A method of purifying tildipirosin

Abstract

The application provides a purification method of tildipirosin, which comprises the following steps: first chromatographic purification: performing gradient elution on a reversed-phase chromatographic column for a tildipirosin crude product solution, and collecting a first fraction with a purity of greater than or equal to 95%; second chromatographic purification: performing gradient elution on a reversed-phase chromatographic column for the first fraction, and collecting a second fraction with a purity of greater than or equal to 99%; crystallization and salt conversion: adjusting the second fraction to have a volume percentage concentration of acetonitrile of not higher than 15%, and adjusting the pH value to be not higher than 5.0, standing for crystallization, and centrifugally collecting a solid; dissolving the obtained solid with a sodium hydroxide solution, and freeze-drying, to obtain a tildipirosin pure product. The method realizes a comprehensive breakthrough in purity, yield and safety of tildipirosin, simplifies production steps, greatly reduces cost, is suitable for low-purity low-quality crude products, and avoids highly corrosive and highly toxic reagents such as perchlorate and trifluoroacetic acid.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug purification technology, mainly to a purification method for tiduglutide, specifically a purification method for high-purity tiduglutide suitable for industrial-scale production. Background Technology

[0002] Teduglutide, a glucagon-like peptide-2 (GLP-2) analog, is a key drug for the treatment of short bowel syndrome in adults. Its chemical structure is a polypeptide containing 33 amino acids (molecular formula C). 164 H 252 N 44 O 55 S (molecular weight 3752).

[0003] Tiduglutide is a glucagon-like peptide-2 (GLP-2) analogue. It is primarily used to treat adult and pediatric patients aged 1 year and older with short bowel syndrome (SBS) who require parenteral support. The recommended dose is 0.05 mg / kg once daily via subcutaneous injection. It works by mimicking the effects of naturally secreted GLP-2, increasing intestinal villus height and crypt depth, promoting intestinal repair and normal growth, thereby maintaining mucosal integrity. This helps accelerate intestinal adaptation after bowel resection, enhances the selective barrier function of the small intestine, increases intestinal absorptive surface area, and consequently improves the absorption of fluids, nutrients, and electrolytes, reducing dependence on parenteral nutrition / intravenous infusion (PN / IV).

[0004] The existing purification technology for tiduglutide has the following drawbacks: 1. The mobile phase contains highly corrosive reagents such as perchloric acid and trifluoroacetic acid (e.g., CN104045707A), resulting in high equipment wear and safety hazards; 2. Low-purity crude products (20%-40%) require multiple reversed-phase high-performance liquid chromatography (RP-HPLC) purifications, with yields below 65% (e.g., CN104045707A yield is less than 70%); 3. The salt conversion process is complex, and there is a high risk of excessive residual solvent in the product.

[0005] The foregoing background information is intended to help those skilled in the art understand prior art that is similar to the present invention, and to facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clearly stated that, in the absence of clear evidence that the above content was disclosed before the filing date of this patent application, the foregoing background information should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0006] Technical issues To address the aforementioned problems, the present invention aims to provide a novel purification method for tiduglutide. This method is based on the unique inventive concept of "differential separation - precise purification - crystallization and salt conversion." Through a three-step synergistic process design, it achieves a comprehensive breakthrough in the purity, yield, and safety of tiduglutide, simplifies production steps, significantly reduces costs, and is suitable for low-purity crude products. It avoids highly corrosive and toxic reagents such as perchlorate and trifluoroacetic acid.

[0007] Technical solution (1) A method for purifying tiduglutide, comprising the following steps: First chromatographic purification: The crude tiduglutide solution was subjected to gradient elution on a reversed-phase column, and the first fraction with a purity ≥95% was collected; wherein, mobile phase A was phosphate-triethylamine buffer at pH 2.0-3.0, and mobile phase B was acetonitrile, and during the gradient elution process, the volume percentage concentration of mobile phase B increased from 30% to 35%; Second chromatographic purification: The first fraction was subjected to gradient elution on a reversed-phase column, and the second fraction with a purity ≥99% was collected; wherein, mobile phase A was phosphate-triethylamine buffer at pH 6.0-7.0, and mobile phase B was acetonitrile, and during the gradient elution process, the volume percentage concentration of mobile phase B increased from 33% to 35%; Crystallization and salt conversion: Adjust the acetonitrile volume percentage concentration of the second fraction to no higher than 15% and adjust its pH value to no higher than 5.0. Allow it to stand to crystallize, and collect the solid by centrifugation. Dissolve the obtained solid in sodium hydroxide solution and freeze-dry to obtain pure teduglutide.

