A method for cation exchange chromatography analysis of GLP-1 class polypeptides

Abstract

The application relates to a cation exchange chromatography analysis method of GLP-1 polypeptides, and belongs to the technical field of polypeptide drug analysis. The method adopts a cation exchange chromatography column, uses a mobile phase system composed of an acidic phosphate buffer solution A phase, a mixed solution B phase of a weakly acidic or weakly alkaline phosphate buffer solution and a sodium chloride solution and a methanol C phase, carries out detection at a wavelength of 210 nm through a specific gradient elution procedure. In the method, the pH of the acidic phosphate buffer solution is 2.0-3.0, the pH of the weakly acidic or weakly alkaline phosphate buffer solution is 6.0-8.0, and the flow rate of the mobile phase is 0.5-1.5 mL / min. The application also provides a sample pretreatment method, that is, a phosphate buffer solution with a pH greater than 7.5 is used for sample dissolution. The method is suitable for detecting GLP-1 polypeptides such as semeglutide, liraglutide and the like and impurities thereof, has the advantages of high specificity, good separation effect, high repeatability and low detection limit, and can effectively solve the problem that there is no analysis method for detecting GLP-1 polypeptides and impurities by using different principles in the prior art.

Description

Technical Field

[0001] This invention relates to the field of peptide analysis methods, specifically to a cation exchange chromatography method for GLP-1 type peptides. Background Technology

[0002] Glucose hormone-like peptide-1 (GLP-1) peptides are an important class of hypoglycemic drugs with various physiological functions, including promoting insulin secretion, inhibiting glucagon release, delaying gastric emptying, and reducing appetite. They have significant application value in the treatment of diabetes. Currently marketed GLP-1 peptide drugs include semaglutide, liraglutide, exenatide, lixisenatide, abiglutide, dulaglutide, and telpoxetine (a dual GLP-1 / GIP target). With the widespread use of GLP-1 peptide drugs, the requirements for their quality control are becoming increasingly stringent, especially the detection and control of impurities.

[0003] In the quality control of peptide drugs, liquid chromatography (LC) is one of the commonly used analytical methods. Invention patent CN116183752B discloses a LC method for detecting peptide impurities. This method uses phosphate buffer as mobile phase A, a mixed solution of organic reagent and water as mobile phase B, and a C18 column as the stationary phase. It employs high-performance liquid chromatography gradient elution of peptide impurities, effectively detecting difficult-to-separate impurities within peptides. However, this method is primarily based on reversed-phase chromatography, and its separation effect is limited for some structurally similar peptide impurities.

[0004] Invention patent CN119104645B discloses a method for the simultaneous detection of six GLP-1RA drugs. This method uses reversed-phase high-performance liquid chromatography-triple quadrupole mass spectrometry to simultaneously determine six GLP-1RA drug components, including semaglutide, in a sample. A single injection analysis takes only 30 minutes to rapidly and effectively separate and detect the six components. However, this method requires mass spectrometry equipment, is costly, and is complex to operate.

[0005] Ion exchange chromatography is another commonly used method for peptide analysis. Patent CN115505035B discloses a purification method for the semaglutide intermediate peptide Arg34GLP-1(9-37), which completes the purification of the semaglutide intermediate peptide through a one-step cation exchange chromatography and a one-step reversed-phase chromatography. Patent application CN104936610A discloses a purification method for the (Aib8,35)-GLP-1(7-36)NH2 peptide, which consists of an ion exchange chromatography purification step and a reversed-phase high-performance liquid chromatography (HPLC) purification step, capable of preparing high-yield, high-purity GLP-1 analog peptides. However, these methods require coupling with reversed-phase chromatography, increasing the cost. Patent CN106290601B discloses a method for detecting racemic amino acids and diastereomer impurities in peptide drugs based on ion exchange chromatography, using two chromatographic columns in series.

[0006] Although there are various analytical methods available for the detection of GLP-1 peptides, the following problems still exist: the combination of ion exchange chromatography with mass spectrometry or reversed-phase chromatography, or the use of multiple chromatographic columns in series, leads to complicated operation and increased cost.

