A preparation method of sterile lanreotide raw material

Abstract

This invention provides a method for preparing sterile lanreotide raw material, comprising the following five steps: (1) preparing a lanreotide cyclic peptide solution; (2) purifying the lanreotide cyclic peptide solution once using HPLC, wherein the mobile phase A is an ammonium salt solution and the mobile phase B is an organic reagent; (3) purifying the once-purified lanreotide cyclic peptide solution a second time using HPLC, wherein the mobile phase A is a sodium salt solution and the mobile phase B is an organic reagent; (4) treating the second-purified lanreotide cyclic peptide solution with an aqueous acetic acid solution to obtain an acetate lanreotide solution; (5) filtering and drying the acetate lanreotide solution to obtain the finished product. The lanreotide raw material obtained by the preparation method of this invention has a purity greater than 99.9% and a single impurity content ≤0.10%.

Description

Technical Field

[0001] This invention relates to the field of preparation of lanreotide active pharmaceutical ingredient, and particularly to a method for preparing sterile lanreotide active pharmaceutical ingredient. Background Technology

[0002] Lanreotide, marketed as semadulin, was originally developed by Ipsen Pharmaceuticals in France. It was first launched in France in 1994, entered the Chinese market in 2002, and was approved by the US FDA in 2007 as a long-acting sustained-release formulation.

[0003] Lanreotide acetate injection is a long-acting, sustained-release somatostatin octapeptide analog that inhibits the secretion of growth hormone, thyroid-stimulating hormone, insulin, and glucagon. Lanreotide acetate was initially used primarily to treat acromegaly and growth hormone-secreting pituitary adenomas that were unresponsive to surgery or radiotherapy. Subsequently, it was approved by the FDA for the treatment of unresectable, moderately or highly differentiated, or metastatic gastrointestinal and pancreatic neuroendocrine tumors.

[0004] Patent CN 10330560A describes a method for synthesizing lanreotide using iodine oxidation to form disulfide bonds. However, iodine oxidation may lead to iodine substitution of Tyr residues, increasing the risk of oxidative and dimer impurities. Patent CN 113480634A discloses a method for preparing lanreotide active pharmaceutical ingredient (API) using 0.1% trifluoroacetic acid / water / acetonitrile as the mobile phase. This mobile phase has a pH of approximately 2, which may increase degradation impurities throughout the production cycle. Lanreotide acetate sustained-release injection (pre-filled) is a sustained-release formulation based on self-assembled nanotube technology. It contains no excipients other than water and is a supersaturated solution with a paste-like viscosity. Achieving sterility through filtration or terminal sterilization during formulation is difficult. Furthermore, the API, being a polypeptide product, is thermally unstable. Therefore, it is essential to invent a sterile lanreotide API that can be produced on an industrial scale with controllable impurities. Summary of the Invention

[0005] Therefore, this invention proposes a method for preparing sterile lanreotide raw material to solve the above problems.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for preparing a sterile lanreotide active pharmaceutical ingredient includes the following steps:

[0008] (1) Dissolve crude linear lanreotide in acetonitrile aqueous solution, adjust the pH value to 6.0-7.0 with ammonia, oxidize with oxidant, close the ring, filter, and obtain lanreotide cyclic peptide solution.

[0009] (2) The crude cyclized peptide solution of lanreotide was purified for the first time using octadecylsilane-bonded silica gel as the stationary phase. The mobile phase A was an ammonium salt solution, and the mobile phase B was acetonitrile, methanol, or a mixture of acetonitrile and methanol. The elution time in the elution program was 40-80 min, and the mobile phase B was 10-50 wt%. The purified lanreotide cyclized peptide solution was collected.

