Preparation method and application of optimized reversed-phase C18 silica gel substrate

Abstract

The invention provides a preparation method and application of an optimized reversed-phase C18 silica gel matrix, and the preparation method comprises the following steps: activating mesoporous silica with hydrochloric acid, washing to be neutral, drying, adding into toluene, sequentially adding pyridine and octadecyl trichlorosilane, stirring for reaction under the nitrogen protection condition, filtering, washing, and drying to obtain the optimized reversed-phase C18 silica gel matrix. Washing with methylbenzene, absolute ethyl alcohol and acetone in sequence, and performing vacuum drying; and taking out the silica gel substrate, replacing octadecyl trichlorosilane with methyl trichlorosilane, repeating the operation, and blocking to obtain the optimized reversed-phase C18 silica gel substrate. According to the application, the optimized reversed-phase C18 silica gel substrate is applied to purification of the lisanatide. The purification operation is simple and convenient, the yield of the obtained lisanatide is high, and the method is suitable for industrial production.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of "2024.09.20", application number "202411313801.3", and invention name "A method for purifying lixisenatide". Technical Field

[0002] The present invention relates to the technical field of polypeptide drug purification, and in particular to a preparation method and application of an optimized reverse phase C18 silica gel matrix. Background Art

[0003] On July 28, 2016, lixisenatide was approved by the U.S. Food and Drug Administration (FDA) for improving the control of blood sugar levels in adults with type 2 diabetes. It became the fifth GLP-1 receptor agonist launched globally after exenatide, liraglutide, albiglutide and dulaglutide, providing patients with a new treatment option.

[0004] As a short-acting GLP-1 receptor agonist administered once a day, lixisenatide can significantly reduce the levels of glycated hemoglobin (HbA1c), postprandial blood glucose (PPG) and fasting blood glucose (FPG), especially the most significant reduction in PPG, whether used alone or in combination with oral hypoglycemic drugs or basal insulin. This drug has a low risk of hypoglycemia, can reduce weight, and is well tolerated, which also makes lixisenatide have broad application prospects in the personalized treatment of type 2 diabetes.

[0005] Lixisenatide is composed of 44 natural amino acids with the sequence of H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-lle-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-NH 2 , due to the long sequence, amino acid condensation is difficult and the crude product purity is poor, resulting in a low purification yield.

[0006] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the invention

[0007] Technical issues In order to solve the above problems, the purpose of the present invention is to provide a method for purifying lixisenatide, which successively performs cation exchange resin purification and reversed-phase liquid chromatography purification on a crude lixisenatide solution, is simple to operate, has a stable process, can significantly improve the purity and yield of the lixisenatide finished product, and the purified finished product has high purity and low impurity content, and has good economic use value and broad application prospects.

[0008] Technical Solution In order to achieve the above objectives, this application provides: A method for purifying lixisenatide, comprising: (1) adding purified water to dissolve crude lixisenatide into a crude solution; (2) using a cation exchange resin to initially purify the crude product solution and collecting the eluate; (3) The eluate is purified for a second time by reverse phase high performance chromatography, and the eluate is collected, concentrated, and freeze-dried to obtain the finished product of lixisenatide.

[0009] For some specific implementation schemes, in step (2), it is necessary to filter through a filter membrane with a pore size of no greater than 0.22 μm before the initial purification; the filter membrane is preferably a 0.22 μm PVDF membrane.

[0010] For some specific embodiments, in step (2), the cation exchange resin is preferably Source 30S or Amberlite CG-120, more preferably Source 30S.

[0011] For some specific embodiments, in step (2), the mobile phase A for initial purification is NaH 2 PO 4 Buffer solution, concentration 50-75mmol / L.

[0012] For some specific embodiments, in step (2), the mobile phase B for the initial purification is NaH 2 PO 4 Buffer solution contains 0.8-1.0 mol / L NaCl.

[0013] For some specific embodiments, in step (2), the initial purification elution procedure is a flow rate of 50 mL / min, and the proportion of mobile phase B increases from 10% to 50% within 60 minutes.

[0014] For some specific embodiments, in step (3), the separation medium of the reverse phase high performance chromatography is a reverse phase C18 silica gel matrix.

[0015] For some specific embodiments, in step (3), the mobile phase A for secondary purification is 1-1.5% (volume content) acetic acid / water solution.