[0007] According to the requirements of drug quality control, especially in the detection of impurities in peptide products such as smegglutide, it is necessary to use two or more methods based on different principles to ensure the reliability and comprehensiveness of the test results. Therefore, developing an analytical method based on different separation principles that is simple to operate and low in cost, especially a cation exchange chromatography method, is of great significance for improving the quality control level of GLP-1 peptide drugs. Summary of the Invention

[0008] The technical problem this invention aims to solve is to address the lack of an analytical detection method for related substances in GLP-1 type peptides that is simple to operate and low in cost, based on different separation principles, particularly the lack of methods that employ two or more different principles for impurity detection in smegglutide products. This invention provides a cation exchange chromatography method for GLP-1 type peptides to meet the requirements for related substance analysis of peptides in the "Technical Guidelines for Pharmaceutical Research of Synthetic Peptide Drugs".

[0009] The technical solution adopted by this invention to solve its technical problem is as follows: A cation exchange chromatography method for the analysis of GLP-1 type peptides is provided, wherein the sample to be tested is a GLP-1 type peptide, and a cation exchange column is used; the mobile phase consists of: phase A being 1-50 mM acidic phosphate buffer, phase B being a mixture of 1-50 mM weakly acidic or weakly basic phosphate buffer and 50-500 mM sodium chloride solution, and phase C being methanol; the mobile phase flow rate is 0.5-1.5 mL / min; a gradient elution method is used, and the gradient elution conditions are as follows:

[0010]

[0011] Detection was performed at a wavelength of 210 nm using a DAD / PDA (photodiode array detector).

[0012] In this invention, the mobile phase system consists of mobile phases A / B / C. The specific selection of these phosphates can provide a stable pH buffer system, ensuring the effectiveness, reproducibility, and reliability of chromatographic separation.

[0013] In this invention, adding the organic solvent methanol to the mobile phase can suppress the hydrophobic interaction between the stationary phase and the linking groups, thereby reducing peak broadening or tailing. When the volume of methanol added is close to 50%, the effect on improving the peak shape is more obvious.

[0014] In one embodiment of the present invention, the GLP-1 class polypeptide is smegglutide, liraglutide, exenatide, lixisenatide, abiglutide, dulaglutide, or telpoglutide.

[0015] In one embodiment of the present invention, the pH of the acidic phosphate buffer is 2.0-3.0, and the pH of the weakly acidic or weakly basic phosphate buffer is 6.0-8.0.

[0016] In one embodiment of the present invention, the acidic phosphate is NaH2PO4 or KH2PO4, and the weakly acidic or weakly basic phosphate is Na2HPO4 or K2HPO4.

[0017] In one embodiment of the present invention, the mobile phase A is a 10 mM NaH2PO4 solution with pH 2.5; the mobile phase B is a mixed solution of 20 mM Na2HPO4 and 300 mM NaCl with pH 7.5.

[0018] In one embodiment of the present invention, the chromatographic column is an Aglient BioSCX NP5 PK.

[0019] As one embodiment of the present invention, the GLP-1 type peptide sample to be tested is first dissolved in an alkaline diluent before injection.

[0020] In one embodiment of the present invention, the alkaline diluent is a mixed solution of dihydrogen phosphate and sodium diphosphate with a pH > 7.5, wherein the salt is a sodium salt or a potassium salt.

[0021] As one embodiment of the present invention, the gradient elution conditions of the mobile phase are as follows:

[0022]

[0023] Compared with existing technologies, this invention has the following advantages: By employing ion exchange chromatography (IEC) as a second analytical method based on a different principle, it forms an orthogonal analytical approach with reversed-phase chromatography, enabling a more comprehensive characterization of related substances in peptides. The cation exchange chromatography method provided by this invention, through optimization of mobile phase composition, pH value and gradient elution conditions, column type, and sample pretreatment conditions, can effectively separate impurities in GLP-1 peptides, particularly for the detection of impurities in smegraglutide products, meeting the requirement in the "Technical Guidelines for Pharmaceutical Research of Synthetic Peptide Drugs" that the analysis of peptide-related substances must employ two or more methods based on different principles. This method is simple to operate, has good reproducibility, and low cost. It can accurately detect and quantify various impurities in GLP-1 peptides, including impurities generated during preparation, degradation products generated during storage, and other related substances. This is of great significance for the quality control and stability studies of GLP-1 peptide drugs. Attached Figure Description

[0024] Figure 1 This is the chromatogram of Example 1.

[0025] Figure 2 This is the chromatogram of Example 2 under gradient 1 conditions.

