A preparation method of semaglutide injection
Through methods such as adsorption purification and pH adjustment of high crosslinking resin, the instability problem of semegglutide injection was solved, its purity and stability were improved, and effective administration of long-acting glucose-like peptides was achieved.
Patent Information
- Application Number
- CN202411357937.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2025-01-17
- Estimated Expiration
- 2044-09-27
AI Technical Summary
Semegglutide in existing liquid polypeptide preparations is prone to aggregation due to physical instability, which affects its effectiveness and safety, resulting in preparation quality problems.
Semegglutide was adsorbed and purified by high crosslinking resin, and the adsorption capacity was improved by modifying the chloromethylated polystyrene resin. Combined with pH adjustment and filtration and sterilization, stable semegglutide injection was prepared.
It effectively improves the purity and stability of semegglutide, slows down its aggregation, obtains effective and safe injections, extends the half-life in the body, and achieves a long-acting effect of administering once a week.
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Figure CN118845644B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a method for preparing semaglutide injection. Background Art
[0002] In modern society, diabetes and its complications have gradually become another chronic non-communicable disease with serious harm after cardiovascular and cerebrovascular diseases and cancer. Diabetes is mainly divided into type 1 diabetes, type 2 diabetes and other special types of diabetes. The cause of type 2 diabetes is that the insulin secreted by the patient cannot play an effective role, and type 2 diabetes accounts for the highest proportion of the disease, reaching more than 90%.
[0003] Semaglutide is a new long-acting glucagon-like peptide-1 (GLP-1) analog that promotes the body's secretion of insulin and inhibits the secretion of glucagon in a glucose concentration-dependent mechanism. Therefore, while semaglutide normalizes the blood sugar level of patients with type 2 diabetes, it also reduces the risk of hypoglycemia caused by overregulation. On the other hand, semaglutide can also reduce the patient's appetite and can be combined with diet and exercise to improve blood sugar control in patients with type 2 diabetes. Semaglutide has a high homology with human GLP-1. It is based on the natural human GLP-1 (7-37) molecule and is obtained by replacing the 8th amino acid (alanine replaced by α-aminobutyric acid) and the 34th amino acid (lysine replaced by arginine), and at the same time connecting the C18 fatty diacid side chain at the 26th lysine through a spacer. Through the above structural adjustment, semaglutide can resist degradation by dipeptidyl peptidase 4, bind to albumin to prolong the half-life in the body, and achieve a long-term effect of once-a-week administration. Due to physical instability, the polypeptides in the polypeptide liquid preparation aggregate to form high molecular weight proteins and lose their activity, which affects the effectiveness and safety of the polypeptides in the polypeptide liquid preparation and affects the quality of the preparation. Therefore, the prior art urgently needs a method for preparing an effective and safe semaglutide injection. Summary of the invention
[0004] The object of the present invention is to provide a method for preparing semaglutide injection to solve the problems of instability of polypeptide liquid preparations.
[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:
[0006] A method for preparing semaglutide injection, comprising:
[0007] The auxiliary material solution and purified semaglutide are mixed, and the pH is adjusted and filtered for sterilization to obtain semaglutide injection; purified semaglutide is obtained by adsorbing crude semaglutide peptide by highly cross-linked resin, and then purified by desorption; the highly cross-linked resin is obtained by reacting chloromethylated polystyrene resin with 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene. Chloromethylated polystyrene resin reacts with 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene to obtain a highly cross-linked resin modified with naphthalene groups, so that the highly cross-linked resin has a high specific surface area and high selectivity, thereby improving the adsorption and purification ability of semaglutide, and the modified resin solves the problem of unfocused desorption peaks and has a good desorption effect.
[0008] Preferably, the excipient solution is prepared by dissolving an antibacterial agent, a pH buffer and an isotonic agent in water for injection; the antibacterial agent is at least one of phenol, benzyl alcohol and m-cresol; the pH buffer is at least one of disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate and sodium phosphate; and the isotonic agent is at least one of propylene glycol and mannitol.
[0009] Preferably, the amount of purified semaglutide added to the excipient solution is 0.001-0.002 g / ml, the pH is adjusted to 7-7.9, and the preparation of semaglutide injection is carried out at 2-15°C.
[0010] Preferably, the preparation of the highly cross-linked resin comprises:
[0011] Chloromethylated polystyrene resin and 1,2-dichloroethane were mixed to obtain a swollen resin, and FeCl was added to the swollen resin. 3 , 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene, and after the reaction is completed, suction filtration and washing are performed to obtain a low cross-linked resin;
[0012] The low cross-linked resin was swelled and FeCl 3 and methylal to react by stirring, and after the reaction is completed, filtration, washing, and drying to obtain a highly cross-linked resin.