[0010] (3) Mix the first-purified lanreotide cyclic peptide solution and water at a volume ratio of 1:1-2. Perform a second purification on the mixed first-purified lanreotide linear peptide solution. The stationary phase is octadecylsilane-bonded silica gel, the mobile phase A is a sodium salt solution, and the mobile phase B is acetonitrile, methanol, or a mixture of acetonitrile and methanol. The elution time in the elution program is 40-80 min, and the mobile phase B is 10-50 wt%. Collect the eluent and adjust the pH of the eluent to 3.0-5.0 using an acidic regulator to obtain the second-purified lanreotide cyclic peptide solution.

[0011] (4) The purified lanreotide cyclic peptide solution was subjected to gradient elution using mobile phase A, which was aqueous acetic acid solution, and mobile phase B, which was acetic acid acetonitrile solution or acetic acid methanol solution. The elution time in the elution program was 25-50 min and the mobile phase B was 10-60 wt%, to obtain lanreotide acetate solution.

[0012] (5) Sterilize and filter the lanreotide acetate and dry it to obtain the finished product.

[0013] Furthermore, in step (1), the concentration of the linear crude lanreotide is 0.5–5 mg / mL, the concentration of the acetonitrile aqueous solution is 5–15% (V / V), and the oxidant is one or more of air, hydrogen peroxide, and DMSO.

[0014] Furthermore, in step (2), the ammonium salt is ammonium acetate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate or ammonium phosphate, and the mobile phase A is an aqueous solution containing 30 mmol / L to 100 mmol / L of ammonium salt, and the pH value is adjusted to 3.7 to 4.7 using an acidic regulator.

[0015] The method for preparing sterile lanreotide raw material as described in claim 1 is characterized in that, further, in step (3), the sodium salt is sodium acetate, sodium dihydrogen phosphate or disodium hydrogen phosphate, the mobile phase A is a 20 mmol / L to 100 mmol / L sodium salt buffer, and the pH value is adjusted to 5.8 to 6.8 using an acid regulator.

[0016] Furthermore, in step (4), the volume concentration of acetic acid in the aqueous acetic acid solution is 0.1% to 1.0%.

[0017] Furthermore, in steps (2) and (3), the volume ratio of acetonitrile or methanol or acetonitrile-methanol mixture to water is 50% to 100%.

[0018] Furthermore, in step (3), the acidity regulator is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, hydrochloric acid, or perchloric acid.

[0019] Furthermore, in step (5), the sterilization filtration includes two-stage or multi-stage sterilization filtration, and the filter element pore size is 0.2μm.

[0020] Furthermore, in step (5), the drying method is spray drying or freeze drying.

[0021] Furthermore, the freeze-drying includes the following steps:

[0022] S1: Refrigeration and heat preservation: Refrigeration temperature is 4-6℃, heat preservation for 1-2 hours.

[0023] S2: Pre-freezing: Pre-freezing temperature is -45~-35℃, vacuum degree is 0.25~0.35mbar, and the temperature is maintained for 3-4 hours.

[0024] S3: Primary drying: drying temperature -20~-10℃, vacuum degree 0.25~0.35mbar, holding time 24-246h.

[0025] S4: Secondary drying: Drying temperature is 10-20℃, vacuum degree is 0.25-0.35mbar, hold for 20-22h, then continue to raise the temperature to 25-35℃, vacuum degree is 0.3-0.5mbar, hold for 12-15h.

[0026] Furthermore, the spray drying inlet air temperature is 100℃~140℃, the outlet air temperature is 50℃~80℃, the carrier gas is sterile nitrogen, and the drying time is 10-15h.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) This invention requires only one reverse-phase packing material, two-step purification, and one salt-changing method to obtain high-purity, high-quality, and high-stability lanreotide acetate raw material. The purity can reach 98% in one-step purification and the purity can reach 99.5% in two-step purification. The single impurity ≤0.10% can be obtained. The number of purification rounds is small, the operation method is simple, the production efficiency is improved, and it can be mass-produced.

[0029] (2) The product is obtained by spray drying, which has the advantages of simple operation and low energy consumption. The aseptic spray drying method is used for drying, which has a short drying time and simple operation. Both drying methods provided by this invention can ensure stable product quality.

