A method for preparing a salt of a polypeptide
The nanofiltration salt formation technology is used to remove the original ions under the conditions of controlling pH and temperature, which solves the problems of sample loss and impurities in the preparation of polypeptide salts, and achieves efficient and stable preparation of polypeptide salts, suitable for polypeptide salts. Industrial production of medicines.
Patent Information
- Application Number
- CN202110043351.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-01-13
- Publication Date
- 2024-12-06
- Estimated Expiration
- 2041-01-13
AI Technical Summary
The existing polypeptide salt preparation methods have problems such as large sample losses, many impurities, complex operating procedures and long production cycles, especially in high-value-added drugs, resulting in economic losses and unstable quality.
Nanofiltration salt-forming technology is adopted to remove the original ions through a nanofiltration system under the condition of controlling pH and temperature, compensate for new ions, and obtain polypeptide salt products after concentration, avoiding the use of organic solvents and the generation of impurities.
It simplifies the operation steps, reduces sample losses and impurities generation, improves sample recovery rate and product quality stability, reduces production costs and environmental impacts, and is suitable for efficient preparation of polypeptide drugs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polypeptide drug preparation, and in particular to a method for preparing polypeptide salts. Background Art
[0002] In recent years, with the development of combinatorial chemistry and high-throughput screening technology, the discovery rate of new active compounds has greatly accelerated. In order to improve their drugability, it is necessary to modify these compounds and optimize their physicochemical properties, pharmacokinetic properties, etc. by salting without changing their chemical structure. Polypeptide drugs are mostly made into products in the form of salts. Peptide salting can adjust the solubility of polypeptides, improve drug compliance, make patients easy to accept, and ensure their release in the body. At the same time, it also plays an extremely important role in improving drug stability and reducing adverse drug reactions. At present, the methods generally used for polypeptide salting are chromatography salting or crystallization salting. The Chinese patent with announcement number CN102993293B discloses a purification method of teripatriptide acetate, using reverse phase filler as a chromatography medium, loading, eluting, and finally concentrating and lyophilizing. The equipment used in the chromatography salt exchange process includes preparative liquid phase, preparative chromatographic column and corresponding fillers, analytical liquid phase, condensation temperature control system, and concentration equipment such as rotary evaporation or nanofiltration equipment. The equipment of this method is expensive and the construction period is long. In addition, the adsorption of the chromatography medium will inevitably lead to sample loss.
[0003] The Chinese patent with publication number CN101955511A discloses a method for synthesizing oxidized glutathione and its alkali metal salt, which uses crystallization to exchange salt after preparing oxidized glutathione to obtain oxidized glutathione alkali metal salt. The crystallization and salt exchange steps include concentrating the sample to a certain concentration, adjusting the pH to near the sample PI value, cooling and crystallizing, filtering, top washing, beating, filtering, and drying to obtain the compound. This method has the following problems: 1. It produces many impurities: Although strong ions have stronger binding ability with compounds, there is a dynamic equilibrium between ions in the solution, and the original salt ions are inevitably present. Organic solvents and inorganic salts are easily wrapped in the crystal lattice, and more impurities will be produced, such as degradation impurities, polymer impurities, etc., which are particularly serious in heat-sensitive polypeptide drugs; 2. Large sample loss: Crystallization, filtration, top washing, pulping and other operations inevitably lead to large sample losses. They can be used on ordinary compounds, but will bring great economic losses to high value-added drugs; 3. Poor sample stability: In the process of crystallization, most of the time, excessive reagents are added, and the added acid and alkali have the risk of causing sample degradation, especially in the salt exchange process of polypeptide drugs, which is easy to cause degradation, racemization and other problems; 4. Small scope of application: The scope of application of the crystallization method for salt formation is narrow, and it is only applicable to compounds with poor solubility in water after salt formation and good stability.