[0016] For some specific embodiments, in step (3), the mobile phase B for secondary purification is 0.5-2% (volume content) acetic acid / acetonitrile solution.

[0017] For some specific embodiments, in step (3), the elution procedure for the secondary purification is a flow rate of 50 mL / min, and the proportion of mobile phase B increases from 10% to 40% within 60 min.

[0018] The lixisenatide product obtained by the above purification method has a purity of more than 99% and a single impurity content of less than 0.1%.

[0019] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0020] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0021] Beneficial Effects According to the present invention, by successively subjecting a crude lixisenatide solution to cation exchange resin purification and reverse phase liquid chromatography purification, the operation is simple and the process is stable, and the purity and yield of the lixisenatide finished product can be significantly improved. The purity of the purified finished product can reach more than 99%, the single impurity is less than 0.1%, and the purification yield reaches 60-70%, which is better than the prior art CN103709243A and CN104844706A, is suitable for industrial production, and has good economic use value and broad application prospects.

[0022] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The maximum single impurity analysis diagram of the finished product lixisenatide is shown; Figure 2 The infrared spectrum of the mesoporous silica obtained in Example 12 is shown; Figure 3 A purity analysis chart showing the finished product lixisenatide; Figure 4 A graph showing the yield analysis of the finished product lixisenatide. DETAILED DESCRIPTION

[0025] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0027] Unless otherwise specified, all percentages, parts, ratios, etc. are by weight; otherwise specified, including but not limited to, "%", "wt%", "mass%" means weight percentage, "mol%" means molar percentage, and "vol%" means volume percentage.

[0028] When a quantity, concentration or other numerical value or parameter is given as a range, a preferred range or a series of upper preferred values ​​and lower preferred values, it should be understood that it specifically discloses all ranges formed by any pair of numerical values ​​of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether the range is disclosed separately. For example, when describing the range of "1 to 5 (1-5)", the described range should be understood to include the range of "1 to 4 (1-4)", "1 to 3 (1-3)", "1 to 2 (1-2)", "1 to 2 (1-2) and 4 to 5 (4-5)", "1 to 3 (1-3) and 5" etc. Unless otherwise stated, where a numerical range is described herein, the range includes the range end values ​​and all integers and fractions within the range.

[0029] Unless specifically stated, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are still described herein.

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0031] In order to facilitate understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention are first explained or defined. For undefined abbreviations or key terms, they are commonly understood by those skilled in the art.

[0032] GLP-1: glucagon-like peptide-1; HbA1c: glycosylated hemoglobin; PPG: postprandial blood glucose; FPG: fasting plasma glucose; PVDF membrane: polyvinylidene fluoride, polyvinylidene fluoride membrane.

[0033] The present invention is described in detail below Embodiment 1: Provided is a method for purifying lixisenatide, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0034] (2) Ion exchange resin purification The ion exchange resin Source 30S was used as the separation medium, and 50 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 3.5 NaH 2 PO 4 The buffer is phase B.

[0035] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0036] (3) Reverse Phase High Performance Chromatography Purification Reverse phase C18 silica gel matrix was used as the separation medium (commercially available spherical C18 chromatographic filler reverse phase chromatography silica gel matrix 100Å 15μm, the same below), 1% acetic acid system was used as mobile phase A, and 0.5% acetic acid / acetonitrile solution system was used as mobile phase B for purification; before loading, 95% A phase was used to balance 3 column volumes before loading, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected, it was concentrated under reduced pressure and then freeze-dried to obtain the finished lixisenatide with a purity of 99.5%, a maximum single impurity of 0.08%, and a yield of 64.5%.

[0037] Embodiment 2: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0038] (2) Ion exchange resin purification Ion exchange resin Source 30S was used as the separation medium, and 50 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0039] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0040] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 1.2% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 2 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.3%, a maximum single impurity of 0.10%, and a yield of 62.5%.

[0041] Embodiment 3: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0042] (2) Ion exchange resin purification The ion exchange resin Source 30S was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 3.5 NaH 2 PO 4 The buffer is phase B.

[0043] Before loading the sample, equilibrate 2 column volumes with 100% phase A and then perform gradient elution. Within 60 minutes, the proportion of mobile phase B increases from 10% to 50%, and the eluate is collected.

