Size exclusion chromatography method for polymer analysis in GHRH analogues

Abstract

The present application relates to a size exclusion chromatography method for polymer analysis in GHRH analogues, belonging to the technical field of biological medicine analysis. The present application develops a size exclusion chromatography method for polymer analysis in GHRH analogues, the sample to be measured is GHRH analogue, and an Agilent AdvanceBio SEC column is used; the mobile phase is a mixed solution of water, acetonitrile and trifluoroacetic acid, and isocratic elution is carried out; the method is especially suitable for tesamorelin or semorelin. The exclusive, efficient, accurate and stable size exclusion chromatography method developed by the present application is used for the analysis of polymer impurities in tesamorelin or semorelin, solves the technical problems of poor separation effect and insufficient detection sensitivity in the prior art, perfects the quality control system of GHRH analogues, comprehensively guarantees the safety and stability of the drug, and solves the technical problems urgently needed to be solved by the technical personnel in the field.

Description

Technical Field

[0001] This invention relates to the field of biomedical analytical technology, and more specifically to a size exclusion chromatography method for polymer analysis in GHRH analogs. Background Technology

[0002] Growth hormone-releasing hormone (GHRH) is a polypeptide hormone secreted by the hypothalamus that specifically stimulates the pituitary gland to secrete growth hormone, regulating various physiological processes such as growth, development, and metabolism. GHRH analogs are polypeptide drugs obtained by modifying the amino acid sequence of natural GHRH. Among them, temorelin and sermorelin are two important ones currently used and researched in clinical practice, each with unique pharmacological properties and therapeutic advantages. Temorelin, as a novel growth hormone-releasing factor analog, can restore normal growth hormone secretion in the body, reduce visceral adipose tissue, and improve dyslipidemia. It has been approved for the treatment of HIV-related lipid metabolism disorder. Sermorelin, with its long-acting growth hormone-promoting activity, shows promising application prospects in the treatment of growth hormone deficiency and anti-aging research.

[0003] GHRH analogs are mostly prepared through chemical synthesis or genetic recombination. During synthesis, purification, storage, and transportation, they are highly susceptible to polymerization reactions due to reaction conditions, impurities, and environmental factors, forming dimers, tetramers, and polymer impurities with higher molecular weights. The formation of these polymer impurities not only reduces the content of the active ingredient and affects the drug's pharmacological activity, but may also trigger immune responses, leading to adverse reactions such as allergies and inflammation, and even increasing the burden on organs, posing a serious threat to drug safety. As studies on impurities in peptide drugs have shown, polymers, as one of the common types of impurities in peptide drugs, are crucial for ensuring drug safety and efficacy when their content is controlled.

[0004] Drug quality control is a core requirement in the pharmaceutical field, and the accurate detection and quantification of impurities is a crucial component of drug quality standards. For peptide drugs, the analysis of polymeric impurities is both a key focus and a challenge in quality control. Currently, analytical methods for polymeric impurities in peptide drugs mainly include gel filtration chromatography, reversed-phase high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry. Among these, size exclusion chromatography (SEC), a chromatographic technique based on molecular size differences, offers advantages such as a simple separation mechanism, convenient operation, and minimal sample damage. It has been widely used in biopharmaceuticals for the detection of monoclonal antibody polymers, peptide polymers, and macromolecular impurities. Through the molecular sieving effect of porous packing material, components of different molecular weights elute sequentially according to their volume. High-molecular-weight polymeric impurities cannot enter the packing pores and are eluted first, while low-molecular-weight target drug molecules can enter the packing pores and elute subsequently, achieving effective separation of polymeric impurities from the target drug.