[0013] Preferably, the amount of chloromethylated polystyrene resin in 1,2-dichloroethane is 0.05-0.2 g / ml, FeCl 3 The mass ratio of the amount added to the swelling resin to the amount of the chloromethylated polystyrene resin used is 1:4-6, the mass ratio of the amount of 1-fluoro-2-iodonaphthalene to the amount of the chloromethylated polystyrene resin used is 1:2-4, the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester to the amount of the chloromethylated polystyrene resin used is 1:2-4.5, and the mass ratio of the amount of 1-bromo-5-chloronaphthalene to the amount of the chloromethylated polystyrene resin used is 1:2-3.5.
[0014] Preferably, when the low cross-linked resin is swollen, the volume ratio of the amount of 1,2-dichloroethane used to the amount of 1,2-dichloroethane used when the chloromethylated polystyrene resin is 1:0.8-1.5, and FeCl 3 The mass ratio of the amount added to the low cross-linked resin to the amount of the low cross-linked resin is 1:0.15-1, and the volume ratio of the amount of methylal used to the amount of 1,2-dichloroethane used when swelling the low cross-linked resin is 1:4.5-7.
[0015] Preferably, the swelling time of the chloromethylated polystyrene resin is 0.5-2h; when preparing low cross-linked resin, the reaction temperature is 40-50°C and the reaction time is 15-25h; the swelling time of the low cross-linked resin is 0.5-2h; when preparing high cross-linked resin, the reaction temperature is 75-85°C and the reaction time is 15-25h.
[0016] Preferably, the preparation of purified semaglutide comprises:
[0017] The highly cross-linked resin and the crude semaglutide peptide aqueous solution were mixed and shaken, and after the shaking, the resin-semaglutide complex was filtered and washed, and then the resin-semaglutide complex was eluted with 65-75% methanol at a flow rate of 0.8-1.2 BV / h, and the eluate was collected in sections until the semaglutide completely flowed out, and the methanol was removed by drying to obtain purified semaglutide.
[0018] Preferably, the shaking reaction temperature is 10-18°C, the shaking time is 20-30h; the amount of crude semaglutide in the crude semaglutide aqueous solution is 0.0002-0.001g / ml, and the mass ratio of crude semaglutide to highly cross-linked resin is 1:20-30.
[0019] Preferably, highly cross-linked resins are used for polypeptide purification.
[0020] A method for preparing semaglutide injection, comprising:
[0021] The auxiliary material solution and purified semaglutide are mixed, and the pH is adjusted and filtered for sterilization to obtain semaglutide injection; purified semaglutide is obtained by adsorbing semaglutide crude peptide on a highly cross-linked resin and then undergoing desorption and purification.
[0022] Preferably, in the preparation of the above-mentioned semaglutide injection, the excipient solution is prepared by dissolving an antibacterial agent, a pH buffer and an isotonic agent in water for injection.
[0023] Preferably, in the preparation of the above-mentioned semaglutide injection, the antibacterial agent is at least one of phenol, benzyl alcohol and m-cresol, the amount of phenol added to the water for injection is 0.001-0.008 g / ml, the concentration of benzyl alcohol added to the water for injection is 0.005-0.01 g / ml, and the amount of m-cresol added to the water for injection is 0.0001-0.005 g / ml.
[0024] Preferably, in the preparation of the above-mentioned semaglutide injection, the pH buffer is at least one of disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, and sodium phosphate, the amount of disodium hydrogen phosphate dihydrate added to the water for injection is 0.001-0.003 g / ml, the amount of sodium dihydrogen phosphate added to the water for injection is 0.0005-0.0015 g / ml, and the amount of sodium phosphate added to the water for injection is 0.001-0.002 g / ml.
[0025] Preferably, in the preparation of the above-mentioned semaglutide injection, the isotonic agent is at least one of propylene glycol and mannitol, the amount of propylene glycol added to the water for injection is 0.01-0.02 g / ml, and the amount of mannitol added to the water for injection is 0.025-0.06 g / ml.
[0026] Preferably, in the preparation of the above-mentioned semaglutide injection, the amount of purified semaglutide added to the excipient solution is 0.001-0.002 g / ml, the pH is adjusted to 7-7.9, and the preparation of semaglutide injection is carried out at 2-15°C.
