Method for reducing microfiltration separation loss of semaglutide

Abstract

The invention discloses a method for reducing microfiltration separation loss of semaglutide, which comprises the following steps: (1) adding inorganic salt into a solution containing semaglutide to obtain a first solution; (2) adjusting the pH value of the first solution obtained in the step (1) to obtain a second solution; and (3) carrying out microfiltration treatment on the second solution obtained in the step (2). According to the method provided by the invention, the average particle size of semaglutide aggregate in the solution is reduced to be less than 100nm, the transmittance of semaglutide penetrating through a micro-filtration membrane during micro-filtration is greatly improved, the adsorption capacity of a membrane material to semaglutide is reduced, the loss amount of semaglutide is remarkably reduced during micro-filtration separation of semaglutide, and the semaglutide separation efficiency is improved. The application of an efficient membrane separation technology in semaglutide separation and purification production is promoted, the separation and purification cost of semaglutide is reduced, and a guarantee is provided for the application of semaglutide in the medical industry.

Description

Technical Field

[0001] The invention belongs to the field of separation and purification of biopharmaceutical products, and specifically relates to a method for reducing the loss of semaglutide microfiltration separation. Background Art

[0002] Semaglutide, also known as semaglutide or semaglutide, is composed of a 31-amino acid peptide backbone connected to a fatty acid side chain. Its chemical formula is C 187 H 291 N 45 O 59 , molecular weight 4113.64. Semaglutide has a hypoglycemic effect and is a long-acting glucagon-like peptide-1 (GLP-1) analog. It shows good therapeutic effects on blood sugar control and other physiological effects in patients with type 2 diabetes. Its unique structure and mechanism of action make it an effective treatment option. In recent years, the main preparation methods of semaglutide are biosynthesis and solid phase synthesis. Among them, the biosynthesis method is easy to prepare semaglutide on a large scale, but it requires complex separation and purification to avoid impurities in the fermentation system affecting the biological activity and safety of polypeptide drugs.

[0003] At present, chromatography is mainly used to separate and purify semaglutide, including ion exchange chromatography, reversed-phase high-performance liquid chromatography, and chromatography. Reverse-phase high-performance liquid chromatography (RP-HPLC) is a commonly used chromatographic method, in which the stationary phase is an organic solvent with a polarity less than that of the solvent, such as an aqueous solution of methanol, isopropanol or acetonitrile, and the components in the mixture are separated due to their different distribution coefficients between the stationary phase and the mobile phase. For example, Chinese invention patent 202011629639.8 discloses a method for purifying high-purity semaglutide using reversed-phase high-performance liquid chromatography, and the resulting finished product has a high purity. Supercritical fluid chromatography (SFC) is a chromatographic method using a supercritical fluid as a mobile phase and a solid adsorbent (such as silica gel) or an organic high-molecular polymer bonded to a carrier (or capillary wall) as a stationary phase. Chinese invention patent 201811473970.8 discloses a method for purifying GLP-1-like polypeptides using supercritical chromatography, but due to a series of reasons such as poor stability of the supercritical fluid itself, harsh conditions of use, and high cost, it is difficult to apply on a large scale in industrial production. Column chromatography uses the difference in adsorption capacity of adsorbents for different components to separate the mixture. China Invention 202010825323.X announced a method of purifying semaglutide by combining column chromatography and membrane separation technology, which has a high yield. However, before chromatographic separation and purification, primary separation is required to remove a large number of impurities and improve the efficiency and selectivity of chromatographic separation.

[0004] Membrane separation technology is a modern separation technology used for material separation, concentration and purification. It uses the sieving effect of semipermeable membranes to separate, purify and concentrate substances in multiple components. Among them, microfiltration membranes with a pore size of 0.2 to 0.5 μm can effectively separate impurities such as particulate matter, cell debris, organelles, colloids and even pathogens in liquids from polypeptides at room temperature, while maintaining the biological activity of polypeptides. Microfiltration separation has the characteristics of low energy consumption, no pollution, high efficiency, sustainability and easy operation, which can greatly reduce the pressure of later chromatographic separation and purification. Therefore, microfiltration has good application prospects in the primary separation of pharmaceutical polypeptides. However, it is worth noting that there are strong hydrophobic regions in the peptide chain structure of semaglutide, which contains 11 hydrophobic amino acids, which makes semaglutide in the solution easy to aggregate and difficult to disperse. The aggregates formed are large in size and easily intercepted by microfiltration membranes. On the other hand, the commonly used materials for microfiltration membranes are mainly PVDF (polyvinylidene fluoride). Due to the strong hydrophobicity of PVDF itself, it is possible to produce hydrophobic adsorption on the highly hydrophobic semaglutide, resulting in poor microfiltration separation performance and reduced separation yield. In addition, the pH of the isoelectric point of semaglutide is between 5.2 and 5.4. The charge characteristics of the membrane material surface at certain pH will also cause the membrane material to electrostatically adsorb semaglutide carrying opposite charges, making it difficult to pass through the microfiltration membrane, resulting in a large loss of polypeptides.