[0026] Figure 3 This is the chromatogram of Example 2 under gradient 2 conditions.

[0027] Figure 4 This is the chromatogram of Example 2 under gradient 3 conditions.

[0028] Figure 5 This is a comparison of the impurities and main peak purity of the company's self-produced semaglutide API and the original semaglutide injection / tablet in Example 7.

[0029] Figure 6 This is the chromatogram of Comparative Example 1.

[0030] Figure 7 This is the chromatogram of Comparative Example 2.

[0031] Figure 8This is the chromatogram of Comparative Example 3. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the experimental materials and instruments used in the embodiments of this invention can be commercially available (Novo Nordisk's reference formulations: semaglutide injection and semaglutide tablets). The semaglutide sample prepared by our company can be prepared by polypeptide synthesis or recombinant biology, as detailed in the published invention patent application CN202411926737.6.

[0034] Example 1

[0035] This invention relates to a cation exchange chromatography method for the analysis of GLP-1 type peptides. In this method, a cation exchange chromatography column is used to analyze GLP-1 type peptide samples.

[0036] The specific steps of the analysis method are as follows:

[0037] Step 1: Prepare the mobile phase.

[0038] Mobile phase A was a 10 mM NaH2PO4 buffer solution (pH 2.5), mobile phase B was a mixture of a 20 mM Na2HPO4 buffer solution (pH 7.5) and a 300 mM sodium chloride solution, and mobile phase C was chromatographically pure methanol.

[0039] The mobile phase must be filtered through a 0.22μm filter membrane and degassed in an ultrasonic cleaner for 15 minutes before use.

[0040] Step 2: Preparation of the chromatographic system.

[0041] A high-performance liquid chromatograph equipped with a DAD / PDA detector was used. The chromatographic column selected was an Aglient BioSCX NP5 PK column with strong cation exchange. The column temperature was set to 40℃ and the mobile phase flow rate was set to 1.0 mL / min.

[0042] Step 3: Sample preparation.

[0043] Take an appropriate amount of smegglutide sample (smegglutide raw material prepared by Hanxin Company), dilute it to a concentration of 1.0 mg / mL with an alkaline diluent composed of potassium dihydrogen phosphate and disodium hydrogen phosphate (pH 7.8), mix well, and filter through a 0.22 μm filter membrane.

[0044] Step 4: Chromatographic analysis.

[0045] The analysis was performed using a gradient elution method, with the following gradient elution conditions:

[0046]

[0047] Specifically, from 0 to 2 minutes, mobile phase A is 55%, mobile phase B is 0%, and mobile phase C is 45%; at 30 minutes, mobile phase A is 30%, mobile phase B is 25%, and mobile phase C is 45%; from 31 to 41 minutes, mobile phase A is 5%, mobile phase B is 50%, and mobile phase C is 45%; and from 42 to 52 minutes, mobile phase A is 55%, mobile phase B is 0%, and mobile phase C is 45%.

[0048] Step 5: Testing.

[0049] Detection was performed using a DAD / PDA detector at a wavelength of 210 nm, and chromatograms were recorded. A typical chromatogram is shown below. Figure 1 As shown, the content of smegglutinin peptide and related impurities was calculated using the peak area normalization method.

[0050] Example 2

[0051] The separation of smegglutide raw material sample prepared by Hanxin Company after the addition of impurity Z14 was investigated under three different elution gradients (gradients 1-3, where gradient 1 is the same as the gradient in Example 1). All other chromatographic conditions were the same as in Example 1. The results are shown in Tables 1 and 2. Figure 2-4 As shown. Under gradient 1 and gradient 2 conditions, the separation degree between the main peak and the preceding and following peaks of both gradients is greater than 1.0, and the tailing factor is less than 1.8. Among them, gradient 1 has a stronger ability to detect impurities, so gradient 1 condition is better.

[0052] Since gradient 3 cannot be effectively separated from the added impurity Z14, with a separation degree of less than 1.0, the tailing factor cannot be calculated, therefore the conditions for gradient 3 are not applicable.