[0004] Therefore, there is an urgent need to find a polypeptide preparation method with simple operation procedures, short working period, small sample loss, few impurities, high sample recovery rate, controllable quality, and salt exchange while removing organic solvents. Summary of the invention
[0005] The present invention aims to solve the problems of large sample loss, large amount of impurities, complicated operation procedures and long production cycle in the prior art, and provides a method for preparing a salt of a polypeptide, the method comprising the following steps:
[0006] After the crude peptide is purified to obtain refined peptide, nanofiltration is used to form salt. The corresponding buffer salt is added to the nanofiltration system, and a certain pH value is adjusted and maintained to free the original ions from the compound. The original ions are dialyzed and removed through the principle of nanofiltration dialysis, and new ions are compensated. Finally, the peptide salt solution is concentrated and freeze-dried. The specific steps are as follows:
[0007] 1) dissolving the refined peptide, filtering and removing impurities to obtain a refined peptide solution;
[0008] 2) Adding the refined peptide solution to the nanofiltration system, adding a buffer solution at the same time, adjusting the pH, controlling the temperature, and concentrating to obtain a polypeptide salt solution;
[0009] 3) The polypeptide salt solution is freeze-dried to obtain the target polypeptide product.
[0010] Preferably, the polypeptides of the present invention include: thymalfasin, terlipressin, desmopressin, semaglutide, atosiban, pramlintide, liraglutide, linaclotide, carbetocin, cilpressin, teduglutide, glucagon, plecanatide, and bivalirudin.
[0011] Preferably, the nanofiltration system in step 2) includes a feed liquid tank, a pressure pump, a pressure regulating valve, a membrane assembly, a flow meter, a pH value adjustment system and a feed liquid temperature control system.
[0012] Further preferably, step 2) is specifically to place the refined peptide solution in a feed liquid tank, control the temperature by a feed liquid temperature control system, control the pH value by a pH adjustment system, and then use a pressure pump to transport the feed liquid to the membrane assembly for dialysis, salt exchange, and concentration, monitor the flow rate by a flow meter, and control the flow rate by a pressure regulating valve.
[0013] In a preferred embodiment of the present invention, the buffer solution in step 2) is selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonium carbonate, ammonium bicarbonate, ammonia water, sodium hydroxide, potassium hydroxide, sodium chloride, potassium chloride, ammonium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium perchlorate, potassium perchlorate, and ammonium perchlorate.
[0014] Further preferably, the concentration of the buffer solution is 0-100 mM, more preferably, the concentration of the buffer solution is selected from 0-50 mM.
[0015] In a preferred embodiment of the present invention, the pH adjuster in step 2) is selected from sodium hydroxide, potassium hydroxide, and ammonia water.
[0016] Further preferably, the pH value ranges from 6 to 10, and more preferably, the pH value ranges from 7 to 9.5. Too high a pH value is not conducive to sample stability, and too low a pH value is not conducive to salt replacement.
[0017] In a preferred embodiment of the present invention, the temperature of the sample during the nanofiltration process in step 2) is controlled to 5-40° C., more preferably, the temperature is controlled at 10-30° C. Temperature control is beneficial to ensuring sample stability, while too low a temperature reduces the solubility of the sample, and too high a temperature easily degrades the sample.
[0018] In a preferred embodiment of the present invention, the polypeptide salt solution obtained in step 2) is a polypeptide salt solution containing sodium ions, potassium ions or ammonium ions.
[0019] The comparison between the present invention and the prior art is shown in the following table:
[0020]
[0021] Note: High molecular weight protein is a type of polymer impurity in polypeptide products, which is mainly formed by hydrogen bonds, ionic bonds, and salt bonds. The higher the concentration, the easier it is to form such impurities. Since crystallization is a kind of aggregation phenomenon caused by the concentration of the sample in the solution exceeding its solubility, it is particularly serious during the crystallization process.