[0030] (3) It provides sterile lanreotide acetate raw material, which simplifies the production process of lanreotide acetate sustained-release injection. Attached Figure Description

[0031] Figure 1 This is the HPLC chromatogram of the crude lanreotide cyclized product from Example 1;

[0032] Figure 2 This is the HPLC chromatogram of the lanreotide product after the first step of purification in Example 2;

[0033] Figure 3 This is the HPLC chromatogram of the lanreotide product after the second step of purification in Example 2;

[0034] Figure 4 This is the HPLC chromatogram of the sterile lanreotide product in Example 3;

[0035] Figure 5 This is an MS image of the lanreotide product from Example 4. Detailed Implementation

[0036] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0039] Example 1

[0040] 1. Oxidation of crude linear lanreotide peptide:

[0041] 3 grams of crude linear lanreotide peptide were dissolved in 3 L of acetonitrile aqueous solution, with a volume ratio of acetonitrile to water of 1:9. The pH of the crude linear peptide solution was adjusted to 6.8 with 25% ammonia solution. The solution was oxidized with 0.3% hydrogen peroxide and detected by HPLC. After ring closure, the solution was filtered through a 0.45 μm filter membrane to obtain a cyclic lanreotide peptide solution.

[0042] 2. First step purification:

[0043] The lanreotide cyclic peptide solution obtained in step 1 was purified by HPLC using C18 chromatography stationary phase. The chromatographic column was 10 μm in diameter and 30 × 250 mm in length. Mobile phase A was a 60 mmol / L ammonium acetate aqueous solution, adjusted to pH 4.5 with 1 mol / L hydrochloric acid. Mobile phase B was 90% acetonitrile:10% purified water (v / v). The flow rate was 20 mL / min, the detection wavelength was 220 nm, the sample loading was 3 g, and a gradient elution of 10–50 wt% mobile phase B was performed for 60 min to obtain a once-purified lanreotide cyclic peptide solution.

[0044] 3. Second step purification:

[0045] The cyclic peptide solution collected in the first step of purification was diluted with purified water at a volume ratio of 1:1, and a C18 chromatography packing material was used. The second purification step used a 10 μm stationary phase. Mobile phase A consisted of a 60 mmol / L sodium acetate aqueous solution, with the pH adjusted to 6.4 using 1 mol / L hydrochloric acid. Mobile phase B was 100% acetonitrile. The flow rate was 20 mL / min, the detection wavelength was 220 nm, and gradient elution was performed using 10–50 wt% mobile phase B for 60 min. The peptide solutions were collected in fractions, and the pH was adjusted to 3.5 with acetic acid to obtain the second purified lanreotide cyclic peptide solution.

[0046] 4. Acetic acid to salt conversion:

[0047] The acetic acid method involves changing the salt, and then using C18 to purify the lanreotide cyclic peptide solution from the second purification process. A 10 μm stationary phase was used for salt replacement. Mobile phase A was a 0.5% (V / V) aqueous acetic acid solution, and mobile phase B was a 0.5% (V / V) acetic acid acetonitrile solution. The flow rate was 20 mL / min, the detection wavelength was 220 nm, and gradient elution was performed using 10–60 wt% mobile phase B for 35 min. This yielded a high-purity and highly stable lanreotide acetate product after salt replacement.

[0048] Example 2

[0049] 1. Oxidation of linear crude lanreotide peptide:

[0050] 400 grams of crude linear lanreotide peptide were dissolved in 400 L of acetonitrile aqueous solution, with a volume ratio of acetonitrile to water of 1:9. The pH of the crude linear peptide was adjusted to 6.7 with 25% ammonia solution, and oxidized with 0.3% hydrogen peroxide solution. After cyclic closure by HPLC, the solution was filtered through a 0.45 μm filter membrane to obtain a lanreotide cyclic peptide solution.

