Smeglutide polymer content detection method
By using a G2000 SWxl column and specific mobile phase conditions, the problem of insufficient separation of smegglutinin polymer was solved, ensuring the accuracy of the detection results and the stability of the column, thus achieving efficient polymer content detection.
Patent Information
- Application Number
- CN202511519417.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-23
- Publication Date
- 2026-01-27
AI Technical Summary
In the existing technology, the polymer of smegglutinin has a resolution of less than 1.5 with the main peak, and the ion pair reagents in the mobile phase affect the retention performance of the chromatographic column, resulting in inaccurate detection results.
A G2000 SWxl column was used with isocratic elution conditions. The mobile phase consisted of a mixture of sodium chloride phosphate solution and isopropanol. The detection wavelength was 280 nm, the flow rate was 0.28–0.32 mL/min, the column temperature was 26–30 °C, the injection volume was 20 ± 0.1 μL, and the injection plate temperature was controlled at 2 °C–8 °C. The mobile phase was a 50:50 mixture of sodium chloride phosphate solution and isopropanol.
The separation degree between the polymer and the main peak was greater than 1.5, the column retention performance was not affected, the detection results were accurate and reliable, and met the requirements of limit of quantitation, linearity, repeatability and stability.
Smart Images
Figure CN121410172A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of peptide detection technology, specifically to a method for detecting the content of smegglutinin polymers. Background Technology
[0002] Smegglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist that lowers blood sugar by mediating GLP-1 receptors in a glucose-dependent manner, promoting insulin secretion and inhibiting glucagon secretion. It is commonly used to treat type 2 diabetes in adults. Smegglutide can be used in diabetic patients whose blood sugar is not controlled with metformin and / or sulfonylureas in addition to diet and exercise. It is suitable for reducing the risk of major adverse cardiovascular events (cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke) in adult patients with type 2 diabetes and cardiovascular disease. Currently, the marketed formulation is mainly an injectable solution, and the original developer is Novo Nordisk.
[0003] During the synthesis and storage of smegraglutide, a series of polymeric impurities of varying molecular weights inevitably arise due to the aggregation of molecules through non-covalent or covalent interactions. Existing technology discloses a liquid chromatography detection method for smegraglutide using an Agilent C8 column. Mobile phase A is triethylamine phosphate, and mobile phase B is an aqueous acetonitrile solution. Triethylamine phosphate is a common ion-pairing reagent. Ion-pairing reagents adsorb onto the surface of the stationary phase, altering its hydrophobicity or charge properties, thus changing the column's retention capacity for the target analyte. This effect may be long-term; even with changes in the mobile phase, residual ion-pairing reagents may still cause the column retention behavior to deviate from its initial state. Furthermore, the resolution between the main peak and the polymer in this detection method does not reach 1.5. Summary of the Invention
[0004] The purpose of this invention is to provide a method for detecting the content of smegglutide polymers, which can increase the resolution between the main peak and the polymer to more than 1.5, without affecting the retention performance of the chromatographic column.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for detecting the content of semaglutide polymers, using liquid chromatography to analyze the semaglutide polymers: Column: G2000 SWxl; Elution conditions: isocratic elution for 40 ± 5 min; The detection wavelength is 280nm; The flow rate was 0.28~0.32 mL / min; Column temperature 26~30℃; The injection volume is 20±0.1μL, and the injection plate temperature is controlled. The mobile phase is a mixture of sodium chloride phosphate solution and isopropanol in a volume ratio of 50:50.
[0006] Preferably, the chromatographic column has the following specifications: inner diameter 7.8 mm, column length 300 mm, and average particle size of packing material 5 μm.
[0007] Preferably, the flow rate is 0.3 mL / min.
[0008] Preferably, the column temperature is 28°C.
[0009] Preferably, the injection volume is 20 μL.
[0010] Preferably, the temperature of the sample inlet plate is controlled between 2°C and 8°C.
[0011] Preferably, the concentration of sodium chloride in the sodium chloride phosphate solution is 0.5 ± 0.005 mol / L.
[0012] Preferably, the preparation process of the mobile phase is as follows: weigh 29.25g of sodium chloride and 1.38g of sodium dihydrogen phosphate, add 500mL of purified water and sonicate to dissolve, adjust the pH with phosphoric acid, filter, and then add 500mL of isopropanol and mix well to obtain the mobile phase.
[0013] Preferably, the pH of the sodium chloride phosphate solution is 2.9 to 3.1.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The mobile phase used in existing technologies can alter the retention performance of the chromatographic column, while the mobile phase selected in this invention will not affect the retention performance of the chromatographic column; the resolution between polymers and the main peak in existing technologies is not high, while the resolution between polymers and the main peak in this invention is greater than 1.5; existing technologies use gradient elution, while this invention uses isocratic elution, which has lower requirements for instruments.