[0005] It can be seen that reducing the loss of semaglutide in microfiltration separation and enhancing its permeability in the microfiltration separation process are of great significance for reducing the preparation cost of high-purity semaglutide and promoting the application of semaglutide in the pharmaceutical field. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for reducing the loss of semaglutide microfiltration separation in view of the shortcomings of the prior art.

[0007] In order to achieve the above object, the present invention discloses a method for reducing the loss of semaglutide microfiltration separation, comprising the following steps:

[0008] (1) adding an inorganic salt to a solution containing semaglutide to obtain a first solution;

[0009] (2) adjusting the pH of the first solution obtained in step (1) to obtain a second solution;

[0010] (3) The second solution obtained in step (2) is subjected to microfiltration treatment.

[0011] In step (1), the inorganic salt is any one of sodium chloride, sodium acetate, sodium sulfate and ammonium sulfate, preferably sodium chloride.

[0012] In step (1), the concentration of the inorganic salt in the first solution is 0.01-0.1 mol / L, preferably 0.01 mol / L.

[0013] In step (2), the pH of the first solution obtained in step (1) is adjusted to 2.0 to 8.0, preferably, the pH of the first solution is adjusted to 2.5 or 8.0.

[0014] In step (2), the pH of the first solution obtained in step (1) is adjusted by using hydrochloric acid or sodium hydroxide to adjust the pH of the first solution obtained in step (1).

[0015] In step (3), when performing the microfiltration, the microfiltration membrane used is a hollow fiber membrane, the membrane pore size of the microfiltration membrane is 0.2 to 0.45 μm, and the membrane material is polyvinylidene fluoride or polyether sulfone.

[0016] Beneficial effects:

[0017] The present invention provides a method for reducing the loss of semaglutide microfiltration separation. By adding low-concentration inorganic salts to an aqueous solution containing semaglutide and adjusting the pH, semaglutide with a strong hydrophobic region structure is promoted to disperse in water, so that the average particle size of semaglutide aggregates is reduced to below 100 nm, the permeability of semaglutide through a microfiltration membrane during microfiltration is greatly improved, and the adsorption amount of semaglutide by the membrane material is reduced. When semaglutide is separated by microfiltration, its loss is significantly reduced, and the application of efficient membrane separation technology in the separation and purification production of semaglutide is promoted, and its separation and purification cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 Schematic diagram of the peptide chain structure of semaglutide.

[0020] Figure 2 This is the particle size distribution diagram of semaglutide in the solution when the pH is 2.5 and no sodium chloride is added in Example 2.

[0021] Figure 3 This is the particle size distribution diagram of semaglutide in the solution when the pH is 2.5 and 0.01 mol / L sodium chloride is added in Example 2.

[0022] Figure 4 This is the particle size distribution diagram of semaglutide in the solution when the pH is 8.0 and no sodium chloride is added in Example 3.

[0023] Figure 5This is the particle size distribution diagram of semaglutide in the solution when the pH is 8.0 and 0.01 mol / L sodium chloride is added in Example 3. DETAILED DESCRIPTION

[0024] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0025] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0026] Figure 1 The diagram is a schematic diagram of the peptide chain structure of semaglutide. The amino acids enclosed in red boxes are hydrophobic amino acids. There are strong hydrophobic regions in the peptide chain structure of semaglutide, including 11 hydrophobic amino acids ( Figure 1 This causes the semaglutide in the solution to be easily aggregated and difficult to disperse. The aggregates formed are large in size and easily intercepted by the microfiltration membrane.

[0027] The average particle size of semaglutide described in the following examples refers to the particle size of semaglutide aggregates corresponding to the cumulative particle size distribution percentage of semaglutide aggregates in the sample reaching 50% (D 50 ), detected by laser particle size analyzer (PSA).

[0028] The microfiltration separation conditions described in the following examples are: 0.5 m 2 Hollow fiber microfiltration membrane was used as the separation medium. The operating pressure of microfiltration separation was 0.05 MPa and the feed flow rate was 29.26 L / h.

[0029] The quantitative detection method of semaglutide in the following examples: the concentration of semaglutide in the raw material solution is detected by high performance liquid chromatography external standard method (concentration detection range 0.01 ~ 0.5g / L), a series of semaglutide standard concentration samples are prepared with ultrapure water, and filtered through a CA (cellulose acetate membrane) filter with a pore size of 2.5μm before detection, and a standard concentration curve is drawn according to the chromatographic peak area and its corresponding semaglutide standard sample concentration, and then the semaglutide peak area in the sample to be tested is detected, and then the semaglutide concentration in the sample to be tested is converted according to the standard curve.