[0005] However, current technologies do not disclose analytical methods for detecting polymers in GHRH analogs such as temorepine and sermorepine. The disclosed methods all involve purification of temorepine or sermorepine using C18 reversed-phase chromatography. For example, prior art CN110818790A discloses purification using high-performance liquid chromatography (HPLC), with a 10 μm reversed-phase C18 column as the chromatographic packing material. Two mobile phase systems are used alternately: the first is 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, and the second is 50 mmol ammonium acetate / water solution-acetonitrile, with a flow rate of 90 mL / min. Gradient elution is used, with cyclic injection for purification. The crude solution is loaded onto the column, and elution is initiated. Prior art CN104177491B discloses purification conditions using a 0.2% TFA / acetonitrile mobile phase and a reversed-phase C18 column.

[0006] Existing methods have several shortcomings: Firstly, most existing methods are general-purpose detection methods and are not specifically optimized for the molecular structural characteristics of GHRH analogs (such as the 3-hexenoyl modification of temorelin), resulting in poor chromatographic separation. Polymer impurities overlap significantly with the target peak (main peak) and other impurity peaks, making accurate separation and quantification impossible. Secondly, the chromatographic conditions of existing methods (including column selection, mobile phase composition, elution program, and detection conditions) are poorly designed, resulting in problems such as low resolution, asymmetrical peak shape, insufficient detection sensitivity, and excessively long analysis cycles. These issues make it difficult to accurately determine the content of trace polymer impurities in GHRH analogs and fail to meet the stringent requirements of drug quality control.

[0007] Furthermore, current research on quality standards for GHRH analogs lacks a unified and specific method for analyzing polymeric impurities, particularly for polymers in temorepinephrine and sermorepinephrine. The absence of targeted size exclusion chromatography (SLC) detection schemes hinders effective quality control during the production process, failing to fully guarantee the safety and stability of these drugs. Therefore, developing a specific, efficient, accurate, and stable SLC method for analyzing polymeric impurities in temorepinephrine and sermorepinephrine, addressing the technical challenges of poor separation and insufficient detection sensitivity in existing technologies, and improving the quality control system for GHRH analogs has become a pressing technical issue for those skilled in the art. Summary of the Invention

[0008] The technical solution adopted by this invention to solve the technical problems existing in the prior art is as follows:

[0009] A size exclusion chromatography method for polymer analysis in GHRH analogs, wherein the sample to be analyzed is a GHRH analog, an Agilent AdvanceBio SEC size exclusion column is used, and the mobile phase is a mixed solution of water, acetonitrile and trifluoroacetic acid, with isocratic elution.

[0010] In one embodiment of the present invention, the GHRH analogue is temorepine or sermorepine.

[0011] In one embodiment of the present invention, the injection volume of the sample to be tested is 10~100 μl. In another embodiment, the injection volume of the sample to be tested is 10~50 μl, further optionally 10~25 μl or optionally 25~50 μl. In a specific embodiment, the injection volume of the sample to be tested is 25 μl. When the injection volume is >25 μl, the signal-to-noise ratio of the polymer peak increases, but the separation between the main peak and the polymer peak gradually decreases; when the injection volume is 10 μl, the separation between the main peak and the polymer peak is >2.0, but the signal-to-noise ratio of the polymer peak is relatively low.

[0012] In one embodiment of the present invention, the volume ratio of water to acetonitrile in the mobile phase is 2:3 to 4:1, and the volume ratio can be further set to 2:3 to 3:2. When the volume ratio of water to acetonitrile in the mobile phase is less than 2:3, neither the main peak nor the polymer peak emerges within a 30-minute elution time. When the volume ratio of water to acetonitrile in the mobile phase is within the range of 2:3 to 3:2, both the separation degree between the main peak and the polymer peak, and the signal-to-noise ratio (SNR) of the polymer peak, increase with the increase in the volume percentage of water in the mobile phase. When the volume ratio of water to acetonitrile in the mobile phase is 4:1, compared to the 3:2 volume ratio, the separation degree between the main peak and the polymer peak remains unchanged, but the SNR of the polymer peak decreases. When the volume ratio of water to acetonitrile in the mobile phase is 3:2, both the separation degree between the main peak and the polymer peak, and the SNR of the polymer peak, are relatively better.