[0027] Preferably, a method for preparing a highly cross-linked resin comprises:
[0028] The pretreated chloromethylated polystyrene resin and 1,2-dichloroethane were mixed and stirred to obtain a swollen resin, and FeCl was added to the swollen resin. 3 , 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene, heated and stirred to react, after the reaction, filtered and washed to obtain the product, and dried the product to obtain a low cross-linked resin. The low cross-linked resin and 1,2-dichloroethane were mixed and stirred to obtain a swollen low cross-linked resin, and FeCl was added to the swollen low cross-linked resin. 3 The mixture is stirred and mixed with methylal, and then heated and refluxed for reaction. After the reaction is completed, the mixture is filtered and washed to obtain a highly cross-linked product. The highly cross-linked crude product is dried to obtain a highly cross-linked resin.
[0029] Preferably, in the preparation of the highly cross-linked resin, the amount of the chloromethylated polystyrene resin in 1,2-dichloroethane is 0.05-0.2 g / ml.
[0030] Preferably, in the preparation of the highly cross-linked resin, FeCl 3The mass ratio of the amount added to the swelling resin to the amount of the chloromethylated polystyrene resin used is 1:4-6.
[0031] Preferably, in the preparation of the highly cross-linked resin, the mass ratio of the amount of 1-fluoro-2-iodonaphthalene to the amount of chloromethylated polystyrene resin is 1:2-4, the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester to the amount of chloromethylated polystyrene resin is 1:2-4.5, and the mass ratio of the amount of 1-bromo-5-chloronaphthalene to the amount of chloromethylated polystyrene resin is 1:2-3.5.
[0032] Preferably, in the preparation of the highly cross-linked resin, the mixing and stirring time of the chloromethylated polystyrene resin and 1,2-dichloroethane is 0.5-2 h.
[0033] Preferably, in the preparation of the highly cross-linked resin, FeCl is added to the swollen resin. 3 , 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene, and the temperature is raised to 40-50°C for reaction.
[0034] Preferably, in the preparation of the highly cross-linked resin, FeCl is added to the swollen resin. 3 , 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene, the reaction time is 15-25h.
[0035] Preferably, in the preparation of the highly cross-linked resin, the product is dried at 45-55° C. to obtain the low cross-linked resin.
[0036] Preferably, in the preparation of the highly cross-linked resin, the volume ratio of the amount of 1,2-dichloroethane used when swelling the low cross-linked resin to the amount of 1,2-dichloroethane used when swelling the chloromethylated polystyrene resin is 1:0.8-1.5.
[0037] Preferably, in the preparation of the highly cross-linked resin, FeCl 3 The mass ratio of the amount added to the low cross-linked resin to the amount of the low cross-linked resin is 1:0.15-1, and the volume ratio of the amount of methylal used to the amount of 1,2-dichloroethane used when swelling the low cross-linked resin is 1:4.5-7.
[0038] Preferably, in the preparation of the highly cross-linked resin, FeCl is added to the swollen low cross-linked resin. 3 After adding methylal, stir and mix for 2-5h.
[0039] Preferably, in the preparation of the highly cross-linked resin, the low cross-linked resin, FeCl 3 The reaction temperature of methylal is 75-85°C.
[0040] Preferably, in the preparation of the highly cross-linked resin, the low cross-linked resin, FeCl 3 and methylal time is 15-25h.
[0041] Preferably, in the preparation of the highly cross-linked resin, the drying temperature of the highly cross-linked crude product is 75-80°C.
[0042] Preferably, a method for preparing purified semaglutide comprises:
[0043] The highly cross-linked resin and the crude semaglutide peptide aqueous solution were mixed and shaken, and after the shaking, the resin-semaglutide complex was filtered and washed, and then the resin-semaglutide complex was eluted with 65-75% methanol at a flow rate of 0.8-1.2 BV / h, and the eluate was collected in sections until the semaglutide completely flowed out, and the methanol was removed by drying to obtain purified semaglutide.
[0044] Preferably, in the method for preparing purified semaglutide, the shaking reaction temperature is 10-18°C.
[0045] Preferably, in the method for preparing purified semaglutide, the shaking time is 20-30 hours.
[0046] Preferably, in the preparation method of purified semaglutide, the amount of crude semaglutide peptide in the aqueous solution of crude semaglutide peptide is 0.0002-0.001 g / ml.
[0047] Preferably, in the method for preparing purified semaglutide, the mass ratio of the amount of crude semaglutide used to the amount of highly cross-linked resin used is 1:20-30.