[0030] Example 1

[0031] (1) Sodium chloride was added to a 0.5 g / L semaglutide solution to adjust the sodium chloride concentrations to 0.01, 0.02, 0.05 and 0.1 mol / L, respectively. The pH of each solution was adjusted to 2.0, 2.5, 6.5, 7.0, 7.5 and 8.0, and the average particle size of semaglutide in the solution under different pH and sodium chloride concentration conditions was investigated. The results are shown in Table 1.

[0032] Table 1 Effect of sodium chloride concentration on the average particle size of semaglutide in solution at different pH

[0033]

[0034] As shown in Table 1, when the pH is 2.5 and the sodium chloride concentration is 0.01 mol / L, the average particle size of semaglutide in the solution is less than 70 nm; when the pH is 8.0 and the sodium chloride concentration is 0.01 mol / L, the average particle size of semaglutide in the solution is less than 60 nm, and the average particle size is reduced by more than 89% compared with the pH of 7.0 and no sodium chloride is added, which is beneficial to enhance the permeability of semaglutide during microfiltration.

[0035] (2) Sodium acetate was added to a 0.5 g / L semaglutide solution to adjust the sodium acetate concentrations to 0.01, 0.02, 0.05 and 0.1 mol / L, respectively. The pH of each solution was adjusted to 2.0, 2.5, 6.5, 7.0, 7.5 and 8.0, and the average particle size of semaglutide in the solution under different pH and sodium acetate concentration conditions was investigated. The results are shown in Table 2.

[0036] Table 2 Effect of sodium acetate concentration on the average particle size of semaglutide in solution at different pH

[0037]

[0038] As shown in Table 2, within the relevant pH range, the addition of sodium acetate cannot effectively promote the dispersion of semaglutide in the solution, and its average particle size is still close to or exceeds 200 nm, which is not conducive to its penetration through the microfiltration membrane.

[0039] (3) Sodium sulfate was added to a 0.5 g / L semaglutide solution to adjust the sodium sulfate concentrations to 0.01, 0.02, 0.05 and 0.1 mol / L, respectively. The pH of each solution was adjusted to 2.0, 2.5, 6.5, 7.0, 7.5 and 8.0, and the average particle size of semaglutide in the solution under different pH and sodium sulfate concentration conditions was investigated. The results are shown in Table 3.

[0040] Table 3 Effect of sodium sulfate concentration on the average particle size of semaglutide in solution at different pH

[0041]

[0042] As shown in Table 3, when the pH is 2.5 and the sodium sulfate concentration is 0.01 mol / L, the average particle size of semaglutide in the solution is about 140 nm, which is more than 75% lower than when the pH is 7.0 and no sodium sulfate is added, which is beneficial to enhance the permeability of semaglutide during microfiltration.

[0043] (4) Ammonium sulfate was added to a 0.5 g / L semaglutide solution to adjust the concentrations of ammonium sulfate to 0.01, 0.02, 0.05 and 0.1 mol / L, respectively. The pH of each solution was adjusted to 2.0, 2.5, 6.5, 7.0, 7.5 and 8.0, and the average particle size of semaglutide in the solution under different pH and ammonium sulfate concentration conditions was investigated. The results are shown in Table 4.

[0044] Table 4 Effect of ammonium sulfate concentration on the average particle size of semaglutide in solution at different pH

[0045]

[0046] As shown in Table 4, when the pH is 2.5 and the ammonium sulfate concentration is 0.1 mol / L, the average particle size of semaglutide in the solution is about 100 nm, which is more than 83% lower than when the pH is 7.0 and no ammonium sulfate is added, which is beneficial to enhance the permeability of semaglutide during microfiltration.

[0047] According to the above results, under certain pH conditions, adding certain low-concentration inorganic salts can promote the dispersion of semaglutide in aqueous solution, thereby significantly reducing the average particle size of semaglutide aggregates, which is beneficial to enhancing the microfiltration permeability of semaglutide and reducing the loss of semaglutide during the separation process. Among them, the best conditions are when the pH is 2.5 or 8.0, the inorganic salt is sodium chloride, and the sodium chloride concentration is 0.01 mol / L.

[0048] Example 2

[0049] PVDF (polyvinylidene fluoride) hollow fiber microfiltration membranes with pore sizes of 0.22 μm and 0.45 μm were used to separate the semaglutide solution, and the membrane area was 0.5 m 2 . 0.8 L of 0.5 g / L semaglutide aqueous solution was prepared, its pH was adjusted to 2.5, and sodium chloride was added to its concentration of 0.01 mol / L. The operating pressure during microfiltration was 0.05 MPa, and the feed flow rate was 29.26 L / h. The permeability and adsorption loss of semaglutide during microfiltration were investigated, and the microfiltration separation effect of the semaglutide aqueous solution without sodium chloride (other conditions were the same) was used as a control. The results are shown in Table 5.