[0008] Preferably, the crude teduglutide solution is a solution obtained by dissolving crude teduglutide in a 0.5-1.0% ammonia solution.

[0009] Preferably, the purity of the crude tiduglutide is as low as 22.5%-52.3%.

[0010] Preferably, the gradient elution time for the first chromatographic purification is 20-40 min.

[0011] Preferably, the mass-to-volume ratio of the crude teduglutide to the ammonia solution is 20g-50g:1L.

[0012] Preferably, the phosphate-triethylamine buffer contains 0.2-0.5% phosphate and 0.2-0.5% triethylamine.

[0013] Preferably, the pH of the phosphate-triethylamine buffer solution is adjusted using phosphoric acid and ammonia.

[0014] Preferably, the gradient elution time for the second chromatographic purification is 30-40 min.

[0015] Preferably, adjusting the acetonitrile volume percentage concentration of the second fraction to no more than 15% is achieved by diluting the second fraction with purified water.

[0016] Preferably, the mobile phase A of the first and / or second chromatographic purification contains 1.7-2.0 mM hydroxybenzaldehyde. The inventors unexpectedly discovered that adding trace amounts of hydroxybenzaldehyde to mobile phase A can increase the single-load volume by approximately 60%-100% while maintaining or even slightly improving separation efficiency and yield. The proposed mechanism is as follows: hydroxybenzaldehyde acts as a dynamic selectivity modifier; its benzene ring provides hydrophobic interaction sites, and its aldehyde and phenolic hydroxyl groups provide hydrogen bond donors / acceptors. This allows for reversible non-covalent interactions between the peptide molecule, the mobile phase, and the stationary phase. The hydrophobic-hydrogen bond synergistic effect selectively alters the apparent hydrophobicity difference between tiduglutide and structurally similar impurities, increasing the separation factor of key impurity pairs. Ultimately, baseline separation is achieved even with higher load volumes, thus ensuring efficiency and yield.

[0017] Preferably, the temperature for static crystallization is 2℃-8℃.

[0018] Preferably, in the crystallization-to-salt step, the second fraction is adjusted to have an acetonitrile volume percentage concentration of 15-5%.

[0019] Preferably, the pH for static crystallization is 4.0-5.0.

[0020] Preferably, the freeze-drying conditions are as follows: pre-freezing at -40°C to -50°C for 2-4 hours, followed by sublimation drying under a vacuum of 10-30 Pa.

[0021] Preferably, the reversed-phase column in the first chromatographic purification step and the reversed-phase column in the second chromatographic purification step are the same or different columns, and their column temperature is controlled at 25℃-35℃.

[0022] (2) A pure tiduglutide product, wherein the pure tiduglutide product is prepared by the method described above, and the HPLC purity of the pure tiduglutide product is ≥99.0%, the maximum single impurity is ≤0.1%, and the total yield is ≥73% based on crude tiduglutide.

[0023] (3) A crystalline form of teduglutide intermediate, said crystalline form of teduglutide intermediate is obtained by the static crystallization and centrifugation steps in the crystallization-saltification step of the aforementioned teduglutide purification method.

[0024] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0025] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0026] Beneficial effects This invention employs a unique process combination, using a two-step purification method involving crystallization and salt-replacement freeze-drying, to obtain teduglutide with excellent yield and purity. This method solves the long-standing problems of purity, yield, safety, and cost in the industrial production of teduglutide from the source, and therefore has the following beneficial effects.

[0027] (1) The purity and yield of the product were significantly improved, achieving a stable HPLC purity of ≥99.0% and a maximum single impurity content of ≤0.1%, which fully meets the technical requirements for registration of human pharmaceutical products. More importantly, the total yield of the present invention can be consistently maintained above 73% and can reach nearly 80% within a wide range of starting crude product purity, which is significantly better than the existing technology level (usually below 70%).

[0028] (2) The production steps are simplified and the cost is greatly reduced. Only two chromatographic purifications and one crystallization freeze drying are required. The three major functions of "purification, desalting and salt conversion" are integrated into three steps, which greatly shortens the production cycle and reduces the cost of solvents, chromatographic packing materials and labor. It is very suitable for industrial scale-up.

[0029] (3) The use of corrosive reagents has been completely eliminated, achieving green production. The entire process uses a mild phosphate-triethylamine buffer system as the mobile phase, avoiding the use of highly corrosive or highly toxic reagents such as perchlorate and trifluoroacetic acid, extending equipment life, ensuring personnel safety, and reducing the difficulty of treating waste.

[0030] (4) The product has excellent stability. The crystallization process effectively removes trace amounts of organic solvents and hygroscopic impurities, resulting in a fluffy final freeze-dried product with good resolubility and significantly improved long-term storage stability.