[0048] Preferably, in the preparation of purified semaglutide, boric acid alkyl alcohol amides can also be added, the crude semaglutide peptide aqueous solution is mixed with boric acid alkyl alcohol amides to obtain a mixed solution, and a highly cross-linked resin is added to the mixed solution for shock adsorption purification. Boric acid alkyl alcohol amides can be adsorbed on the surface of highly cross-linked resins, reduce the surface tension of highly cross-linked low resins, improve the stability of the surface of highly cross-linked resins, and reduce the aggregation between highly cross-linked resin particles, so that the crude semaglutide peptide is better adsorbed on the highly cross-linked resin and the adsorption rate is improved. At the same time, the addition of boric acid alkyl alcohol amides can reduce the tailing phenomenon during methanol elution and improve the desorption rate.
[0049] Preferably, in the preparation of the purified semaglutide, the mass ratio of the amount of boric acid alkylolamide used to the amount of crude semaglutide peptide used is 1:8-15.
[0050] Preferably, in a preferred embodiment of the present invention, purified semaglutide is prepared by mixing a crude semaglutide peptide aqueous solution with boric acid alkyl alcohol amide to obtain a mixed solution, adding a highly cross-linked resin to the mixed solution, and then shaking at 10-18°C for 20-30 hours, filtering and washing after the shaking to obtain a resin-semaglutide complex, and then using 65-75% methanol as an eluent to elute the resin-semaglutide complex at a flow rate of 0.8-1.2 BV / h, collecting the eluate in sections until the semaglutide is completely out, and drying to remove the methanol to obtain purified semaglutide.
[0051] Preferably, in the above preferred embodiment, the amount of semaglutide in the semaglutide aqueous solution is 0.0002-0.001 g / ml, the mass ratio of the amount of semaglutide used to the amount of high cross-linked resin used is 1:20-30, and the mass ratio of the amount of boric acid alkyl alcohol amide used to the amount of semaglutide used is 1:8-15.
[0052] Preferably, the present invention also provides a semaglutide injection prepared by the above method.
[0053] Compared with the prior art, the present invention has the following beneficial effects:
[0054] The present invention obtains a highly cross-linked resin by modifying and cross-linking chloromethylated polystyrene resin; and uses the highly cross-linked resin to purify crude semaglutide peptide with a high adsorption rate to obtain purified semaglutide with high purity; and uses purified semaglutide as a raw material to prepare semaglutide injection. By controlling the temperature at 2-15°C, adding purified semaglutide to the auxiliary material solution, and undergoing pH adjustment, filtration and sterilization, the aggregation of semaglutide is effectively slowed down, and an effective and safe semaglutide injection is obtained. BRIEF DESCRIPTION OF THE DRAWINGS
[0055] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.
[0056] Figure 1 is the HPLC spectrum of purified semaglutide in Example 1;
[0057] Figure 2 is the HPLC spectrum of semaglutide injection in Example 1;
[0058] Figure 3 is the HPLC spectrum of semaglutide injection in Example 7;
[0059] Figure 4 is the HPLC spectrum of semaglutide injection in Example 8;
[0060] Figure 5 is the HPLC spectrum of semaglutide injection in Example 9;
[0061] Figure 6 is the HPLC spectrum of semaglutide injection in Comparative Example 4;
[0062] Figure 7 This is the HPLC spectrum of semaglutide injection in Comparative Example 5. DETAILED DESCRIPTION
[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0064] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.
[0065] Embodiment 1:
[0066] Preparation of highly cross-linked resin: The pretreated chloromethylated polystyrene resin and 1,2-dichloroethane were mixed and stirred for 1 h to obtain a swollen resin. FeCl 3 , 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene, heated to 45 °C and stirred for 18 hours. After the reaction, the product was filtered and washed to obtain the product, and the product was dried at 50 °C to obtain a low cross-linked resin. The low cross-linked resin and 1,2-dichloroethane were mixed and stirred for 1 hour to obtain a swollen low cross-linked resin. FeCl was added to the swollen low cross-linked resin. 3 and methylal for 3 hours, then heated to 80°C for reflux reaction for 18 hours. After the reaction, the highly cross-linked product was obtained by filtration and washing, and the highly cross-linked crude product was dried at 80°C to obtain a highly cross-linked resin. The amount of chloromethylated polystyrene resin in 1,2-dichloroethane was 0.1 g / ml, and FeCl 3The mass ratio of the amount added to the swelling resin to the amount of the chloromethylated polystyrene resin is 1:5, the mass ratio of the amount of 1-fluoro-2-iodonaphthalene to the amount of the chloromethylated polystyrene resin is 1:2.5, the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester to the amount of the chloromethylated polystyrene resin is 1:2.7, the mass ratio of the amount of 1-bromo-5-chloronaphthalene to the amount of the chloromethylated polystyrene resin is 1:2.1, the volume ratio of the amount of 1,2-dichloroethane used when swelling the low cross-linked resin to the amount of 1,2-dichloroethane used when swelling the chloromethylated polystyrene resin is 1:1, FeCl 3 The mass ratio of the amount added to the low cross-linked resin to the amount of the low cross-linked resin is 1:0.34, and the volume ratio of the amount of methylal used to the amount of 1,2-dichloroethane used when swelling the low cross-linked resin is 1:5.68.