[0022] In summary, the salt preparation method of a polypeptide provided by the present invention adopts nanofiltration technology for salt formation, uses a nanofiltration system to concentrate while changing salt, and does not use organic solvent elution, which greatly reduces the risk of organic solvent residues, reduces sample losses, and greatly reduces production and waste liquid treatment costs, and has obvious advantages in environmental protection. By controlling the various parameters of the nanofiltration process and the nanofiltration endpoint, the purpose of controllable product quality is achieved, and then the data before freeze-drying of batch samples is guaranteed to be consistent, so that the batch difference is small and the product quality is stable. The method has simple operating steps, high efficiency, high sample recovery rate, more accurate control of final product quality, higher sample quality, and is easy to industrially amplify, which brings great convenience to the field of salt formation of compounds, and has broad application prospects in the field of polypeptide drug production with high product quality requirements. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the flow chart of the nanofiltration system;
[0024] Figure 2 This is a diagram of the dialysate content detection in Example 1;
[0025] Figure 3 This is the high molecular protein spectrum of the nanofiltration salt-forming sample in Example 2;
[0026] Figure 4 This is the organic residue spectrum of the nanofiltration sample in Example 2;
[0027] Figure 5 This is the residual spectrum of anions after nanofiltration in Example 2;
[0028] Figure 6 This is the high molecular protein spectrum of the nanofiltration salt-forming sample in Example 6;
[0029] Figure 7 This is the organic residue spectrum of the nanofiltration salt-forming sample in Example 6;
[0030] Figure 8 This is the high molecular protein spectrum of the nanofiltration salt-forming sample in Example 11;
[0031] Fig. 9 This is the organic residue spectrum of the nanofiltration salt-forming sample in Example 11;
[0032] Fig.10 This is the spectrum of high molecular weight protein in Comparative Example 2;
[0033] Fig.11 This is the high molecular protein spectrum of the crystallized salt sample in Comparative Example 3;
[0034] Fig.12 This is the organic residue spectrum of the crystallized sample in Comparative Example 3;
[0035] Fig.13 This is the residual spectrum of anions after crystallization in Comparative Example 3;
[0036] Fig.14 This is the organic residue spectrum in Comparative Example 4. DETAILED DESCRIPTION
[0037] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.
[0038] Example 1: A method for preparing a salt of pramlintide using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0039] 1) Passing the pramlintide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0040] 2) Add the solution from which insoluble impurities have been removed to the feed liquid tank, add buffer salt, the buffer is 10 mM potassium dihydrogen phosphate, and adjust the pH to 7.5 with potassium hydroxide through the pH adjustment system; cool it to 5° C. through the feed liquid temperature control system;
[0041] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure at 1.3 MPa and the flow rate at 12 L / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a pramlintide sample solution containing potassium ions at a concentration of 30 mg / mL;
[0042] 4) After freeze-drying, the sample detected high molecular weight protein was 0.05%, and organic residue was basically undetectable. The dialysate content was 0.0003-0.0006g / L, and the loss was negligible. The calculated yield after freeze-drying was 99.27%. Dialysate content detection diagram is shown in Figure 2 .
[0043] Example 2: A method for forming a salt of thymosin using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0044] 1) Passing the thymosin mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0045] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 50 mM ammonium bicarbonate, and the pH is adjusted to 9.5 with ammonia water through the pH adjustment system; the temperature is lowered to 10° C. through the feed liquid temperature control system;
[0046] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.5 MPa and the flow rate to 16 liters / hour. The buffer solution is continuously replenished during the nanofiltration process; a thymosin sample solution containing ammonium ions is obtained, with a concentration of 60 mg / mL;
[0047] 4) After freeze-drying, the high molecular weight protein in the sample was 0.05% (see Figure 3 ), the residue on ignition is 0.04%, and the organic residue is negligible (see Figure 4 ), the residual anions are negligible (see Figure 5 ). The calculated yield after freeze-drying was 99.21%.
[0048] Example 3: A method for forming a salt of thymosin using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0049] 1) Passing the thymosin mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0050] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, and a buffer salt is added, the buffer solution is an ammonia solution with a pH of 9.5, and the pH is adjusted to 9.5 by a pH control system; the temperature is lowered to 20° C. by a feed liquid temperature control system;
[0051] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure at 1.3Mpa and the flow rate at 12L / hour. The buffer solution is continuously replenished during the nanofiltration process; a thymosin sample solution containing ammonium ions is obtained, with a concentration of 70mg / mL;
[0052] 4) After freeze-drying, the sample detected high molecular weight protein was 0.04%, the ignition residue was 0.04%, and the organic residue and ion residue were negligible. Figure 3 Similarly, the organic residue spectrum is Figure 4 Similarly, the anion residue spectrum is Figure 5 The calculated yield after freeze drying was 99.45%.