[0015] The method specificity (resolution between polymer and main component not less than 1.5, peak purity of main peak not less than 990), limit of quantitation, linearity and range, precision, solution stability and robustness of the present invention all meet the acceptable standards, indicating that accurate and reliable experimental results can be obtained by determining the polymer in smegglutinin using this method. Attached Figure Description
[0016] Figure 1 This is a blank solvent chromatogram of Example 1 of the present invention.
[0017] Figure 2 This is a chromatogram of a solution demonstrating the system suitability of Example 1 of the present invention.
[0018] Figure 3This is a chromatogram of the polymer-containing smegglutinin sample of Comparative Example 1 of the present invention. Detailed Implementation
[0019] Example 1 Diluent: Weigh 29.25g sodium chloride and 1.38g sodium dihydrogen phosphate, add 500mL purified water and sonicate to dissolve. Adjust the pH to 3.0 with phosphoric acid, filter, and then add 500mL isopropanol and mix well to obtain the diluent.
[0020] System suitability solution: Weigh approximately 20 mg of the smegglutinin high-temperature degraded sample, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.
[0021] Column: Tosoh G2000 SWxl (7.8mm×300mm, 5μm); Flow rate: 0.3 mL / min; Detection wavelength: 280nm (ultraviolet detector); Column temperature: 28℃; Injection volume: 20 μL; Sample inlet temperature: 6℃ Mobile phase: 0.5 mol / L sodium chloride phosphate solution and isopropanol are mixed at a volume ratio of 50:50 to form a solution with pH=3.0. The preparation process is as follows: Weigh 29.25 g of sodium chloride and 1.38 g of sodium dihydrogen phosphate, add 500 mL of purified water and sonicate to dissolve. Adjust the pH to 3.0 with phosphoric acid, filter, and then add 500 mL of isopropanol and mix well to obtain the final product.
[0022] Isocratic elution run time: 40 min.
[0023] Chromatograms of blank and system suitability solutions are shown below. Figure 1 He Ru Figure 2 The results showed that there was no interference from the blank, and the separation degree between the main peak and the polymer reached more than 1.5, achieving baseline separation and meeting the requirements for quantitative detection. The purity of the main peak of smegglutinin was 997, which is greater than 990 (see Table 1), and it meets the standard.
[0024] Table 1: Peak purity of Example 1 of the present invention
[0025] Comparative Example 1 Chromatographic column: Agilent C8 column, 4.6 mm × 150 mm, 3.5 μm; Flow rate: 0.7 mL / min; Detection wavelength: 220nm; Column temperature: 30℃; Injection volume: 20 μL; Mobile phase A: 50 mmol / L triethylamine phosphate (pH 4.5); Mobile phase B: acetonitrile:water = 8:2; Running time: 46 minutes.
[0026] Elution conditions: as shown in Table 2 below Table 2: Gradient elution
[0027] like Figure 3 As shown in Table 3, the separation degree between the polymer and smegglutinin was 1.3, which is less than 1.5.
[0028] Table 3: Liquid Chromatography Detection Results of Comparative Example 1
[0029] Comparative Example 2 Diluent: Mobile phase.
[0030] System suitability solution: Weigh approximately 20 mg of the smegglutinin high-temperature degraded sample, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.
[0031] Column: Tosoh G2000 SWxl (7.8mm×300mm, 5μm); Flow rate: 0.7 mL / min; Detection wavelength: 280nm (ultraviolet detector); Column temperature: 30℃; Injection volume: 40 μL; Sample inlet temperature: 6℃ Mobile phase: 0.7% TFA acetonitrile: water = 25:75 (v / v) Isocratic elution run time: 30 min.
[0032] Under the above liquid chromatography conditions, compared with the results of Example 1, the peak purity of the main peak of smegglutinin was 964, which is less than 990 (see Table 4), and does not meet the standard.
[0033] Table 4: Peak purity of Comparative Example 2 of the present invention
[0034] Example 1 Methodological Validation I. Exclusivity Reference solution: Weigh about 20 mg of smegglutinin reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.
[0035] Test solution: Weigh about 20 mg of smegglutinin test sample, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.
[0036] Specific solution: Weigh about 20 mg of the sample of semaglutide that has been destroyed by high temperature, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well.
[0037] Table 5: Specificity Validation Results
[0038] The validation results showed that the separation degree between the polymer and smegglutinin was greater than 1.5, which met the acceptable standard, indicating that the method has good specificity.
[0039] II. Limit of Quantification The reference solution is serially diluted until the signal-to-noise ratio (S / N) is ≥10, which is the limit of quantitation solution.
[0040] Table 6: Results of Limit of Quantitation Validation
[0041] The validation results showed that the RSD (n=6) of the main peak area in the chromatograms of the 6 consecutive injection limit of quantitation solutions was 5.0% (<10%), and the S / N was greater than 10, which met the acceptable standard.