[0051] 2. First step purification:

[0052] The lanreotide cyclic peptide solution obtained in step 1 was purified by HPLC using C18 chromatography stationary phase. The chromatographic column was 10 μm in diameter and 200 × 250 mm in length. Mobile phase A consisted of a 40 mmol / L ammonium acetate aqueous solution, adjusted to pH 4.3 with acetic acid. Mobile phase B was 90% acetonitrile:10% purified water (v / v). The flow rate was 700 mL / min, the wavelength was 220 nm, the sample loading was 200 g, and a gradient elution of 10–50 wt% mobile phase B was performed for 60 min to obtain a once-purified lanreotide cyclic peptide solution.

[0053] 3. Second step purification:

[0054] The purified lanreotide cyclic peptide solution was diluted with purified water at a volume ratio of 1:1, and the chromatographic packing material was C18. The second purification step was performed using a 10 μm stationary phase. Mobile phase A consisted of a 30 mmol / L sodium acetate aqueous solution, with the pH adjusted to 6.5 using 1 mol / L hydrochloric acid. Mobile phase B was acetonitrile, with a flow rate of 700 mL / min and a detection wavelength of 220 nm. Gradient elution was performed using 10–50 wt% mobile phase B for 60 min. The peptide solutions were collected in fractions, and the pH of the peptide solutions was adjusted to 4.0 using acetic acid to obtain the second-purified lanreotide cyclic peptide solution.

[0055] 4. Using acetic acid to replace salt:

[0056] The acetic acid method involves changing the salt, and then using C18 to purify the lanreotide cyclic peptide solution in a second step. A 10 μm stationary phase was used for salt replacement. Mobile phase A was a 0.3% (V / V) aqueous acetic acid solution, and mobile phase B was a 0.3% (V / V) acetic acid acetonitrile solution. The flow rate was 700 mL / min, the detection wavelength was 220 nm, and gradient elution was performed: 10–60 wt% mobile phase B was eluted for 35 min to obtain a high-purity and high-stability lanreotide acetate solution after salt replacement.

[0057] Example 3

[0058] A method for drying sterile lanreotide raw material, comprising the following steps:

[0059] 1. Take 5L of lanreotide acetate solution (net peptide 100g) from Example 2, first use a pressure pump to pass it through a primary 0.2μm filter for sterilization, and then through a secondary 0.2μm filter for sterilization to obtain a sterile lanreotide acetate solution.

[0060] 2. In the sterile area, directly pour the sterile filtered lanreotide acetate solution into the sterile freeze-drying tray, with a thickness not exceeding 1.5 cm;

[0061] 3. Aseptic freeze-drying:

[0062] Refrigeration and insulation: Lower the temperature of the partition from room temperature to 5°C and keep it constant for 1 hour;

[0063] Pre-freezing: The pre-freezing temperature is -45℃, and it is kept constant for 5 hours under a vacuum of 0.3mbar;

[0064] First-stage drying: heating from -45℃ to -15℃ and maintaining a constant temperature under a vacuum of 0.3mbar for 24 hours;

[0065] Secondary drying: Heat from -15℃ to 15℃ and maintain a constant temperature under a vacuum of 0.3mbar for 24 hours, then continue heating to 35℃ and maintain a constant temperature under a vacuum of 0.4mbar for 18 hours;

[0066] After drying, 95g of sterile lanreotide lyophilized powder with high purity and high stability (≥99.5% purity, ≤0.10% single impurity) was obtained, with a yield of 95%.

[0067] Example 4

[0068] A method for drying sterile lanreotide raw material, comprising the following steps:

[0069] 1. Take 2L of lanreotide acetate solution (net peptide 40g) from Example 2, first use a peristaltic pump to pass it through a first-stage 0.2μm filter for sterilization, and then pass it through a second-stage 0.2μm filter for sterilization to obtain a sterile lanreotide acetate solution;

[0070] 2. The sterile lanreotide acetate solution was directly pumped into the spray drying equipment in a sterile area. Sterile nitrogen was used as the carrier gas, with an inlet temperature of 120°C and an outlet temperature of 60°C. Spray drying was carried out under sterile conditions for 4 hours, yielding 32.2 g of sterile active pharmaceutical ingredient (API), with a yield of 80.5%. The MS chromatogram of the final lyophilized product is shown below. Figure 4 .