[0013] In one embodiment of the present invention, the volume percentage of trifluoroacetic acid in the mobile phase is 0.01% to 0.2%, and more preferably 0.01% to 0.1%. When the volume percentage of trifluoroacetic acid in the mobile phase is less than 0.01%, the polymer peak and the main peak are not separated. When the volume percentage of trifluoroacetic acid in the mobile phase is greater than 0.1%, both the separation degree between the main peak and the polymer peak and the signal-to-noise ratio of the polymer peak decrease. In a specific embodiment of the present invention, the volume percentage of trifluoroacetic acid in the mobile phase is 0.05%, at which point the separation degree between the main peak and the polymer peak and the signal-to-noise ratio of the polymer peak are relatively better.

[0014] In one embodiment of the present invention, the flow rate of the mobile phase is 0.3~1.0 mL / min, and more preferably 0.5~1.0 mL / min. When the flow rate of the mobile phase is less than 0.3 mL / min, neither the main peak nor the polymer peak elutes within a 30 min elution time. When the flow rate of the mobile phase is 0.3 mL / min, the elution times of both the main peak and the polymer peak are greater than 20 min. Increasing the flow rate of the mobile phase can reduce the elution times of the main peak and the polymer peak. In a specific embodiment of the present invention, the flow rate of the mobile phase is 0.5 mL / min, at which point the separation between the main peak and the polymer peak and the signal-to-noise ratio of the polymer peak are relatively better.

[0015] In one embodiment of the present invention, an ultraviolet detector is used for detection at a wavelength of 214 nm, the column temperature is 25°C, and the sample tray temperature is 4°C.

[0016] Compared with existing technologies, this invention has the following beneficial effects: For the research on quality standards of GHRH analogs, a unified and specific method for polymer impurity analysis has been developed. In particular, for the polymer analysis of GHRH analogs such as temorepine or sermorepine, a targeted size exclusion chromatography detection scheme has been developed, forming an effective means of quality control in the production process of these drugs, comprehensively ensuring the safety and stability of the drugs. This specific, efficient, accurate, and stable size exclusion chromatography method developed in this invention, used for the analysis of polymer impurities in temorepine or sermorepine, solves the technical problems of poor separation effect and insufficient detection sensitivity in existing technologies, improves the quality control system of GHRH analogs, and solves the technical problems that urgently need to be solved by those skilled in the art. Attached Figure Description

[0017] Figure 1 This is the chromatogram corresponding to Example 1.

[0018] Figure 2 This is the chromatogram corresponding to Example 2.

[0019] Figure 3 This is the chromatogram corresponding to the injection volume of the sample to be tested in Example 3 being 10 μl.

[0020] Figure 4 This is the chromatogram corresponding to the injection volume of the sample to be tested in Example 3 being 50 μl.

[0021] Figure 5 This is the chromatogram corresponding to the injection volume of the sample to be tested in Example 3 being 100 μl.

[0022] Figure 6 This is the chromatogram corresponding to the volume ratio of water to acetonitrile in the mobile phase of Example 3 being 2:8.

[0023] Figure 7 This is the chromatogram corresponding to the volume ratio of water to acetonitrile in the mobile phase of Example 3 being 4:6.

[0024] Figure 8 This is the chromatogram corresponding to the volume ratio of water to acetonitrile in the mobile phase of Example 3 being 5:5.

[0025] Figure 9 This is the chromatogram corresponding to the volume ratio of water to acetonitrile in the mobile phase of Example 3 being 8:2.

[0026] Figure 10 This is the chromatogram corresponding to the volume percentage of trifluoroacetic acid in the mobile phase of Example 3 being 0.005%.

[0027] Figure 11 This is the chromatogram corresponding to the volume percentage of trifluoroacetic acid in the mobile phase of Example 3 being 0.01%.

[0028] Figure 12 This is the chromatogram corresponding to the volume percentage of trifluoroacetic acid in the mobile phase of Example 3 being 0.1%.