[0044] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 1.8% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 3 column volumes were equilibrated with 95% phase A and then loaded, followed by gradient elution. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.2%, a maximum single impurity of 0.09%, and a yield of 67.5%.

[0045] Embodiment 4: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0046] (2) Ion exchange resin purification The ion exchange resin Source 30S was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0047] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0048] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 1.5% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 2 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.6%, a maximum single impurity of 0.07%, and a yield of 60.5%.

[0049] Embodiment 5: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0050] (2) Ion exchange resin purification The ion exchange resin Source 30S was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0051] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0052] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1.5% acetic acid system as the mobile phase A, and a 1% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 3 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.4%, a maximum single impurity of 0.11%, and a yield of 66.5%.

[0053] Embodiment 6: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0054] (2) Ion exchange resin purification Ion exchange resin Amberlite CG-120 was used as the separation medium, and 50 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 3.5 NaH 2 PO 4 The buffer is phase B.

[0055] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0056] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 1.2% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 2 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.2%, a maximum single impurity of 0.11%, and a yield of 62.5%.

[0057] Embodiment 7: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0058] (2) Ion exchange resin purification Ion exchange resin Amberlite CG-120 was used as the separation medium, and 50 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0059] Before loading the sample, equilibrate 2 column volumes with 100% phase A and then perform gradient elution. Within 60 minutes, the proportion of mobile phase B increases from 10% to 50%, and the eluate is collected.

[0060] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 0.5% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 2 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.5%, a maximum single impurity of 0.09%, and a yield of 66.7%.

[0061] Embodiment 8: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0062] (2) Ion exchange resin purification Ion exchange resin Amberlite CG-120 was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 3.5 NaH 2 PO 4 The buffer is phase B.

[0063] Before loading the sample, equilibrate 2 column volumes with 100% phase A and then perform gradient elution. Within 60 minutes, the proportion of mobile phase B increases from 10% to 50%, and the eluate is collected.

[0064] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 1.5% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 3 column volumes were equilibrated with 95% phase A and then loaded, followed by gradient elution. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.3%, a maximum single impurity of 0.08%, and a yield of 62.2%.

[0065] Embodiment 9: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0066] (2) Ion exchange resin purification Ion exchange resin Amberlite CG-120 was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0067] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0068] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1% acetic acid system as the mobile phase A, and a 2% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 3 column volumes were equilibrated with 95% phase A and then loaded, followed by gradient elution. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.5%, a maximum single impurity of 0.12%, and a yield of 68.8%.

[0069] Embodiment 10: Another method for purifying lixisenatide is provided, comprising the following steps: (1) Treatment of crude lixisenatide peptide Weigh 2.5 g of crude lixisenatide, add 100 mL of purified water to dissolve until clear, and then filter with a 0.22 μm PVDF membrane.

[0070] (2) Ion exchange resin purification Ion exchange resin Amberlite CG-120 was used as the separation medium, and 75 mmol / L NaH 2 PO 4 The buffer is phase A, containing 1 mol / L NaCl, pH 4.5 NaH 2 PO 4 The buffer is phase B.

[0071] Before loading the sample, 100% phase A was used to equilibrate 3 column volumes, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B was increased from 10% to 50%, and the eluate was collected.

[0072] (3) Reverse Phase High Performance Chromatography Purification The purification was carried out using a reversed phase C18 silica gel matrix as the separation medium, a 1.5% acetic acid system as the mobile phase A, and a 2% acetic acid / acetonitrile solution system as the mobile phase B. Before loading the sample, 2 column volumes were equilibrated with 95% phase A and then loaded, and then gradient elution was performed. Within 60 minutes, the proportion of mobile phase B increased from 10% to 40%. After the eluate was collected and concentrated under reduced pressure, it was lyophilized to obtain the finished product lixisenatide with a purity of 99.6%, a maximum single impurity of 0.08%, and a yield of 61.4%.

[0073] Embodiment 11: Based on the above examples, the ion exchange resin purification step was optimized, specifically, NaH 2 PO 4 The buffer solution contains a trace amount of 1-4 mmol / L thioethyl-β-cyclodextrin, and a comparative example is set according to the above specific example without changing the other steps and parameters.

