Preparation process for solid-phase synthesis of insulin glargine
By constructing disulfide bonds for glargine insulin in three steps using a solid-phase synthesis method, selective oxidation with NCS, DTDP, and iodine, and monitoring by LC-MS and HPLC, the problems of insufficient purity and low yield in traditional processes were solved, and high-purity and high-yield glargine insulin was prepared.
Patent Information
- Application Number
- CN202511502831.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-21
- Publication Date
- 2025-12-26
AI Technical Summary
The traditional production process of glargine insulin in the present technology has problems such as host cell protein residue, endotoxin contamination, complex purification process, and non-directional disulfide bond formation, resulting in high product heterogeneity, insufficient purity and low yield.
A solid-phase synthesis method was adopted to construct disulfide bonds in three steps through selective oxidation with NCS, DTDP and iodine. The reaction was purified using Wang resin and C18 column, and the reaction process was monitored by LC-MS and HPLC to ensure the directional formation and high purity of disulfide bonds.
It achieves high purity (99.2%) and high yield (28%) of glargine insulin, meeting pharmaceutical standards, suitable for industrial production, and reducing by-product generation and impurity content.
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Figure CN121202995A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biopharmaceuticals, and particularly relates to a preparation process of solid-phase synthesis of insulin glargine. BACKGROUND
[0002] As a long-acting insulin analogue, the traditional production process of insulin glargine mainly relies on biological fermentation method, but this method has problems such as host cell protein residues, endotoxin pollution, and a complex purification process. Although the solid-phase synthesis method can avoid biological impurities, the correct folding of the three pairs of disulfide bonds in the insulin glargine molecule is a key difficulty in the synthesis process.
[0003] In the prior art, non-directional oxidation is often used to form disulfide bonds, resulting in high heterogeneity of the product and a purity of less than 95%, which cannot meet the pharmaceutical standards. In addition, one-pot oxidation is often used in the traditional synthesis process, which is difficult to control the reaction process, resulting in an increase in by-products and a low yield. SUMMARY
[0004] The present application relates to the technical field of biopharmaceuticals, and particularly relates to a preparation process of solid-phase synthesis of insulin glargine.
[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A preparation process of solid-phase synthesis of insulin glargine, comprising the following steps: S1, synthesizing an insulin A chain by Wang resin solid phase, and forming a first pair of disulfide bonds by using NCS after removing the protective group: NCS (N-chlorosuccinimide) is used as an oxidizing agent, Wang resin (Rink Amide Wang Resin) is used as a solid phase carrier, amino acids are sequentially connected according to the Fmoc solid phase synthesis method to construct the A chain sequence; 0.5 times the molar amount of NCS is added, and the reaction is carried out in a DMF solution at room temperature for 1 h to form the first pair of disulfide bonds, and LC-MS shows that the oxidation rate is 98.3%; the resin is treated with 95% TFA / 2.5% TIS / 2.5% H2O cutting solution for 2 h, the filtrate is collected, dissolved in DMSO, and purified by C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile, gradient elution), and then freeze-dried to obtain the A chain pure product with a purity of 95.6%; S2, synthesizing an insulin B chain by Wang resin solid phase, and grafting DTDP after cutting and purifying: After treatment with the cutting solution (95% TFA / 2.5% TIS / 2.5% H2O) and freeze-drying, the crude peptide is obtained; the crude peptide is purified by C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile), and then the target peak is collected and freeze-dried, and the purity is 91.2% S3, connecting the A chain and the B chain solution to form a second pair of disulfide bonds: C18 column purification (mobile phase: 10 mM ammonium bicarbonate-acetonitrile, gradient elution), and then the target peak was collected and freeze-dried, with a purity of 97.8%, and the formation of the second pair of disulfide bonds was confirmed by LC-MS; S4, constructing a third pair of disulfide bonds by iodine oxidation.
[0006] Preferably, the amino acid sequence of the insulin A chain is: GIVEQC(mmt)C(Acm)TSICC(mmt)SLYQLENYCG, and the MMT protecting group was removed by treating with 2% TFA / 2% TIS / DCM solution for 3 times, 20 min each time, and the deprotection rate reached 99.5% by LC-MS detection.
[0007] Preferably, the amino acid sequence of the insulin B chain is: FVNQHLC(Acm)GSHLVEALYLVCGERGFFYTPKTRR, and the grafting molar ratio of DTDP to the crude peptide was 1:1, and the reaction was carried out in PBS buffer at pH 7.0 at room temperature for 4 h, and the grafting rate was 92.7% by LC-MS detection.