[0053] Gradient 2

[0054]

[0055] Gradient 3

[0056]

[0057] Table 1. Detection results for different elution gradients

[0058]

[0059] Example 3

[0060] The resolution of the self-made smegglutide raw material sample from Hanxin Company under different pH elution mobile phase B conditions was investigated. All other chromatographic conditions were the same as in Example 1. The results are shown in Table 2 below:

[0061] Table 2 Results under different pH elution mobile phase B conditions

[0062]

[0063] As shown in the table above, the chromatographic results are comparable when mobile phase B is at pH 6.5 or 7.5, achieving the technical effects of this invention in both cases. When mobile phase B is at pH 7.5, the resolution with subsequent peaks is higher, and the tailing factor is smaller. However, the resolution with preceding peaks is not meaningful due to the absence of significant impurities at the peak tip. Since the chromatographic purity is comparable under both pH conditions, pH 7.5 is relatively superior.

[0064] Example 4

[0065] The main peak characteristics of the test sample (similar to simecrolimide prepared by Hanxin Company) after dissolution with diluents under different pH conditions were examined. All other chromatographic conditions were the same as in Example 1. The results are shown in Table 3 below:

[0066] Table 3. Main peak results after dissolution with different pH dilution solutions

[0067] 2.5 19.726 547.899 3.72 7.8 19.768 1251.747 1.15

[0068] As can be seen from the table above, the main peak shape is better under alkaline conditions. Therefore, it is better to dissolve and dilute the test sample in a diluent with pH 7.8 before testing.

[0069] Example 5

[0070] The effect of mobile phase A / B buffer concentration on the results was investigated. For different test samples (liraglutide, exenatide, lixisenatide, abiglutide, dulaglutide, or telpopotide, etc.), the buffer concentration can be adjusted adaptively. All other chromatographic conditions are the same as in Example 1.

[0071] Mobile phase A is a 1 mM NaH₂PO₄ buffer solution (pH 2.0), and mobile phase B is a mixture of a 1 mM Na₂HPO₄ buffer solution (pH 6.0) and a 50 mM sodium chloride solution. This mobile phase formulation offers higher sensitivity for detecting acidic impurities in GLP-1 peptides or is suitable for analyzing pH-sensitive GLP-1 peptide samples.

[0072] Mobile phase A is a 50 mM NaH₂PO₄ buffer (pH 3.0), and mobile phase B is a mixture of a 50 mM Na₂HPO₄ buffer (pH 8.0) and a 500 mM sodium chloride solution. This mobile phase formulation is suitable for analyzing high concentrations of GLP-1 peptide samples.

[0073] Example 6

[0074] The analytical method of Example 1 was validated (including specificity, repeatability, linearity, limit of detection (DL), and limit of quantitation (QL).

[0075] The verification criteria for specificity are shown in Table 4 below:

[0076] Table 4 Specificity Verification

[0077]

[0078] The results showed that the peak of smegglutinin in the chromatogram of the test sample solution after high-temperature degradation was pure and met the acceptable criteria. Therefore, this method is specific.

[0079] The analytical method meets the repeatability requirements: among the repeatability test results of the 6 test samples, the RSD% of impurities higher than 0.10% is ≤15%; the difference in impurity content lower than 0.10% is within 0.03%; and the RSD% of the total impurities is ≤8%, which meets the acceptable standard.

[0080] This analytical method exhibits good linearity; the correlation coefficients of the regression lines obtained for semaglutide concentrations ranging from 0.462 μg / mL to 46.20 μg / mL (equivalent to 0.05%–5% of the detection concentration) meet the NLT standard of 0.990. Furthermore, the Y-intercept is within 4% of the 0.5% response value, and the RSD% of the response factor is 5%. Therefore, this method is linear.

[0081] The limit of quantitation for this method is 0.924 μg / mL, equivalent to 0.10% of the sample concentration; the limit of detection for this method is 0.462 μg / mL, equivalent to 0.05% of the sample concentration.

[0082] Method validation results show that this method can effectively detect impurities in GLP-1 peptides, including deamidation impurities, oxidative impurities, truncated fragments, and polymers, with a detection limit of 0.05%. Compared with traditional reversed-phase chromatography, this method has higher selectivity and resolution for charge-changing impurities in GLP-1 peptides, making it particularly suitable for quality control analysis of GLP-1 peptide drugs.