[0074] Based on the above Example 2, the ion exchange resin purification step was optimized, specifically, NaHCO3 with pH 4.5 containing 1 mmol / L thioethyl-β-cyclodextrin. 2 PO 4 The buffer was phase A, and the remaining steps and parameters were the same as those in Example 2 to prepare the finished product lixisenatide, and its maximum single impurity was counted.

[0075] Based on the above Example 5, the ion exchange resin purification step was optimized, specifically, NaHCO3 at pH 4.5 containing 4 mmol / L thioethyl-β-cyclodextrin. 2 PO 4 The buffer was phase A, and the remaining steps and parameters were the same as those in Example 5 to prepare the finished product lixisenatide, and its maximum single impurity was counted.

[0076] The final statistical maximum single impurity analysis diagram of the finished lixisenatide obtained in Example 2, Example 5, Comparative Example 2 and Comparative Example 5 is shown in the figure below. Figure 1 As shown by Figure 1 It can be seen that compared with the maximum single impurity of not less than 0.1% in the lixisenatide finished product of Example 2 and Example 5, by adding a trace amount of 1-4mmol / L thioethyl-β-cyclodextrin in phase A in the ion exchange resin purification step, the maximum single impurity content in the purified product can be significantly reduced to not more than 0.05%, effectively reducing the maximum single impurity content and improving the purification efficiency. The possible reason is that the high water solubility and polar groups of thioethyl-β-cyclodextrin are more conducive to separating impurities in the lixisenatide solution, thereby reducing the maximum single impurity content. Based on this, the introduction of thioethyl-β-cyclodextrin in the ion exchange resin purification step can significantly reduce the content of the maximum single impurity in the finished product and improve the purity of the finished product.

[0077] Embodiment 12: On the basis of the foregoing embodiments, the separation medium of the modified reverse-phase high performance chromatography is optimized, specifically, mesoporous silica microspheres are modified with C18 to prepare an optimized reverse-phase C18 silica gel matrix, and the preparation steps specifically include preparing mesoporous silica and preparing an optimized reverse-phase C18 silica gel matrix.

[0078] Preparation of mesoporous silica: 100 mL of tetraethyl orthosilicate was uniformly dispersed in 50 mL of anhydrous ethanol, and then 15 mL of 0.05 mol / L hydrochloric acid was injected at a rate of 1 mL / min, and the reaction was stirred at 35 ° C for 2 h, and the intermediate was obtained by rotary evaporation at 60 ° C for 1 h; 10 g of the intermediate was mixed with 0.5 mL of N, N-dimethylformamide, and a large amount of ultrapure water and isopropanol mixed solution (V 超纯水 :V 异丙醇 =3:1), fully emulsify at room temperature and 2100r / min for 30min; inject 1mL of ammonia water into the emulsion at a speed of 1mL / min, continue stirring for 30min, centrifuge, wash with deionized water, isopropanol, and methanol three times respectively, filter and dry to obtain mesoporous silica. The infrared spectrum of mesoporous silica is shown in Figure 2 As shown, about 808cm -1 The symmetrical stretching vibration absorption peak of Si-O is shown at about 1064 cm -1 The antisymmetric stretching vibration absorption peak of Si-O-Si is shown at , indicating that pure mesoporous silica microspheres were successfully synthesized.

[0079] Preparation of optimized reverse C18 silica gel matrix: activate mesoporous silica with 1 mol / L hydrochloric acid, wash to neutral and then dry, take 5 g and add to 100 mL of toluene, then add 1 mL of pyridine and 10 mL of octadecyltrichlorosilane in turn, stir and react for 24 hours at 85°C and 300 r / min under nitrogen protection, wash with toluene, anhydrous ethanol and acetone in turn and dry in vacuum at 60°C; take out 5 g and repeat the above operation with 6 mL of methyltrichlorosilane instead of octadecyltrichlorosilane for end-capping to obtain the optimized reverse C18 silica gel matrix. The results of elemental analysis showed that the C content of the microspheres was about 1.6%, indicating that C18 was successfully modified on the surface of the silica microspheres.

