqueous solution and methanol (1: 9, v/v) as a reaction solvent, pH 6.07, C 14 The molar ratio MAL/LOX is 5: 1, and the reaction is carried out at 20 ℃ for 4 h.
The reaction process is as follows: 2mg/mL LOX solution (aqueous PBS-methanol, 1: 9) and 0.70mg/mL C were prepared 14 MAL solutions (PBS aqueous solution-methanol, 1: 9), 2mL each, into a syringe, which is mounted in a syringe pump. The syringe pump was first run at a flow rate of 0.22mL/min for 1min to allow the reaction fluid to flow to the micromixer, and then at a flow rate of 0.19mL/h to ensure 4h of reaction. A2 mL tube of 50. mu.L DTT solution (1M) was placed at the exit of the channel and 1mL of the reaction mixture was collected for detection.
Example 4 comparison of the reaction efficiency of microreactors of varying internal diameter dimensions with conventional batch reactors
The reaction was carried out using microchannels with two dimensions of 0.04 inch (0.1cm) and 0.02 inch (0.5mm) internal diameter, and the other reaction conditions were unchanged, and the reactions were carried out for 0.5, 1, 2, 4, 6, 8, 10, and 12 hours, respectively (the reaction time was controlled by controlling the flow rate). The difference of reaction efficiency in microreactors with different inner diameter sizes was examined by comparing the reactions in conventional batch reactors. Specific experimental methods refer to examples 2 and 3. As shown in FIG. 4, the yield of the reaction product was 94.5% when the reaction was carried out in a conventional batch reactor for 10 hours; the reaction is carried out for 8 hours in a microreactor with 0.04 inch, and the yield of reaction products is up to 96.0 percent; the reaction is carried out for 4 hours in a 0.02 inch micro-reactor, and the yield of the reaction product reaches 95.7 percent. It can be seen that the smaller the inner diameter of the microreactor under the same conditions, the shorter the modification reaction time and the higher the reaction efficiency can be maintained.
Example 5 continuous Synthesis of C in microreactor 14 -LOX
As can be seen from example 4, the reaction was substantially complete in 4 hours in a 0.02 inch microreactor with a 95.7% yield of reaction product, and therefore the continuous synthesis of C in a 0.02 inch microreactor was chosen 14 -LOX. Referring to the experimental conditions in example 3, a 2mg/mL LOX solution (aqueous PBS-methanol, 1: 9) and 0.70mg/mL C were prepared 14 MAL solutions (aqueous PBS-methanol, 1: 9), 8mL each, were put into syringes, which were mounted in syringe pumps. The syringe pump was first operated at a flow rate of 0.22mL/min for 1min to flow the reaction fluid to the micromixer, and then operated at a flow rate of 0.19mL/h for 40 hours, as shown in FIG. 5, listing 4Strength profile was produced within 0 hour.
As can be seen from FIG. 5, after 4 hours of operation, the microreactor has a C content 14 The average productivity of-LOX is 0.42mg/h, and the integral shows that 16.36mg C can be produced in the micro-reactor within 40 hours 14 -LOX. Compared with the conventional batch reactor in example 2, the batch reaction was completed in 10 hours, the average productivity was 0.16mg/h, and 6.51mgC could be produced in 40 hours 14 -LOX. The capacity of the 0.02 inch micro-reactor after stable operation within 40 hours is 2.6 times of that of the traditional batch reactor, and the C is obviously improved 14 -production efficiency of LOX.
EXAMPLE 6 isolation of the reaction product by precipitation
C was collected from the continuous synthesis in the microreactor of example 5 14 Crude LOX solution, C achieved by increasing the proportion of the aqueous phase 14 -LOX and C 14 Isolation of MAL and its hydrolysates. The reaction products were collected in a 2mL tube at the microreactor outlet and the water phase ratio in the tube was increased to 30%, 50%, 70% and 90% (v/v), respectively, as shown in FIG. 6. When the proportion of the aqueous phase exceeds 70%, the solution precipitates. After centrifugation at 12000rpm for 10min and concentration by distillation under reduced pressure, liquid chromatography was performed, and the results are shown in FIG. 7. The precipitate fraction is C 14 MAL and its hydrolysate, supernatant fraction C 14 -LOX in a yield of 94.0% and a purity of 93.1%, achieving reaction product C 14 Efficient separation of LOX.