[0008] Preferably, when the insulin A chain and the insulin B chain are docked to form the second pair of disulfide bonds, the insulin A chain is dissolved in DMSO, the insulin B chain is dissolved in water, the reaction process is monitored by MS, the dissolved insulin B chain solution is slowly added to the insulin A chain solution, and the reaction is carried out at 37°C for 2 h, during which the liquid is taken out every 30 min for MS detection, and after the reaction is completed, the reaction is diluted by 10 times with water.
[0009] Preferably, the iodine oxidation conditions of the third pair of disulfide bonds are: iodine concentration 20 mg / ml, reaction at room temperature for 30 min, reaction process monitored by MS, reaction terminated by VC after completion, and after termination of the reaction, an excess of VC (10 mM) is added, and the product is purified by C18 column (mobile phase: 10 mM ammonium bicarbonate-acetonitrile), and then freeze-dried to obtain the pure insulin glargine.
[0010] Preferably, when the insulin A chain is cut and purified, DMSO is used for dissolution, and after two preparation purifications, the insulin B chain is grafted with DTDP and then purified to a purity of more than 90%.
[0011] Compared with the prior art, the beneficial effects of the present application are: 1. Directional formation of disulfide bonds: the disulfide bonds are constructed in three steps, and through selective oxidation of NCS, DTDP and iodine, non-specific crosslinking is avoided, the correct folding of the molecule is ensured, and the total yield of the three-step disulfide bond formation process reaches 28%, which is 13% higher than that of one-pot oxidation, and the yield is improved; 2. Process controllability: each reaction step is monitored in real time by MS, the reaction process can be accurately controlled, the generation of by-products is reduced, the content of main impurities such as oxidized peptides and missing peptides is less than 0.1%, and the USP pharmacopoeia standard is met; 3. Industrial adaptability: the solid phase synthesis steps can be realized by automatic polypeptide synthesizer for large-scale production, and the purification process adopts conventional HPLC column, and the equipment compatibility is strong; 4. High quality standard: the purity of the product detected by HPLC can reach 99.2% or more, which is increased by 4.2% compared with the traditional process, the impurity content is lower than the pharmacopoeia standard, and the requirements of insulin preparation for injection are met. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 The molecular formula of the solid phase synthesis insulin preparation process provided by the application. DETAILED DESCRIPTION
[0013] REFERENCE Figure 1 A solid phase synthesis insulin preparation process, comprising the following steps: S1, synthesizing insulin A chain by Wang resin solid phase, and forming the first pair of disulfide bond by using NCS after removing the protection group: NCS (N-chlorosuccinimide) is used as an oxidizing agent, Wang resin (Rink Amide Wang Resin) is used as a solid phase carrier, amino acids are sequentially connected according to the Fmoc solid phase synthesis method to construct the A chain sequence; 0.5 times the molar amount of NCS is added, and the reaction is carried out in a DMF solution at room temperature for 1 h to form the first pair of disulfide bond, and LC-MS shows that the oxidation rate is 98.3%; the resin is treated with 95% TFA / 2.5% TIS / 2.5% H2O cutting solution for 2 h, the filtrate is collected, dissolved in DMSO, and purified by C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile, gradient elution), and then freeze-dried to obtain the A chain pure product with a purity of 95.6%; NCS (N-chlorosuccinimide) is used as an oxidizing agent, and the cysteine in the A chain is selectively oxidized on the resin solid phase to form the first pair of disulfide bond, the oxidation efficiency of this method is more than 98%, and non-specific oxidation is avoided S2, synthesizing insulin B chain by Wang resin solid phase, grafting DTDP after cutting and purifying: After treatment with the cutting solution (95% TFA / 2.5% TIS / 2.5% H2O), the crude peptide is freeze-dried; the crude peptide is purified by C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile), and then freeze-dried to obtain the target peak with a purity of 91.2%; S3, docking the A chain and B chain solution to form the second pair of disulfide bond: C18 column purification (mobile phase: 10 mM ammonium bicarbonate-acetonitrile, gradient elution), and then the target peak was collected and freeze-dried, with a purity of 97.8%, and the formation of the second pair of disulfide bonds was confirmed by LC-MS; By grafting B chain mercapto with DTDP (3,4-dithiopyridine), and using the thioester bond characteristics of DTDP to react with A chain mercapto in solution to form the second pair of disulfide bonds, the reaction selectivity can reach 95% S4, using iodine oxidation method to construct the third pair of disulfide bonds; After the formation of the first two pairs of disulfide bonds, the remaining mercapto was oxidized with iodine solution, and the reaction process was monitored in real time by MS to ensure the correct formation of the