[0050] Table 5 Effect of low concentration sodium chloride (0.01 mol / L) on PVDF membrane microfiltration separation of semaglutide

[0051]

[0052] The results showed that when the pH was 2.5 and no sodium chloride was added, the average particle size of positively charged semaglutide was about 500-600 nm. At this time, semaglutide could not pass through the positively charged PVDF microfiltration membrane. At this time, the particle size distribution of semaglutide was as follows: Figure 2 When the pH is 2.5 and 0.01 mol / L sodium chloride is added, the average particle size of semaglutide has dropped below 100 nm. At this time, about 20-33% of semaglutide passes through the PVDF microfiltration membrane, and the membrane separation flux is significantly improved, indicating that the precipitate on the membrane surface has been reduced and the permeability has been improved. At this time, the particle size distribution of semaglutide is as follows: Figure 3 However, the adsorption of semaglutide by the hydrophobic PVDF membrane material is still high, which is the reason for the low permeability and high loss of semaglutide in PVDF membrane microfiltration separation.

[0053] Example 3

[0054] Semaglutide solution was separated using a PES (polyethersulfone) hollow fiber microfiltration membrane with a pore size of 0.2 μm and a membrane area of ​​0.5 m 2 . 0.8 L of 0.5 g / L semaglutide aqueous solution was prepared, the pH was adjusted to 8.0, and sodium chloride was added to a concentration of 0.01 mol / L. The operating pressure during microfiltration was 0.05 MPa, and the feed flow rate was 29.26 L / h. The permeability and adsorption loss of semaglutide during microfiltration were investigated, and the microfiltration separation effect of the semaglutide aqueous solution without sodium chloride (other conditions were the same) was used as a control. The results are shown in Table 6.

[0055] Table 6 Effect of low concentration sodium chloride (0.01 mol / L) on the separation of semaglutide by PES membrane microfiltration

[0056]

[0057] The results showed that when the pH was 8.0 and no sodium chloride was added, the average particle size of semaglutide was about 300 nm. Negatively charged semaglutide could partially pass through the negatively charged PES microfiltration membrane. At this time, the particle size distribution of semaglutide was as follows: Figure 4 When the pH was 8.0 and 0.01 mol / L sodium chloride was added, the average particle size of semaglutide was below 100 nm, and 96% of semaglutide passed through the PES microfiltration membrane. The permeability increased by 58.9% compared with the case when 0.01 mol / L sodium chloride was not added. At the same time, the adsorption of semaglutide by the microfiltration membrane was reduced by 83%, which greatly reduced the microfiltration separation loss of semaglutide. At this time, the particle size distribution of semaglutide is as follows: Figure 5 shown.

[0058] Therefore, the optimal conditions for separating semaglutide by microfiltration membrane are as follows: the pH of the semaglutide solution is 8.0, the concentration of sodium chloride in the semaglutide solution is 0.01 mol / L, the microfiltration membrane is a PES membrane, and the membrane pore size is not less than 0.2 μm.

[0059] The present invention provides a thought and method for reducing the loss of semaglutide microfiltration separation. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for reducing the loss of semaglutide microfiltration separation, characterized in that: The following steps are involved: (1) adding an inorganic salt to a solution containing semaglutide to obtain a first solution; (2) adjusting the pH of the first solution obtained in step (1) to obtain a second solution; (3) The second solution obtained in step (2) is subjected to microfiltration treatment.

2. The method according to claim 1, characterized in that In step (1), the inorganic salt is any one of sodium chloride, sodium acetate, sodium sulfate and ammonium sulfate.

3. The method according to claim 1, characterized in that In step (1), the concentration of the inorganic salt in the first solution is 0.01-0.1 mol / L.

4. The method according to claim 1, characterized in that: In step (2), the pH of the first solution obtained in step (1) is adjusted as follows: the pH of the first solution obtained in step (1) is adjusted to 2.0 to 8.

0.

5. The method according to claim 1, characterized in that In step (2), the pH of the first solution obtained in step (1) is adjusted by using hydrochloric acid or sodium hydroxide to adjust the pH of the first solution obtained in step (1).

6. The method according to claim 1, characterized in that In step (3), when performing the microfiltration, the microfiltration membrane used is a hollow fiber membrane, the membrane pore size of the microfiltration membrane is 0.2 to 0.45 μm, and the membrane material is polyvinylidene fluoride or polyether sulfone.

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