[0083] Example 7

[0084] Using the ion chromatography method described in Example 1, appropriate amounts of semaglutide samples (semaglutide raw material manufactured by Hanxin Company, catalog number HX 01-05; semaglutide injection from Novo Nordisk, catalog number RLD 01-03; and semaglutide tablets from Novo Nordisk, catalog number RLD 04-12 (3mg specification number RLD 04-06, 7mg specification number RLD 07-09, and 14mg specification number RLD 10-12), totaling 17 samples, were diluted to a concentration of 1.0 mg / mL with a pH 7.8 diluent composed of potassium dihydrogen phosphate and disodium hydrogen phosphate, and filtered through a 0.22 μm filter membrane. Detection was performed using a DAD / PDA detector at a wavelength of 210 nm, and chromatograms were recorded. The content of semaglutide peptide and related impurities was calculated using the peak area normalization method.

[0085] The results are shown in Table 5 and Figure 5 As shown, in the ion chromatography results, the highest purity of the original semaglutide injection was 99.35%, and the highest purity of the original semaglutide tablets was 98.87%. The purity of our self-produced semaglutide API is not less than 99.95%, and no impurities exceeding the detection limit (DL) were detected, indicating that the impurity residue in our prepared semaglutide API is superior to that of the original.

[0086] Table 5. Comparison of impurities and main peak purity between the company's self-produced simegromide API and the original simegromide injection / tablets.

[0087]

[0088] Comparative Example 1

[0089] In mobile phase B, sodium perchlorate was used instead of sodium chloride in Example 1 to destroy the self-made smegglutinin sample at 80°C. All other chromatographic conditions were the same as in Example 1. The chromatographic results are as follows: Figure 6 As shown, no obvious main peak was found, indicating that the elution capacity of sodium perchlorate is insufficient, and that this method is not applicable.

[0090] Comparative Example 2

[0091] In mobile phase B, sodium acetate was used instead of sodium chloride in Example 1 to destroy the smegglutinin sample prepared by Hanxin at a high temperature of 80°C. All other chromatographic conditions were the same as in Example 1. The chromatographic results are as follows: Figure 7 As shown, peak purity = 100%, the integrated small peak is the baseline perturbation peak, not an impurity peak, impurity number = 0, and main peak tailing factor = 1.2. Sodium acetate, due to its large self-absorption in the low-band, causes baseline jitter and impurity masking during gradient elution, making it impossible to effectively identify relevant impurities, indicating that this method is not applicable.

[0092] Comparative Example 3

[0093] In mobile phase B, sodium phosphate was used instead of sodium chloride in Example 1 to destroy the smegglutinin sample prepared by Hanxin at a high temperature of 80°C. All other chromatographic conditions were the same as in Example 1. The chromatographic results are as follows: Figure 8 As shown, the elution power is too strong, and the main peak is eluted directly, indicating that this method is not applicable.

[0094] Comparative Example 4

[0095] The chromatographic column Waters Spherisorb SCX was used instead of the column in Example 1 to destroy the self-made smegglutinin sample at 80°C. All other chromatographic conditions were the same as in Example 1. Although the chromatographic purity was comparable, it could not be detected when the concentration of the test sample was diluted to 0.1%.

Claims

1. A cation exchange chromatography method for the analysis of GLP-1 type peptides, characterized in that: The sample to be tested was a GLP-1 type peptide, specifically smegglutinin. A cation exchange column, specifically an AglientBioSCX NP5 PK, was used. The mobile phase consisted of: Phase A, 10 mM NaH₂PO₄ solution, pH 2.5; Phase B, a mixture of 20 mM Na₂HPO₄ and 300 mM NaCl, pH 7.5; and Phase C, methanol. The flow rate was 0.5–1.5 mL / min. Gradient elution was employed under the following conditions: Detection was performed using a DAD / PDA detector at a wavelength of 210 nm.

2. The cation exchange chromatography method for GLP-1 type peptides according to claim 1, characterized in that: Before injecting the GLP-1 type peptide sample, it should be dissolved in an alkaline diluent.

3. The cation exchange chromatography method for GLP-1 type peptides according to claim 2, characterized in that: The alkaline diluent is a mixed solution of dihydrogen phosphate and sodium diphosphate with a pH > 7.5, wherein the salt is a sodium salt or a potassium salt.

4. The cation exchange chromatography method for GLP-1 type peptides according to claim 1, characterized in that: The gradient elution conditions are as follows:

5. The application of the cation exchange chromatography method for GLP-1 type peptides according to any one of claims 1-4 in the detection of impurities in GLP-1 type peptides.

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