[0029] Figure 13 This is the chromatogram corresponding to the volume percentage of trifluoroacetic acid in the mobile phase of Example 3 being 0.2%.

[0030] Figure 14 The chromatogram is the one corresponding to the flow rate of the mobile phase in Example 3 when the flow rate is 0.1 mL / min.

[0031] Figure 15 The chromatogram is the one corresponding to the flow rate of the mobile phase in Example 3 when the flow rate is 0.3 mL / min.

[0032] Figure 16 The chromatogram is the one corresponding to the flow rate of the mobile phase in Example 3 being 0.7 mL / min.

[0033] Figure 17 The chromatogram is the one corresponding to the flow rate of the mobile phase in Example 3 being 1.0 mL / min.

[0034] Figure 18 The chromatogram is for Comparative Example 1.

[0035] Figure 19 This is the chromatogram corresponding to Comparative Example 2.

[0036] Figure 20 This is the chromatogram corresponding to Comparative Example 3. Detailed Implementation

[0037] 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.

[0038] Unless otherwise specified, the experimental materials and instruments used in the embodiments of this invention can be commercially available.

[0039] Mobile phase: Take water, acetonitrile and trifluoroacetic acid, mix them, and degas by ultrasonication to obtain the mobile phase.

[0040] Diluent / blank solution: same as mobile phase.

[0041] Test sample (sample) solution: Take an appropriate amount of the test sample (sample), add diluent (same as mobile phase) to dissolve and dilute to prepare a solution containing about 1 mg per 1 ml.

[0042] Example 1

[0043] The specific steps of the analysis method are as follows:

[0044] The sample to be tested was temorepinephrine. The HPLC instrument used was an Agilent AdvanceBio SEC 130Å, 2.7 µm, 300 mm * 7.8 mm column (SEC size exclusion principle). The mobile phase consisted of water and acetonitrile at a volume ratio of 60:40, with trifluoroacetic acid comprising 0.05% of the mobile phase. The mobile phase flow rate was 0.5 ml / min; the column temperature was 25℃; the detection wavelength was 214 nm; the injection volume was 25 μl; the injection plate temperature was 4℃; and the isocratic elution time was 30 min. The chromatographic results are shown below. Figure 1 As shown, the separation degree between the main peak and the polymer is 2.1, which meets the requirement of a separation degree of at least 1.5; at the same time, the signal-to-noise ratio (S / N) of the polymer peak is 868, which is a high response value.

[0045] Example 2

[0046] The specific steps of the analysis method are as follows:

[0047] The sample to be tested was sermorelin. The HPLC instrument used was an Agilent AdvanceBio SEC 130Å, 2.7 µm, 300 mm * 7.8 mm column (SEC size exclusion principle). The mobile phase consisted of water and acetonitrile in a volume ratio of 60:40:=, with trifluoroacetic acid comprising 0.05% of the mobile phase. The mobile phase flow rate was 0.5 ml / min; the column temperature was 25℃; the detection wavelength was 214 nm; the injection volume was 25 μl; the injection plate temperature was 4℃; and the isocratic elution time was 30 min. The chromatographic results are shown below. Figure 2 As shown, the separation degree between the main peak and the polymer is 2.2, which meets the requirement of a separation degree of at least 1.5; at the same time, the signal-to-noise ratio (S / N) of the polymer peak is 396, and the response value also meets the requirements.

[0048] Example 3

[0049] The sample to be tested was temorepinephrine. Compared to the chromatographic conditions of Example 1, one parameter was adjusted (such as the injection volume of the sample, the volume ratio of water to acetonitrile in the mobile phase, the volume percentage of trifluoroacetic acid in the mobile phase, and the flow rate of the mobile phase). The resolution between the main peak and the polymer peak and the method sensitivity under different conditions were investigated. The results are shown in Table 1 below. Figures 3-17 As shown.

[0050] Table 1. Separation degree and method sensitivity of the main peak and polymer peak under different conditions.