[0053] Example 4: A method for forming a salt of thymosin using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0054] 1) Passing the thymosin mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0055] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 20 mM potassium chloride, and the pH is adjusted to 7.5 with potassium hydroxide through the pH adjustment system; the temperature is lowered to 25° C. through the feed liquid temperature control system;
[0056] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.4Mpa and the flow rate to 14L / hour. The buffer solution is continuously replenished during the nanofiltration process; a thymosin sample solution containing potassium ions is obtained with a concentration of 50mg / mL;
[0057] 4) The polymer protein content of the freeze-dried sample was 0.06%, and the organic residue was negligible. Figure 3 Similarly, the organic residue spectrum is Figure 4 The calculated yield after freeze drying was 99.04%.
[0058] Example 5: A method for preparing a salt of carbetocin using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0059] 1) Passing the carbetocin mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0060] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, and a buffer salt is added, wherein the buffer is 10 mM sodium dihydrogen phosphate and the pH is adjusted to 8.5 with sodium hydroxide through a pH adjustment system; the temperature is lowered to 25° C. through a feed liquid temperature control system;
[0061] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.3Mpa and the flow rate to 12L / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a carbetocin sample solution containing sodium ions with a concentration of 200mg / mL;
[0062] 4) After freeze-drying, the sample detected high molecular weight protein was 0.05%, and organic residue was negligible. The calculated yield after freeze-drying was 99.18%.
[0063] Example 6: A method for preparing liraglutide salt by using nanofiltration salt formation technology, wherein the specific steps are as follows:
[0064] 1) Passing the liraglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0065] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 30 mM ammonium bicarbonate, and the pH is adjusted to 9.0 with ammonia water through the pH adjustment system; the temperature is lowered to 25° C. through the feed liquid temperature control system;
[0066] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.5 MPa and the flow rate to 16 liters / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a liraglutide sample solution containing ammonium ions with a concentration of 80 mg / mL;
[0067] 4) After freeze-drying, the high molecular weight protein in the sample was 0.04% (see Figure 6 ), organic residues are negligible (see Figure 7 ), the calculated yield after freeze-drying was 99.37%.
[0068] Example 7: A method for preparing liraglutide salt by using nanofiltration salt formation technology, wherein the specific steps are as follows:
[0069] 1) Passing the liraglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0070] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, and a buffer salt is added, wherein the buffer is 20 mM sodium chloride and the pH is adjusted to 10.0 with sodium hydroxide through a pH adjustment system; the temperature is lowered to 20° C. through a feed liquid temperature control system;
[0071] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.5 MPa and the flow rate to 16 liters / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a liraglutide sample solution containing sodium ions with a concentration of 40 mg / mL;
[0072] 4) After freeze-drying, the high molecular weight protein in the sample was 0.03% and the organic residue was negligible. Figure 6 Similarly, the organic residue spectrum is Figure 7 The calculated yield after freeze-drying was 99.28%.
[0073] Example 8: A method for preparing liraglutide salt using nanofiltration salt formation technology, wherein the specific steps are as follows:
[0074] 1) Passing the liraglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0075] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, and a buffer salt is added, wherein the buffer is 40 mM potassium dihydrogen phosphate and the pH is adjusted to 7.0 with potassium hydroxide through a pH adjustment system; the temperature is lowered to 30° C. through a feed liquid temperature control system;
[0076] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.5 MPa and the flow rate to 16 liters / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a liraglutide sample solution containing potassium ions at a concentration of 100 mg / mL;
[0077] 4) After freeze-drying, the sample detected high molecular weight protein 0.07%, and organic residues were negligible. High molecular weight protein spectrum and Figure 6 Similarly, the organic residue spectrum is Figure 7 Similar. The calculated yield after freeze-drying is 98.91%
[0078] Example 9: A method for preparing liraglutide salt by using nanofiltration salt formation technology, wherein the specific steps are as follows:
[0079] 1) Passing the liraglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0080] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, and a buffer salt is added, the buffer solution is an ammonia solution with a pH of 9.5, and the pH is adjusted to 9.5 by a pH control system; the temperature is lowered to 10° C. by a feed liquid temperature control system;
[0081] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.4 MPa and the flow rate to 14 L / h. The buffer solution is continuously replenished during the nanofiltration process to obtain a liraglutide sample solution containing ammonium ions with a concentration of 50 mg / mL;
[0082] 4) After freeze-drying, the sample detected high molecular weight protein 0.04%, and organic residues were negligible. High molecular weight protein spectrum and Figure 6 Similarly, the organic residue spectrum is Figure 7 The calculated yield after freeze drying was 99.15%.