[0067] Preparation of purified semaglutide: The highly cross-linked resin and the crude semaglutide peptide aqueous solution are mixed and shaken at 15°C for 24 hours. After the shaking, the resin-semaglutide complex is filtered and washed to obtain the resin-semaglutide complex, and then 70% methanol is used as an eluent to elute the resin-semaglutide complex at a flow rate of 1BV / h. The eluate is collected in sections until the semaglutide completely flows out, and the methanol is dried to remove the purified semaglutide. The amount of crude semaglutide peptide in the crude semaglutide peptide aqueous solution is 0.0003g / ml, and the mass ratio of the amount of crude semaglutide peptide used to the amount of highly cross-linked resin used is 1:26.67.
[0068] Preparation of semaglutide injection: Phenol, disodium hydrogen phosphate dihydrate and propylene glycol are mixed with water for injection to obtain an excipient solution, and then purified semaglutide is added to the excipient solution to obtain a semaglutide solution, and then HCl solution is added to the semaglutide solution to adjust the pH to 7.4, constant volume, and after filtration and sterilization, it is filled into a 3mL borosilicate glass cartridge bottle, sealed with a stopper, and semaglutide injection is obtained. Among them, the amount of phenol added to the water for injection is 0.00692g / ml, the amount of disodium hydrogen phosphate dihydrate added to the water for injection is 0.00175g / ml, the amount of propylene glycol added to the water for injection is 0.0175g / ml, the amount of purified semaglutide added to the excipient solution is 0.00185g / ml, the concentration of the HCl solution is 0.1mol / L, and the entire preparation process is carried out at 15°C.
[0069] Embodiment 2:
[0070] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that the mass ratio of the amount of 1-fluoro-2-iodonaphthalene used to the amount of chloromethylated polystyrene resin used is 1:3, and other conditions and parameters are the same as in Example 1.
[0071] The preparation of purified semaglutide was the same as in Example 1.
[0072] The preparation of semaglutide injection is the same as that in Example 1.
[0073] Embodiment 3:
[0074] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester used to the amount of chloromethylated polystyrene resin used is 1:3.2, and other conditions and parameters are the same as in Example 1.
[0075] The preparation of purified semaglutide was the same as in Example 1.
[0076] The preparation of semaglutide injection is the same as that in Example 1.
[0077] Embodiment 4:
[0078] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that the mass ratio of the amount of 1-bromo-5-chloronaphthalene used to the amount of chloromethylated polystyrene resin used is 1:2.8, and other conditions and parameters are the same as in Example 1.
[0079] The preparation of purified semaglutide was the same as in Example 1.
[0080] The preparation of semaglutide injection is the same as that in Example 1.
[0081] Embodiment 5:
[0082] The preparation of highly cross-linked resin is the same as in Example 1.
[0083] Preparation of purified semaglutide: A crude semaglutide peptide aqueous solution is mixed with boric acid alkyl alcohol amide to obtain a mixed solution, a highly cross-linked resin is added to the mixed solution, and then the mixture is shaken at 15°C for 24 hours. After the shaking, the resin-semaglutide complex is filtered and washed to obtain the resin-semaglutide complex, and then 70% methanol is used as an eluent to elute the resin-semaglutide complex at a flow rate of 1BV / h, and the eluate is collected in sections until the semaglutide completely flows out, and the methanol is dried to remove the purified semaglutide. The amount of crude semaglutide peptide in the crude semaglutide peptide aqueous solution is 0.0003g / ml, the mass ratio of the amount of crude semaglutide peptide used to the amount of highly cross-linked resin used is 1:26.67, the mass ratio of the amount of boric acid alkyl alcohol amide used to the amount of crude semaglutide peptide used is 1:10, and the boric acid alkyl alcohol amide is purchased from Guangzhou Zhonghai Chemical Co., Ltd.
[0084] The preparation of semaglutide injection is the same as that in Example 1.
[0085] Embodiment 6:
[0086] The preparation of highly cross-linked resin is the same as in Example 1.