[0042] III. Linearity and Range Smegglutide reference standard stock solution: Weigh about 200 mg of smegglutide reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well.
[0043] Linear solution: Accurately transfer an appropriate amount of semaglutide reference standard stock solution and dilute quantitatively according to the table below to prepare a linear solution.
[0044] Table 7: Preparation of Linear Solutions
[0045] Table 8: Linearity Validation Results
[0046] The validation results showed that the content of smegglutide was in the range of 0.0017 mg / mL to 2.2504 mg / mL. The linear regression equation was y = 98.4222x - 0.5558, the correlation coefficient r was 0.9999 (>0.998), the ratio of the Y-axis intercept to the 100% concentration point response value (peak area) was 0.3% (<2%), and the sum of squared residuals was 3.90778, which met the acceptable standard. The method showed good linearity.
[0047] IV. Repeatability Reproducible solution: Accurately weigh approximately 20 mg of the smegglutinin high-temperature destroyed sample, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well. Prepare 6 parallel solutions.
[0048] Table 9: Repeatability Validation Results
[0049] The validation results showed that the RSD (n=6) of the polymer content in the reproducible solution was 3.8% (<10%). All validation results met the acceptable criteria, indicating good reproducibility of the method.
[0050] V. Solution Stability System suitability solution: Weigh approximately 20 mg of the smegglutinin high-temperature degraded sample, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.
[0051] Reference solution: Weigh about 20 mg of smegglutinin reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.
[0052] Take the reference solution and the system suitability solution and let them stand at 2-8℃ for 48 hours.
[0053] Table 10: Results of Solution Stability Verification
[0054] The results showed that the peak area of the main peak in the working reference solution chromatogram at each time point within 48 hours was less than 2.0% compared with the peak area at the initial time point; the polymer content detection results in the system suitability solution at each time point within 48 hours were less than 20% compared with the results at the initial time point.
[0055] The verification results all met the acceptable standards, and the working control solution and system suitability solution showed good stability within 48 hours of storage at 2-8℃.
[0056] VI. Durability Fine-tune the chromatographic conditions according to the table below (adjust one condition at a time), inject one blank solution and one system suitability solution.
[0057] Table 11: Durability Parameter Adjustment Range
[0058] Table 12: Durability Verification Results
[0059] The validation results showed that after fine-tuning the chromatographic conditions, the separation results of the polymer and smegglutinin were both greater than 1.5, which met the requirements; the method has good robustness.
[0060] The foregoing has shown and described the basic principles, main features and advantages of this invention. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of this invention. Various changes and modifications can be made to this invention without departing from the spirit and scope of this invention. All such changes and modifications fall within the scope of this invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.
Claims
1. A method for detecting the content of smegglutinin polymer, characterized in that, The smegglutinin polymer was analyzed by liquid chromatography: Column: G2000 SWxl; Elution conditions: isocratic elution for 40 ± 5 min; The detection wavelength is 280nm; The flow rate was 0.28~0.32 mL / min; Column temperature 26~30℃; The injection volume is 20±0.1μL, and the injection plate temperature is controlled. The mobile phase is a mixture of sodium chloride phosphate solution and isopropanol in a volume ratio of 50:
50.
2. The method for detecting the content of smegglutinin polymer according to claim 1, characterized in that: The specifications of the chromatographic column are: inner diameter 7.8 mm, column length 300 mm, and average particle size of packing material 5 μm.
3. The method for detecting the content of smegglutinin polymer according to claim 1, characterized in that: The flow rate is 0.3 mL / min.
4. The method for detecting the content of smegglutinin polymer according to claim 1, characterized in that: The column temperature is 28°C.
5. The method for detecting the content of smegglutinin polymer according to claim 1, characterized in that: The injection volume is 20 μL.
6. The method for detecting the content of smegglutinin polymers according to claim 1, characterized in that: The temperature of the sample inlet plate is controlled between 2℃ and 8℃.
7. The method for detecting the content of smegglutinin polymer according to claim 1, characterized in that: The concentration of sodium chloride in the sodium chloride phosphate solution is 0.5 ± 0.005 mol / L.
8. The method for detecting the content of smegglutinin polymers according to claim 7, characterized in that: The preparation process of the mobile phase is as follows: Weigh 29.25g of sodium chloride and 1.38g of sodium dihydrogen phosphate, add 500mL of purified water and sonicate to dissolve, adjust the pH with phosphoric acid, filter, and then add 500mL of isopropanol and mix well to obtain the mobile phase.
9. The method for detecting the content of smegglutinin polymer according to claim 7, characterized in that: The pH of the sodium chloride phosphate solution is 2.9 to 3.1.