[0051]

[0052] Example 4

[0053] The analytical method of Example 1 (test sample: temorepinephrine) was validated (including specificity, sensitivity, system suitability, repeatability, linearity, limit of quantitation (QL), limit of detection (DL), and accuracy), and the validation results are as follows.

[0054] Specificity: The analytical method blank was free of interference, the minimum resolution between the main peak and the polymer peak was 2.0, and the peak purity of the main peak was 991, which met the requirements.

[0055] Sensitivity: The S / N ratio of the main peak in the sensitivity solution is 22, which meets the requirements.

[0056] System suitability: The peak area RSD of the main peak in the system suitability solution is 0.1%~0.5%, the resolution between the main peak and the polymer peak is 2.1, and the tailing factor of the main peak is 1.0, which meets the acceptable standard, proving that the system suitability of this method is good.

[0057] Repeatability: Six parallel test solutions were prepared, and the polymer content RSD was 10.7%, which meets the NMT 20% acceptable standard, demonstrating that the method has good repeatability.

[0058] Linearity: The reported linear equation has a correlation coefficient R of 0.9981, a Y-intercept of 1.2% of the 100% response value, and an RSD% of 6.6% for the response factor (A / C). The product exhibits good linearity in the concentration range of 0.43 μg / ml to 13.78 μg / ml.

[0059] Limit of Quantitation (LOQ): The recoveries of the LOQ solutions ranged from 88% to 108%, with an RSD of 8.0% and an S / N value of 16 to 39, all meeting the acceptable criteria. The LOQ concentration for this method is 0.43 μg / ml, equivalent to 0.05% of the sample concentration.

[0060] Limit of Detection (DL): The signal-to-noise ratio of the solution at the limit of detection is 7, which meets the requirements. Therefore, the limit of detection concentration is 0.14 μg / ml, equivalent to 0.017% of the sample concentration.

[0061] Accuracy: The recovery rate of the solution was between 87.7% and 102.7%, with an RSD of 5.1%, both meeting the acceptable standard requirements. Therefore, the accuracy of this method is good.

[0062] Comparative Example 1

[0063] The Agilent AdvanceBio SEC column from Example 1 was replaced with a Waters XBridge BEH 125 Å SEC 3.5 μm column (7.8 mm x 300 mm). All other conditions were the same as in Example 1. The chromatogram results are as follows: Figure 18 As shown, the separation degree between the main peak and the polymer is 1.2, which does not meet the requirement of a separation degree of at least 1.5; at the same time, the signal-to-noise ratio (S / N) of the polymer peak is only 147, which is a low response value.

[0064] Comparative Example 2

[0065] The Agilent AdvanceBio SEC column from Example 1 was replaced with a Shodex Protein KW-802.5 5μm, 8.0mm*300mm column. The injection volume was 100 μl, and all other conditions were the same as in Example 1. The chromatogram results are as follows. Figure 19 As shown, the resolution between the main peak and the polymer peak is 1.8. Although the resolution meets the requirement of at least 1.5, the signal-to-noise ratio (S / N) of the polymer peak is only 105, indicating a low response value. Experimental results show that even when the injection volume reaches 100 μl, the S / N of the polymer peak still fails to meet the requirements.

[0066] Comparative Example 3

[0067] The injection volume of the sample to be tested was 5 μl, and all other conditions were the same as in Example 1. The resolution between the main peak and the polymer peak was 2.1, and all other conditions were the same as in Example 1. The chromatographic results are as follows. Figure 20 As shown: Although the resolution meets the requirement of at least 1.5, the polymer peak height is only 3.9, and the polymer peak signal-to-noise ratio (S / N) is only 96, which is too low.

[0068] Comparative Example 4

[0069] Using the reversed-phase C18, 10μm, 77mm*250mm column disclosed in CN110818790A, purification was performed using two mobile phase systems alternately. The first mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, and the second mobile phase system was 50mmol ammonium acetate / water solution-acetonitrile. All other conditions were the same as in Example 1. The chromatographic results showed that the polymer peak overlapped with the main peak, and separation could not be achieved.