[0087] Preparation of purified semaglutide: The preparation of purified semaglutide in this example is different from that in Example 5, except that the mass ratio of the amount of boric acid alkylolamide used to the amount of crude semaglutide peptide used is 1:7, and the other conditions and parameters are the same as in Example 5.
[0088] The preparation of semaglutide injection is the same as that in Example 1.
[0089] Embodiment 7:
[0090] The preparation of highly cross-linked resin is the same as in Example 1.
[0091] The preparation of purified semaglutide was the same as in Example 1.
[0092] Preparation of semaglutide injection: The preparation of semaglutide injection in this example is compared with that in Example 1, except that the entire preparation process is carried out at 8° C., and other conditions and parameters are the same as in Example 1.
[0093] Embodiment 8:
[0094] The preparation of highly cross-linked resin is the same as in Example 1.
[0095] The preparation of purified semaglutide was the same as in Example 1.
[0096] Preparation of semaglutide injection: The preparation of semaglutide injection in this example is compared with that in Example 1, except that the entire preparation process is carried out at 5° C., and other conditions and parameters are the same as in Example 1.
[0097] Embodiment 9:
[0098] The preparation of highly cross-linked resin is the same as in Example 1.
[0099] The preparation of purified semaglutide was the same as in Example 1.
[0100] Preparation of semaglutide injection: The preparation of semaglutide injection in this example is compared with that in Example 1, except that the entire preparation process is carried out at 2° C., and other conditions and parameters are the same as in Example 1.
[0101] Comparative Example 1:
[0102] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that 1-fluoro-2-iodonaphthalene is not added to the swelling resin for reaction, and other conditions and parameters are the same as in Example 1.
[0103] The preparation of purified semaglutide was the same as in Example 1.
[0104] The preparation of semaglutide injection is the same as that in Example 1.
[0105] Comparative Example 2:
[0106] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that 5-bromo-2-naphthoic acid methyl ester is not added to the swelling resin for reaction, and other conditions and parameters are the same as in Example 1.
[0107] The preparation of purified semaglutide was the same as in Example 1.
[0108] The preparation of semaglutide injection is the same as that in Example 1.
[0109] Comparative Example 3:
[0110] Preparation of highly cross-linked resin: The preparation of highly cross-linked resin in this embodiment is compared with that in Example 1, except that 1-bromo-5-chloronaphthalene is not added to the swelling resin for reaction, and other conditions and parameters are the same as in Example 1.
[0111] The preparation of purified semaglutide was the same as in Example 1.
[0112] The preparation of semaglutide injection is the same as that in Example 1.
[0113] Comparative Example 4:
[0114] The preparation of highly cross-linked resin is the same as in Example 1.
[0115] The preparation of purified semaglutide was the same as in Example 1.
[0116] Preparation of semaglutide injection: The preparation of semaglutide injection in this example is compared with that in Example 1, except that the entire preparation process is carried out at 20° C., and other conditions and parameters are the same as in Example 1.
[0117] Comparative Example 5:
[0118] The preparation of highly cross-linked resin is the same as in Example 1.
[0119] The preparation of purified semaglutide was the same as in Example 1.
[0120] Preparation of semaglutide injection: The preparation of semaglutide injection in this example is compared with that in Example 1, except that the entire preparation process is carried out at 30° C., and other conditions and parameters are the same as in Example 1.
[0121] Experimental Example 1:
[0122] Determination of adsorption rate: The filtrate after filtering out the resin-semaglutide complex in Examples 1-6 and Comparative Examples 1-3 was collected, and a biuret reagent was added to the filtrate for color development. After the reaction was protected from light for 30 minutes, the mass fraction was measured and the adsorption rate was calculated. Adsorption rate (%) = (C 0 −C 1 ) / C 0 , C 0 represents the concentration of semaglutide in the solution before adsorption, mg / g; C 1 It represents the concentration of semaglutide in the filtrate after adsorption, mg / g.
[0123] Table 1 Adsorption results of highly cross-linked resin for semaglutide
[0124]
[0125] The results are shown in Table 1. The adsorption rate of semaglutide by the highly cross-linked resin in Example 1 is higher than that in Comparative Example 1, Comparative Example 2 and Comparative Example 3, indicating that the joint use of 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and methyl and 1-bromo-5-chloronaphthalene can well modify the chloromethylated polystyrene resin, thereby improving the adsorption rate of semaglutide by the highly cross-linked resin. The highly cross-linked resin obtained by modifying the chloromethylated polystyrene resin with any two of 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and methyl and 1-bromo-5-chloronaphthalene alone has no good effect on the adsorption of semaglutide. The adsorption rate of semaglutide by the highly cross-linked resin in Example 5 is higher than that in Example 1, indicating that on the basis of using 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and methyl and 1-bromo-5-chloronaphthalene to modify the resin, adding boric acid alkyl alcohol amide during the purification process can further promote the adsorption of semaglutide by the highly cross-linked resin.