[0083] Example 10: A method for forming a salt of teduglutide using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0084] 1) Passing the teduglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0085] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 100 mM sodium chloride, and the pH is adjusted to 6.0 with sodium hydroxide through the pH adjustment system; the temperature is lowered to 20° C. through the feed liquid temperature control system;
[0086] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.4 MPa and the flow rate to 14 L / h. The buffer solution is continuously replenished during the nanofiltration process to obtain a teduglutide sample solution containing sodium ions with a concentration of 10 mg / mL;
[0087] 4) After freeze-drying, the sample detected high molecular weight protein of 0.06%, and organic residue was negligible. The calculated yield after freeze-drying was 99.44%.
[0088] Example 11: A method for preparing semaglutide by using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0089] 1) Passing the semaglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0090] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 50 mM sodium perchlorate, and the pH is adjusted to 8.5 with sodium hydroxide through the pH adjustment system; the temperature is lowered to 40° C. through the feed liquid temperature control system;
[0091] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure at 1.3 MPa and the flow rate at 12 liters / hour. The buffer solution is continuously replenished during the nanofiltration process; a semaglutide sample solution containing sodium ions is obtained with a concentration of 70 mg / mL;
[0092] 4) After freeze-drying, the sample was tested for high molecular weight protein 0.04% (see Figure 8 ), organic residues are negligible (see Fig. 9 ). The calculated yield after freeze-drying was 99.07%.
[0093] Example 12: A method for forming a salt of semaglutide using nanofiltration salt-forming technology, wherein the specific steps are as follows:
[0094] 1) Passing the semaglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0095] 2) The solution from which insoluble impurities have been removed enters the feed liquid tank, a buffer salt is added, the buffer is 50 mM sodium sulfate, and the pH is adjusted to 7.5 with sodium hydroxide through the pH adjustment system; the temperature is lowered to 30° C. through the feed liquid temperature control system;
[0096] 3) The pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange and concentration, and controls the pressure to 1.5 MPa and the flow rate to 16 liters / hour. The buffer solution is continuously replenished during the nanofiltration process to obtain a semaglutide sample solution containing sodium ions with a concentration of 60 mg / mL;
[0097] 4) After freeze-drying, the high molecular weight protein in the sample was 0.05%, and the organic residue was negligible. Figure 8 Similarly, the organic residue spectrum is Fig. 9 The calculated yield after freeze-drying was 99.06%.
[0098] Comparative Example 1: A method for preparing a salt of teduglutide using chromatography concentration technology, wherein the specific steps are as follows:
[0099] 1) Prepare a 100mm preparative chromatography system, including a chromatography system and a 100mm preparative column filled with C8. Clean the chromatography system and balance the chromatography system before loading the sample;
[0100] 2) passing the teduglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0101] 3) The solution from which insoluble impurities have been removed is loaded onto a chromatography column, and gradient eluted with 50 mM ammonium bicarbonate / acetonitrile to collect fractions;
[0102] 4) Analyze the liquid phase detection fractions and combine the qualified samples;
[0103] 5) using rotary evaporation or nanofiltration for concentration, and controlling the temperature of the rotary evaporation or nanofiltration by condensation;
[0104] 6) Finally, the compound was concentrated to 70 mg / mL;
[0105] 7) After freeze-drying, a teduglutide sample containing ammonium ions was obtained. No organic residue was detected in the sample, and the high molecular weight protein was 0.07%. The calculated sample yield was 90.64%.