third pair of disulfide bonds and avoid over-oxidation; The amino acid sequence of the insulin A chain is: GIVEQC(mmt)C(Acm)TSICC(mmt)SLYQLENYCG, and the deprotection rate of MMT protective group reached 99.5% after being treated with 2% TFA / 2% TIS / DCM solution for 3 times, 20 min each time, and LC-MS detection; The amino acid sequence of the insulin B chain is: FVNQHLC(Acm)GSHLVEALYLVCGERGFFYTPKTRR, and the grafting molar ratio of DTDP to crude peptide was 1:1, and the reaction was carried out in PBS buffer at pH 7.0 at room temperature for 4 h, and the grafting rate was 92.7% detected by LC-MS; When the insulin A chain and the insulin B chain were docked to form the second pair of disulfide bonds, the insulin A chain was dissolved in DMSO, and the insulin B chain was dissolved in water, and the reaction process was monitored by MS. The dissolved insulin B chain solution was slowly added to the insulin A chain solution, and the reaction was carried out at 37°C for 2 h, and the liquid was taken out every 30 min for MS detection during the reaction. After the reaction was completed, water was added to dilute it by 10 times; The iodine oxidation conditions of the third pair of disulfide bonds were as follows: iodine concentration 20 mg / ml, room temperature reaction for 30 min, reaction process monitored by MS, reaction terminated by VC after completion of the reaction, and then excess VC (10 mM) was added. After the termination of the reaction, the product was purified by C18 column (mobile phase: 10 mM ammonium bicarbonate-acetonitrile), and then freeze-dried to obtain the pure insulin glargine; When the insulin A chain was cut and purified, DMSO was used for dissolution, and after two preparation purifications, the insulin B chain was grafted with DTDP and purified to a purity of more than 90%.
[0014] In the present application, the insulin A chain is first synthesized by Wang resin solid phase synthesis, and after removing the protecting group, the first pair of disulfide bond is formed by using NCS as the oxidizing agent. The NCS (N-chlorosuccinimide) is used as the oxidizing agent, and the Wang resin (Rink Amide Wang Resin) is used as the solid phase carrier. The amino acids are sequentially connected according to the Fmoc solid phase synthesis method to construct the A chain sequence. 0.5 times the molar amount of NCS is added, and the reaction is carried out in the DMF solution at room temperature for 1 h to form the first pair of disulfide bond. The LC-MS shows that the oxidation rate is 98.3%. The removal of the MMT protecting group is carried out by treating the solution of 2% TFA / 2% TIS / DCM for 3 times, each time for 20 min. The LC-MS detection shows that the deprotection rate is 99.5%. The grafting molar ratio of DTDP and the crude peptide is 1:1. The reaction is carried out in the PBS buffer solution at pH 7.0 at room temperature for 4 h. The LC-MS detection shows that the grafting rate is 92.7%. The resin is treated with the cutting solution of 95% TFA / 2.5% TIS / 2.5% H2O for 2 h. The filtrate is collected, dissolved in DMSO, and purified by the C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile, gradient elution). The A chain pure product is obtained by freeze-drying. The purity is 95.6%. Then the insulin B chain is synthesized by Wang resin solid phase synthesis. After cutting, the DTDP is grafted and purified. The crude peptide is obtained by freeze-drying after the treatment of the cutting solution (95% TFA / 2.5% TIS / 2.5% H2O). The crude peptide is purified by the C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile). The target peak is collected and freeze-dried. The purity is 91.2%. Then the A chain and the B chain solution are docked to form the second pair of disulfide bond. When the insulin A chain and the insulin B chain are docked to form the second pair of disulfide bond, the insulin A chain is dissolved in DMSO, and the insulin B chain is dissolved in water. The reaction process is monitored by MS. The dissolved insulin B chain solution is slowly added to the insulin A chain solution. The reaction is carried out at 37℃ for 2 h. During the reaction, the liquid is taken out every 30 min for MS detection. After the reaction is completed, water is added for dilution by 10 times. The C18 column is used for purification (mobile phase: 10 mM ammonium bicarbonate-acetonitrile, gradient elution). Then the target peak is collected and freeze-dried. The purity is 97.8%. The formation of the second pair of disulfide bond is confirmed by LC-MS. Finally, the third pair of disulfide bond is constructed by iodine oxidation method. The iodine oxidation conditions of the third pair of disulfide bond are as follows: the iodine concentration is 20 mg / ml, the reaction is carried out at room temperature for 30 min, the reaction process is monitored by MS, and the reaction is terminated by VC after the reaction is completed. After the termination of the reaction, excess VC (10 mM) is added. The C18 column is used for purification (mobile phase: 10 mM ammonium bicarbonate-acetonitrile). The insulin pure product is obtained by freeze-drying. The insulin A chain is dissolved in DMSO, freeze-dried after two times of preparation and purification. The purity of the insulin B chain after grafting DTDP is more than 90%.