[0126] Experimental Example 2:
[0127] Determination of desorption rate: Determine the mass fraction of semaglutide in the desorption solution collected by methanol elution in Examples 1-6 and Comparative Examples 1-3, and calculate the desorption rate, desorption rate (%) = C 2 V 1 / (C 0 −C 1 )V 0 , C 0 represents the concentration of semaglutide in the solution before adsorption, mg / g; C 1 represents the concentration of semaglutide in the filtrate after adsorption, mg / g; C 2 represents the concentration of semaglutide in the filtrate after desorption, mg / g; V 1 Indicates the volume of the analytical solution, mL; V 0 represents the volume of semaglutide aqueous solution, mL.
[0128] Table 2 Desorption rate results of semaglutide by highly cross-linked resin
[0129]
[0130] The results are shown in Table 2. The desorption rate of semaglutide by the highly cross-linked resin in Example 1 is higher than that in Comparative Example 1, Comparative Example 2 and Comparative Example 3, indicating that the highly cross-linked resin obtained by using 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene together to modify the chloromethylated polystyrene resin can more effectively desorb semaglutide. The highly cross-linked resin obtained by modifying the chloromethylated polystyrene resin with any two of 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and methyl and 1-bromo-5-chloronaphthalene alone has no good effect on the desorption of semaglutide. The desorption rate of semaglutide by the highly cross-linked resin in Example 5 is higher than that in Example 1, indicating that on the basis of using 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and methyl and 1-bromo-5-chloronaphthalene to modify the resin, adding boric acid alkyl alcohol amide in the purification and adsorption process can improve the subsequent methanol desorption of semaglutide.
[0131] Experimental Example 3:
[0132] Determination of high molecular weight protein: The high molecular weight protein of the semaglutide injection prepared in Example 1, Examples 7-9 and Comparative Examples 4-5 was determined by HPLC, and the trend of high molecular weight protein was calculated and compared with the purified semaglutide prepared in Example 1.
[0133] Table 3 Results of high molecular weight protein determination and high molecular weight protein growth trend of semaglutide injection
[0134]
[0135] The HPLC spectra of purified semaglutide, Example 1, Examples 7-9 and Comparative Examples 4-5 are as follows: Figure 1-7 As shown in the figure, the results are shown in Table 3. Compared with Comparative Examples 4-5, the high molecular weight protein in Examples 1 and Examples 7-9 did not change significantly, and the growth trend of the high molecular weight protein was significantly lower than that in Comparative Examples 4-5, indicating that when the temperature is controlled within a certain low temperature range, the aggregation of semaglutide can be slowed down and the content of high molecular weight protein can be reduced, thereby improving the effectiveness and safety of semaglutide injection.
[0136] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.
[0137] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.
Claims
1. A method for preparing semaglutide injection, comprising: The excipient solution and purified semaglutide are mixed, the pH is adjusted, and filtration is performed for sterilization to obtain semaglutide injection; Purified semaglutide is prepared by adsorbing crude semaglutide peptide on a highly cross-linked resin and then undergoing desorption purification; the highly cross-linked resin is obtained by reacting and modifying chloromethylated polystyrene resin with 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene; the preparation of semaglutide injection is carried out at 2-15°C; The preparation of the highly cross-linked resin comprises: mixing chloromethylated polystyrene resin and 1,2-dichloroethane to obtain a swollen resin, adding FeCl3, 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene to the swollen resin for reaction, and filtering and washing after the reaction to obtain a low cross-linked resin; swelling the low cross-linked resin, adding FeCl3 and methylal to stir and react, filtering and washing after the reaction, and drying to obtain a highly cross-linked resin; The amount of the chloromethylated polystyrene resin in 1,2-dichloroethane is 0.05-0.2 g / ml, the mass ratio of the amount of FeCl3 added to the swollen resin to the amount of the chloromethylated polystyrene resin is 1:4-6, the mass ratio of the amount of 1-fluoro-2-iodonaphthalene to the amount of the chloromethylated polystyrene resin is 1:2-4, the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester to the amount of the chloromethylated polystyrene resin is 1:2-4.5, and the mass ratio of the amount of 1-bromo-5-chloronaphthalene to the amount of the chloromethylated polystyrene resin is 1:2-3.5; The volume ratio of the amount of 1,2-dichloroethane used when the low cross-linked resin is swollen to the amount of 1,2-dichloroethane used when the chloromethylated polystyrene resin is 1:0.8-1.5, the mass ratio of the amount of FeCl3 added to the low cross-linked resin to the amount of the low cross-linked resin is 1:0.15-1, and the volume ratio of the amount of methylal used to the amount of 1,2-dichloroethane used when the low cross-linked resin is 1:4.5-7; The swelling time of the chloromethylated polystyrene resin is 0.5-2h; when preparing low cross-linked resin, the reaction temperature is 40-50°C and the reaction time is 15-25h; the swelling time of the low cross-linked resin is 0.5-2h; when preparing high cross-linked resin, the reaction temperature is 75-85°C and the reaction time is 15-25h.