[0106] Comparative Example 2: A method for preparing liraglutide by using chromatography concentration technology, wherein the specific steps are as follows:
[0107] 1) Prepare a 100mm preparative chromatography system, including a chromatography system and a 100mm preparative column filled with C8. Clean the chromatography system and balance the chromatography system before loading the sample;
[0108] 2) Passing the liraglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0109] 3) applying the solution from which insoluble impurities have been removed to a chromatography column, eluting with a 100 mM ammonium bicarbonate / acetonitrile gradient, and collecting fractions;
[0110] 4) Analyze the liquid phase detection fractions and combine the qualified samples;
[0111] 5) using rotary evaporation or nanofiltration for concentration, and controlling the temperature of the rotary evaporation or nanofiltration by condensation;
[0112] 6) Finally, the compound was concentrated to 50 mg / mL;
[0113] 7) After freeze-drying, a liraglutide sample containing ammonium ions was obtained. No organic residue was detected in the sample, and the high molecular protein content was 0.15% (see Fig.10 ), the sample yield was calculated to be about 91.48%.
[0114] Comparative Example 3: Using crystallization technology, according to the PI value of thymosin 4.2, salt was formed by crystallization method, and the specific operation was as follows:
[0115] 1) Passing the thymosin mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0116] 2) concentrating to remove part of the organic solvent, adjusting the pH to 4.2 with hydrochloric acid, and cooling for crystallization;
[0117] 3) After centrifugal sedimentation, use purified water to pulp until there is no obvious lump, and repeat the centrifugal sedimentation and pulping operation;
[0118] 4) Dissolve the solid with ammonia water and then freeze-dry;
[0119] 5) After freeze-drying, a thymus alpha-sin sample containing ammonium ions was obtained, and the sample recovery rate was calculated to be 81.53%; sample detection: two impurities appeared in the high molecular weight protein, accounting for 0.23% and 0.9% respectively (see Fig.11 ); acetonitrile residue 2.17% (see Fig.12 , acetonitrile limit is 0.041%); ion residues show multiple ion peaks (see Fig.13 ).
[0120] Comparative Example 4: Using crystallization technology, according to the PI value of semaglutide of about 4.9, salt was formed by crystallization method, and the specific operation was as follows:
[0121] 1) Passing the semaglutide mother solution through a 0.22 μm microporous filter membrane to remove insoluble impurities in the solution;
[0122] 2) concentrating to remove part of the organic solvent, adjusting the pH to 4.9 with hydrochloric acid, and cooling for crystallization;
[0123] 3) After centrifugal sedimentation, use purified water to pulp until there is no obvious lump, and repeat the centrifugal sedimentation and pulping operation;
[0124] 4) Dissolve the solid with ammonia water and then freeze-dry;
[0125] After freeze-drying, a semaglutide sample containing ammonium ions was obtained, and the sample recovery rate was calculated to be 82.47%; sample detection: an unknown solvent peak appeared in the organic residue (see Fig.14 ).
Claims
1. A method for preparing a salt of a polypeptide, wherein the polypeptide comprises semaglutide and liraglutide; Features: After the crude peptide is purified to obtain refined peptide, nanofiltration is used to form salt, and the nanofiltration salt formation specifically includes the following steps: 1) Dissolve the refined peptide and filter it through a 0.22 μm microporous membrane to remove insoluble impurities in the solution to obtain a refined peptide solution; 2) Add the refined peptide solution to the nanofiltration system, wherein the nanofiltration system includes a feed liquid tank, a pressure pump, a pressure regulating valve, a membrane assembly, a flow meter, a pH value adjustment system and a feed liquid temperature control system. The specific operation of nanofiltration is as follows: The refined peptide solution is placed in a feed liquid tank, the pH is adjusted by a pH adjustment system, the temperature is controlled by a feed liquid temperature control system, and then a pressure pump transports the feed liquid to the membrane assembly for dialysis, salt exchange, and concentration. The flow rate is monitored by a flow meter, and the flow rate is controlled by a pressure regulating valve. After concentration, a polypeptide salt solution is obtained; 3) The polypeptide salt solution is freeze-dried to obtain the target polypeptide product; In step 2), the buffer solution is selected from ammonia water, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, and sodium perchlorate, and the concentration of the buffer solution is 0-100 mM; In step 2), the temperature is controlled to 5-40°C.
2. The method for preparing a polypeptide salt according to claim 1, characterized in that: The polypeptide salt solution obtained in step 2) is a polypeptide salt solution containing sodium ions, potassium ions or ammonium ions.