[0071] Comparative Example 1

[0072] Based on Example 2, the column packing material was replaced with C4-HG to prepare lanreotide acetate solution, and the drying method was the same as in Example 3.

[0073] Comparative Example 2

[0074] Based on Example 2, the mobile phase of the purification step was adjusted as follows:

[0075] 1. Oxidation of linear crude lanreotide peptide:

[0076] 400 grams of crude linear peptide of Lanreitide were dissolved in 400 L of acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water was 1:9. The pH of the crude linear peptide was adjusted to 6.7 with 25% ammonia solution. It was oxidized with 0.3% hydrogen peroxide solution. After HPLC detection and ring closure, it was filtered through a 0.45 μm filter membrane for later use.

[0077] 2. First step purification:

[0078] The chromatographic packing material is C18. The chromatographic column was 10 μm in diameter and 200*250 mm in length. Mobile phase A was a 0.1% (v / v) aqueous solution of trifluoroacetic acid, and mobile phase B was a 0.1% (v / v) trifluoroacetic acid-acetonitrile solution. The flow rate was 700 mL / min, the wavelength was 220 nm, the sample loading was 200 g, and a gradient elution of 10–50 wt% mobile phase B was performed for 60 min to obtain the purified lanreotide cyclic peptide solution.

[0079] 3. Acetic acid method for replacing salt:

[0080] The acetic acid method involves changing the salt, and then using C18 to purify the lanreotide cyclic peptide solution. A 10 μm stationary phase was used for salt replacement. Mobile phase A was a 0.3% (V / V) aqueous acetic acid solution, and mobile phase B was a 0.3% (V / V) acetic acid acetonitrile solution. The flow rate was 700 mL / min, the detection wavelength was 220 nm, and gradient elution was performed: 10–60 wt% mobile phase B was eluted for 35 min to obtain a high-purity and high-stability lanreotide acetate solution after salt replacement.

[0081] The drying method is the same as in Example 3.

[0082] Test Example 1

[0083] The purity, total impurities, and yield of the sterile lanreotide active pharmaceutical ingredient in Examples 3-4 and Comparative Examples 1-2 were determined. Yield (%) = (Weight of finished product / Weight of net peptide) × 100%

[0084] A reversed-phase octadecylsilane-bonded silica gel column (4.6*250mm, 3μm) was used. Mobile phase A was 17.6mM disodium hydrogen phosphate aqueous solution, with the pH adjusted to 3 by phosphoric acid. Mobile phase B was acetonitrile. The detection wavelength was 220nm, and the flow rate was 0.6mL / min. The % gradient of mobile phase A was 72% isocratic from 0min to 35min, and 72%-40% gradient from 35min to 45min.

[0085] name purity(%) Total impurities (%) Maximum single impurity (%) Yield (%) Example 3 99.78 0.22 0.03 40.7 Example 4 99.83 0.17 0.03 32.8 Comparative Example 1 99.16 0.84 0.47 --- Comparative Example 2 99.54 0.46 0.23 ---

[0086] Experimental results show that the preparation method of lanreotide active pharmaceutical ingredient of the present invention can improve the yield and purity of the product and reduce the impurity content; comparative examples 1 and 2 could not obtain active pharmaceutical ingredient with single impurity ≤0.10%.

[0087] See Figure 2 In Example 2, the purity of the product after the first step of purification was 98.8%.