[0070] By comparing the examples and Comparative Examples 1 and 2, it was found that, although both are based on the principle of size exclusion chromatography, the choice of packing material in the chromatographic column has a significant impact on the analytical results. The chromatographic method of the present invention has achieved unexpected technical effects: the separation degree of the main peak and the polymer peak and the signal-to-noise ratio of the polymer peak are both ideal, meeting the separation requirements.

[0071] The core principle of size exclusion chromatography (SOC) is based on the difference in molecular volume (or hydrodynamic volume) of the analyte components to achieve separation. The packing material within the column forms a three-dimensional network structure with different pore sizes. When the sample solution flows through the column, components with larger molecular volumes cannot enter the micropores of the packing material and can only elute rapidly along the gaps between the packing particles (i.e., interparticle volume), resulting in a shorter peak elution time. Components with smaller molecular volumes, on the other hand, can enter the micropores of the packing material, undergoing permeation and diffusion within the micropores, resulting in a longer elution path and a relatively longer elution time, thus achieving the sequential separation of components of different volumes. In this separation process, the choice of packing material in the column has a decisive impact on the accuracy of the analytical results and the stability of the separation effect. Key parameters such as pore size, pore size distribution uniformity, particle size, and specific surface area directly determine the column's ability to sieve components of different volumes and its separation efficiency. If the packing parameters do not match the molecular volume of the analyte, problems such as insufficient resolution, peak broadening, and low signal-to-noise ratio are likely to occur. The chromatographic method of this invention achieves unexpected technical results by optimizing the selection of packing material suitable for the molecular volume range of the target components and controlling the pore size distribution and particle morphology of the packing material: the separation degree of the main peak and the polymer peak, as well as the signal-to-noise ratio of the polymer peak, are both ideal, meeting the separation requirements. From the perspective of possible mechanisms, on the one hand, the suitable pore size distribution of the packing material effectively distinguishes the molecular volume difference between the component corresponding to the main peak and the polymer impurities, so that the two form a clear difference in elution path during the elution process, avoiding peak overlap and significantly improving the separation degree; on the other hand, the uniform particle morphology of the packing material reduces eddy diffusion and mass transfer resistance when the sample flows through the chromatographic column, reducing baseline noise. At the same time, the optimized specific surface area of ​​the packing material reduces non-specific adsorption of components on the packing surface, reduces peak tailing, and thus improves the signal-to-noise ratio of the polymer peak, ensuring the reliability and accuracy of the analytical results, and ultimately achieving a separation effect that is difficult to achieve with conventional chromatographic methods.

Claims

1. A size exclusion chromatography method for polymer analysis in GHRH analogs, characterized in that: The sample to be tested was a GHRH analogue, and an Agilent AdvanceBio SEC column with size exclusion chromatography was used; the mobile phase was a mixed solution of water, acetonitrile and trifluoroacetic acid, and isocratic elution was performed.

2. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The GHRH analogues are temorepine or semolorepine.

3. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The injection volume of the sample to be tested is 10~100 μl.

4. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 3, characterized in that: The injection volume of the sample to be tested was 25 μl.

5. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The volume ratio of water to acetonitrile in the mobile phase is 2:3 to 4:

1.

6. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The volume ratio of water to acetonitrile in the mobile phase is 3:

2.

7. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The volume percentage of trifluoroacetic acid in the mobile phase is 0.01% to 0.2%.

8. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The volume percentage of trifluoroacetic acid in the mobile phase is 0.05%.

9. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The flow rate of the mobile phase is 0.3~1.0 mL / min, and the flow rate of the mobile phase is further optionally 0.5 mL / min.

10. The size exclusion chromatography method for polymer analysis in GHRH analogs according to claim 1, characterized in that: The detection was performed using an ultraviolet detector at a wavelength of 214 nm, with a column temperature of 25℃ and a sample tray temperature of 4℃.

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