Claims
1. A solid-phase synthesis process for preparing glargine insulin, characterized in that, Includes the following steps: S1. Insulin A chain is synthesized via solid-phase synthesis using royal jelly resin. After removing the protecting group, the first pair of disulfide bonds is formed by oxidation using NCS. Using NCS (N-chlorosuccinimide) as the oxidant and RinkAmide Wang Resin as the solid-phase support, amino acids were sequentially added according to the Fmoc solid-phase synthesis method to construct the A-chain sequence. 0.5 molar amounts of NCS were added, and the reaction was carried out in DMF solution at room temperature for 1 h to form the first pair of disulfide bonds. LC-MS showed an oxidation rate of 98.3%. The resin was treated with a cleavage solution of 95% TFA / 2.5% TIS / 2.5% H2O for 2 h. The filtrate was collected, dissolved in DMSO, and purified by a C18 column (mobile phase: 0.1% TFA aqueous solution-acetonitrile, gradient elution). Lyophilization yielded the pure A-chain with a purity of 95.6%. S2. Insulin B chain was synthesized in a solid-phase manner using royal resin, cleaved, grafted with DTDP, and purified. The crude peptide was obtained by lyophilization after treatment with the cleavage buffer (95% TFA / 2.5% TIS / 2.5% H2O); it was then purified by C18 column purification (mobile phase: 0.1% TFA aqueous solution-acetonitrile), and the target peak was collected and lyophilized to a purity of 91.2%. S3. Form a second pair of disulfide bonds by solution docking of chain A and chain B: C18 column purification (mobile phase: 10mM ammonium bicarbonate-acetonitrile, gradient elution), followed by collection and lyophilization of the target peak, with a purity of 97.8%, and confirmation of the formation of the second disulfide bond by LC-MS. S4. The third disulfide bond is constructed using the iodine oxidation method.
2. The solid-phase synthesis process for preparing glargine insulin according to claim 1, characterized in that, The amino acid sequence of the insulin A chain is as follows: The MMT protecting group was removed by treating GIVEQC(mmt)C(Acm)TSICC(mmt)SLYQLENYCG with 2% TFA / 2% TIS / DCM solution three times for 20 min each time. The deprotection rate was 99.5% as determined by LC-MS.
3. The solid-phase synthesis process for preparing glargine insulin according to claim 1, characterized in that, The amino acid sequence of the insulin B chain is as follows: FVNQHLC(Acm)GSHLVEALYLVCGERGFFYTPKTRR, with a grafting molar ratio of DTDP to crude peptide of 1:1, was reacted in PBS buffer at pH 7.0 at room temperature for 4 h. The grafting rate was 92.7% as determined by LC-MS.
4. The solid-phase synthesis process for preparing glargine insulin according to claim 1, characterized in that, When the insulin A chain and insulin B chain dock to form the second pair of disulfide bonds, the insulin A chain is dissolved in DMSO and the insulin B chain is dissolved in water. The reaction process is monitored by MS. The dissolved insulin B chain solution is slowly added dropwise to the insulin A chain solution and reacted at 37°C for 2 hours. During this period, the solution is taken every 30 minutes for MS detection. After the reaction is completed, it is diluted 10 times with water.
5. The solid-phase synthesis process for preparing glargine insulin according to claim 1, characterized in that, The iodine oxidation conditions for the third disulfide bond were as follows: iodine concentration 20 mg / ml, reaction at room temperature for 30 min, the reaction process was monitored by MS, the reaction was terminated with VC after the reaction was complete, excess VC (10 mM) was added after the reaction was terminated, and the product was purified by C18 column (mobile phase: 10 mM ammonium bicarbonate-acetonitrile), and lyophilized to obtain pure insulin glargine.
6. The solid-phase synthesis process for preparing glargine insulin according to claim 1, characterized in that, The insulin A chain was dissolved in DMSO during cleavage and purification, and then freeze-dried after two preparation and purification processes. The insulin B chain was branched with DTDP and purified to a purity of over 90%.