[0088] See Figure 3 In Example 2, the purity of the product after the second purification step was 99.9%, with single impurities ≤0.10%.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a sterile lanreotide active pharmaceutical ingredient, characterized in that, Includes the following steps: (1) Dissolve the crude linear peptide of Lanrei peptide in an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:

9. Adjust the pH value to 6.7~6.8 with ammonia water, oxidize with 0.3% hydrogen peroxide, filter, and obtain Lanrei peptide cyclic peptide solution. (2) The crude lanreotide cyclic peptide solution was purified for the first time using octadecylsilane-bonded silica gel as the stationary phase. Mobile phase A was an aqueous solution of ammonium acetate with a mass concentration of 30 mmol / L to 100 mmol / L. The pH value was adjusted to 3.7 to 4.7 using an acid regulator. Mobile phase B was an aqueous solution of acetonitrile with a mass concentration of 10%:90% (V / V). The elution time in the elution program was 40-80 min. Mobile phase B was 10-50 wt%. The purified lanreotide cyclic peptide solution was collected. (3) Mix the first-purified lanreotide cyclic peptide solution and water at a volume ratio of 1:1-2. Then, perform a second purification on the mixed first-purified lanreotide linear peptide solution. The stationary phase is octadecylsilane-bonded silica gel, the mobile phase A is a sodium acetate aqueous solution with a mass concentration of 20 mmol / L to 100 mmol / L, and the pH value is adjusted to 5.8 to 6.8 using an acidic regulator. The mobile phase B is 100% acetonitrile. The elution time in the elution program is 40 to 80 min, and the mobile phase B is 10 to 50 wt%. Collect the eluent and adjust the pH value of the eluent to 3.0 to 5.0 using an acidic regulator to obtain the second-purified lanreotide cyclic peptide solution. (4) The purified lanreotide cyclic peptide solution was subjected to salt exchange using octadecylsilane-bonded silica gel as the stationary phase. Mobile phase A was an aqueous acetic acid solution with a volume concentration of 0.3% to 0.5%. Mobile phase B was a 0.3% (V / V) acetic acid acetonitrile solution or a 0.5% (V / V) acetic acid acetonitrile solution for gradient elution. The elution time in the elution program was 25-50 min, and the mobile phase B was 10-60 wt%, to obtain the lanreotide acetate solution. (5) Sterilize and filter the lanreotide acetate and dry it to obtain the finished product.

2. The method for preparing sterile lanreotide active pharmaceutical ingredient as described in claim 1, characterized in that, In step (1), the mass concentration of the linear crude lanreotide is 0.5~5 mg / mL.

3. The method for preparing sterile lanreotide active pharmaceutical ingredient as described in claim 1, characterized in that, In step (3), the acidity regulator is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, hydrochloric acid or perchloric acid.

4. The method for preparing sterile lanreotide raw material as described in claim 1, characterized in that, In step (5), the sterilization filtration includes two-stage or multi-stage sterilization filtration, and the filter element pore size is 0.2μm.

5. The method for preparing sterile lanreotide active pharmaceutical ingredient as described in claim 1, characterized in that, In step (5), the drying method is spray drying or freeze drying.

6. The method for preparing sterile lanreotide active pharmaceutical ingredient as described in claim 5, characterized in that, The freeze-drying process includes the following steps: S1: Refrigeration and heat preservation: Refrigeration temperature is 4~6℃, heat preservation for 1-2 hours. S2: Pre-freezing: Pre-freezing temperature is -45~-35℃, vacuum degree is 0.25~0.35mbar, and the temperature is maintained for 3-4 hours. S3: Primary drying: drying temperature -20~-10℃, vacuum degree 0.25~0.35mbar, holding time 24-246h. S4: Secondary drying: Drying temperature is 10~20℃, vacuum degree is 0.25~0.35mbar, keep warm for 20-22h, continue to raise the temperature to 25~35℃, vacuum degree is 0.3~0.5mbar, keep warm for 12-15h.

7. The method for preparing sterile lanreotide active pharmaceutical ingredient as described in claim 5, characterized in that, The spray drying process involves an inlet air temperature of 100℃~140℃, an outlet air temperature of 50℃~80℃, nitrogen as the carrier gas, and a drying time of 10-15 hours.

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