[0080] Without changing other operating steps, the optimized reversed-phase C18 silica gel matrix was used to replace the reversed-phase C18 silica gel matrix in the above Examples 1, 6, 8 and 9 as the separation matrix for Comparative Examples 1, 6, 8 and 9, respectively, and the purity and yield of the final product lixisenatide were statistically analyzed, as shown in the following table. Figure 3 and 4 As shown, Figure 3The purity of lixisenatide in different embodiments and comparative examples is shown, and it can be seen that the use of the optimized reverse phase C18 silica gel matrix as the separation matrix can obtain a higher purity of lixisenatide pure product compared to the conventional C18 separation medium; Figure 4 The lixisenatide yields of different embodiments and comparative examples are shown. It can be seen that the yield can be increased to 68-70.2% after the optimized reverse phase C18 silica gel matrix is ​​used as the separation matrix, thus avoiding the loss of the product. Figure 3 and attached Figure 4 It can be seen that when the mesoporous silica with uniform and stable particle size is prepared by the method of the present invention and then modified with C18, and used for the reverse phase high performance chromatography to purify lixisenatide, it can exert a more excellent separation performance than the conventional C18 separation medium, and the purity and yield of the finished lixisenatide are improved to varying degrees, and it has a high economic value.

[0081] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0082] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0083] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0084] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0085] Matters not covered in the present invention are all known technologies.

Claims

1. A method for preparing an optimized reverse phase C18 silica gel matrix, characterized in that: Activate mesoporous silica with 1 mol / L hydrochloric acid, wash until neutral and then dry, take 5 g and add to 100 mL of toluene, then add 1 mL of pyridine and 10 mL of octadecyltrichlorosilane in turn, stir and react for 24 hours at 85°C and 300 r / min under nitrogen protection, wash with toluene, anhydrous ethanol and acetone in turn and dry in vacuum at 60°C; take out 5 g and repeat the above operation with 6 mL of methyltrichlorosilane instead of octadecyltrichlorosilane for end-capping to obtain the optimized reversed-phase C18 silica gel matrix.

2. The method according to claim 1, characterized in that: The mesoporous silica is prepared by the following method: 100 mL of tetraethyl orthosilicate is uniformly dispersed in 50 mL of anhydrous ethanol, and then 15 mL of 0.05 mol / L hydrochloric acid is injected at a rate of 1 mL / min, stirred at 35°C for 2 h, and placed at 60°C for 1 h to obtain an intermediate; 10 g of the intermediate is mixed with 0.5 mL of N,N-dimethylformamide, and quickly added to a mixed solution of a large amount of ultrapure water and isopropanol, wherein V 超纯水 :V 异丙醇 =3:1, fully emulsify at room temperature and 2100r / min for 30min; inject 1mL of ammonia water into the emulsion at a speed of 1mL / min, continue stirring for 30min, centrifuge, wash with deionized water, isopropanol and methanol three times respectively, filter and dry to obtain mesoporous silica.

3. The optimized reverse phase C18 silica gel matrix prepared by the method of claim 1 or 2.

4. Use of the optimized reverse phase C18 silica gel matrix according to claim 3 in purifying lixisenatide.

5. The use according to claim 4, characterized in that: Specifically, the optimized reverse phase C18 silica gel matrix described in claim 3 is used as the separation matrix, and the eluate collected after the primary purification of the crude lixisenatide solution by cation exchange resin is subjected to secondary purification by reverse phase high performance chromatography, and the collected eluate is concentrated and freeze-dried to obtain the finished lixisenatide.

6. The use according to claim 5, characterized in that: When the cation exchange resin is used for the initial purification of the crude lixisenatide solution, the mobile phase A is a NaH2PO4 buffer solution with a pH of 3.5-4.5 and a concentration of 50-75 mmol / L, and the NaH2PO4 buffer solution contains 1-4 mmol / L thioethyl-β-cyclodextrin.

7. A method for purifying lixisenatide, characterized in that include: (1) adding purified water to dissolve crude lixisenatide into a crude solution; (2) using a cation exchange resin to perform a primary purification on the crude product solution and collecting the eluate; (3) performing secondary purification on the eluate by reverse phase high performance chromatography, collecting the eluate, concentrating and freeze-drying to obtain the finished product of lixisenatide; The reverse phase high performance chromatography method adopts the optimized reverse phase C18 silica gel matrix as the separation matrix.

8. The method according to claim 7, characterized in that: The mobile phase A for the initial purification is a NaH2PO4 buffer with a pH of 3.5-4.5 and a concentration of 50-75 mmol / L, and the NaH2PO4 buffer contains 1-4 mmol / L thioethyl-β-cyclodextrin.

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