2. The method for preparing semaglutide injection according to claim 1, characterized in that: The auxiliary material solution is prepared by dissolving an antibacterial agent, a pH buffer and an isotonic agent in water for injection; the antibacterial agent is at least one of phenol, benzyl alcohol and m-cresol; the pH buffer is at least one of disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate and sodium phosphate; and the isotonic agent is at least one of propylene glycol and mannitol.
3. The method for preparing semaglutide injection according to claim 1, characterized in that: The amount of the purified semaglutide added to the auxiliary material solution is 0.001-0.002 g / ml, and the pH is adjusted to 7-7.
9.
4. The method for preparing semaglutide injection according to claim 1, characterized in that: The preparation of purified semaglutide comprises: The highly cross-linked resin and the crude semaglutide peptide aqueous solution were mixed and shaken, and after the shaking, the resin-semaglutide complex was filtered and washed, and then the resin-semaglutide complex was eluted with 65-75% methanol at a flow rate of 0.8-1.2 BV / h, and the eluate was collected in sections until the semaglutide completely flowed out, and the methanol was removed by drying to obtain purified semaglutide.
5. The method for preparing semaglutide injection according to claim 4, characterized in that: The shaking reaction temperature is 10-18° C., and the shaking time is 20-30 h. The amount of crude semaglutide in the crude semaglutide aqueous solution is 0.0002-0.001 g / ml, and the mass ratio of the amount of crude semaglutide to the amount of highly cross-linked resin is 1:20-30.
6. Use of a highly cross-linked resin in the preparation of purified semaglutide; the highly cross-linked resin is obtained by reacting and modifying a chloromethylated polystyrene resin with 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene; The preparation of the highly cross-linked resin comprises: The chloromethylated polystyrene resin and 1,2-dichloroethane are mixed to obtain a swollen resin, FeCl3, 1-fluoro-2-iodonaphthalene, 5-bromo-2-naphthoic acid methyl ester and 1-bromo-5-chloronaphthalene are added to the swollen resin for reaction, and after the reaction, the resin is filtered and washed to obtain a low cross-linked resin; the low cross-linked resin is swollen, FeCl3 and methylal are added to stir and react, after the reaction, the resin is filtered and washed, and dried to obtain a high cross-linked resin; The amount of the chloromethylated polystyrene resin in 1,2-dichloroethane is 0.05-0.2 g / ml, the mass ratio of the amount of FeCl3 added to the swollen resin to the amount of the chloromethylated polystyrene resin is 1:4-6, the mass ratio of the amount of 1-fluoro-2-iodonaphthalene to the amount of the chloromethylated polystyrene resin is 1:2-4, the mass ratio of the amount of 5-bromo-2-naphthoic acid methyl ester to the amount of the chloromethylated polystyrene resin is 1:2-4.5, and the mass ratio of the amount of 1-bromo-5-chloronaphthalene to the amount of the chloromethylated polystyrene resin is 1:2-3.5; The volume ratio of the amount of 1,2-dichloroethane used when the low cross-linked resin is swollen to the amount of 1,2-dichloroethane used when the chloromethylated polystyrene resin is 1:0.8-1.5, the mass ratio of the amount of FeCl3 added to the low cross-linked resin to the amount of the low cross-linked resin is 1:0.15-1, and the volume ratio of the amount of methylal used to the amount of 1,2-dichloroethane used when the low cross-linked resin is 1:4.5-7; The swelling time of the chloromethylated polystyrene resin is 0.5-2h; when preparing low cross-linked resin, the reaction temperature is 40-50°C and the reaction time is 15-25h; the swelling time of the low cross-linked resin is 0.5-2h; when preparing high cross-linked resin, the reaction temperature is 75-85°C and the reaction time is 15-25h.
Citation Information
Patent Citations
Preparation method of semeglutide preparation
CN114146163A