Claims (7)
1. A method for continuously preparing aliphatic chain-GLP-1 receptor agonist polypeptide by using a microreactor is characterized by mainly comprising the following steps:
(i) determining the technological parameters of GLP-1 receptor stimulant polypeptides modified by the fatty chain modifier in a traditional batch reactor; the fatty chain modifier is medium-long chain fatty acid, fatty alcohol or fatty amine with activated carboxyl, hydroxyl or amino, the number of carbon atoms of the fatty acid, the fatty alcohol or the fatty amine is 8-36, and the activated group comprises maleimide, succinimidyl and aldehyde group; the GLP-1 receptor agonist polypeptide comprises natural GLP-1, exenatide, linatide, lixisenatide, abilutamide and loxapide;
(ii) designing and constructing a micro-reactor system consisting of a pump, a micro-mixer and a micro-channel, and continuously synthesizing the fat chain-GLP-1 receptor agonist polypeptide in the micro-reactor under the process parameters determined in the step (i) to obtain a crude product solution; the specific steps for synthesizing the fatty chain-GLP-1 receptor stimulant polypeptide in the step (ii) are as follows: dissolving a fatty chain modifier in a reaction solvent to prepare a homogeneous solution A; dissolving GLP-1 receptor agonist polypeptide in a reaction solvent to prepare a homogeneous solution B, respectively and simultaneously pumping the homogeneous solution A and the homogeneous solution B into a micro mixer of a micro reactor, uniformly mixing, and then introducing into a micro channel;
(iii) and (3) adding a buffer solution into the crude product solution prepared in the step (ii) by utilizing the solubility difference of the aliphatic chain modifier and the aliphatic chain-GLP-1 receptor agonist polypeptide in the buffer solution, precipitating the aliphatic chain modifier, carrying out solid-liquid separation, taking supernatant, and concentrating to obtain the aliphatic chain-GLP-1 receptor agonist polypeptide.
2. The method according to claim 1, wherein the reaction solvent is a mixed solution of a buffer solution and an organic solvent, wherein the buffer solution comprises a phosphate buffer solution, a citric acid-sodium citrate buffer solution and a glycine-hydrochloric acid buffer solution, and the organic solvent comprises methanol, ethanol, acetonitrile and dimethyl sulfoxide; the volume percentage of the buffer solution in the reaction solvent is 10-60%.
3. The method according to claim 1, wherein the concentration of the aliphatic chain modifier in the homogeneous solution A is 0.1-5 mg/mL; the concentration of GLP-1 receptor agonist polypeptide in the homogeneous solution B is 0.5-10 mg/mL; the reaction molar ratio of the fatty chain modifier to the GLP-1 receptor agonist polypeptide is 20: 1-1: 1.
4. The method according to claim 1, wherein the flow rate of the homogeneous solution A pumped into the microreactor is 0.05-1.5 mL/h; the flow rate of pumping the homogeneous solution B into the microreactor is 0.05-1.5 mL/h.
5. The method of claim 1, wherein the pump comprises a syringe pump, a peristaltic pump; the micro mixer is a Y-shaped micro mixer or a T-shaped micro mixer, and the inner diameter of the micro mixer is 0.01-0.2 cm; the inner diameter of the micro-channel ranges from 0.01 cm to 0.2 cm.
6. The method according to claim 1, wherein the continuous synthesis is carried out under the conditions of a reaction temperature of 0-50 ℃ and a reaction residence time of 2-10 h.
7. The method according to any one of claims 1 to 6, wherein the buffers added in step (iii) comprise phosphate buffer, citric acid-sodium citrate buffer and glycine-hydrochloric acid buffer, and the final buffer accounts for 70% to 95% of the total system by volume; the solid-liquid separation comprises centrifugation, membrane filtration, filter pressing or suction filtration; the concentration comprises reduced pressure distillation and dialysis.
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