[0031] (5) The method is robust and has a wide range of applications. The method of this invention can still efficiently purify low-quality crude products with a purity as low as 22.5% to obtain finished products that meet quality standards, demonstrating strong process tolerance and wide applicability.

[0032] The present invention adopts the above-mentioned technical solution to achieve the above objectives, which makes up for the shortcomings of the prior art, is reasonably designed, and is easy to operate. Attached Figure Description

[0033] To make the above and / or other objects, features, advantages and examples of the present invention more apparent and understandable, the accompanying drawings used in the specific embodiments 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 from these drawings without creative effort.

[0034] Figure 1 This shows the chromatogram of the finished product, tiduglutide. Detailed Implementation

[0035] Those skilled in the art can refer to the content of this document and appropriately replace and / or modify the process parameters to achieve the desired results. 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 this invention. The products and preparation methods described in this invention have been described through preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0036] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. This 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, patent cases, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0037] Unless otherwise stated, all percentages, parts, proportions, etc. are by weight; other statements include, but are not limited to, “%”, “wt%”, “mass%” meaning weight percentage, “mol%” meaning mole percentage, and “vol%” meaning volume percentage.

[0038] When quantities, concentrations, or other numerical values ​​or parameters are given as ranges, preferred ranges, or a series of upper and lower preferred values, it should be understood that they specifically disclose all ranges formed by any pair of values ​​of any larger or preferred range limit and any smaller or preferred range limit, regardless of whether the ranges are disclosed separately. For example, when describing a range of “1 to 5 (1-5)”, the described range should be understood to include ranges such as “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, wherever numerical ranges are described herein, the ranges include the range endpoints as well as all integers and fractions within that range.

[0039] Unless otherwise specified, the materials, methods, and examples described herein are exemplary and not limiting. While similar or equivalent methods and materials can be used to implement or test the invention, suitable methods and materials are described herein.

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0041] Overview of this invention First, this invention provides a novel "two-step reversed-phase chromatography" + "crystallization-to-salt" strategy. Through in-depth research on the dissociation states and hydrophobicity differences of tiduglutide and its main impurities under different pH conditions, the inventors unexpectedly discovered: The first step, acid separation: Under acidic conditions of pH 2.0-3.0, a unique phosphate-triethylamine / acetonitrile system is used. At this stage, tiduglutide is effectively separated from most impurities with significantly different hydrophobicities. This step eliminates trifluoroacetic acid and perchlorate, making it not only environmentally friendly, but more importantly, the ionic environment provided by the phosphate-triethylamine system unexpectedly results in a higher separation degree between the target peak and impurity peaks compared to the traditional trifluoroacetic acid system. Comparative analysis of Examples 1 and 2 shows that the purity of the crude product can be increased from 52.3% to over 95% in one step, with a yield as high as 84%, while the yield of the trifluoroacetic acid system is only 78.5%.

[0042] The second step, neutral purification: The fraction obtained in the first step with a purity of not less than 95% is subjected to a second chromatographic purification in a neutral phosphate-triethylamine / acetonitrile system at pH 6.0-7.0. This is the key to the invention. In a near-neutral environment, the dissociation state of the acidic / basic amino acid side chains on the teduglutide molecule is significantly different from that under acidic conditions. This exposes new differences in charge and hydrophobicity in structurally very similar "stubborn" impurities (such as racemic aspartic acid, differences in amidation degree, etc.) that co-elute with teduglutide under acidic conditions, allowing them to be easily separated. This allows the product purity to easily exceed the 99% barrier. Surprisingly, when the mobile phase pH deviates from the neutral range (such as pH 7.0-8.0 in Example 4), the yield of the second purification step drops sharply from 89.2% to 75.5%, proving that this neutral pH range is the preferred range for achieving high-yield purification.

[0043] Secondly, after completing the two-step chromatographic purification, this invention creatively introduces a "crystallization-to-salt" step, simultaneously solving the problems of solvent replacement, salt form conversion, and further purification. Through systematic condition screening, the inventors discovered that by precisely controlling three key physicochemical parameters of the chromatographic fraction, tiduglutide can be induced to crystallize and precipitate in a specific form, while a large number of impurities are suppressed in the mother liquor. Parameter 1: Organic phase ratio. The acetonitrile concentration in the second-step fraction is diluted with water to less than 15% by volume. Below this threshold (in Example 5, the yield of 85.1% with an acetonitrile ratio of 15-20% is much lower than the 98% in Example 1 of this invention), the solubility of tiduglutide decreases significantly, driving its crystallization.

[0044] Parameter 2: pH value. Adjust the pH value of the system to 4.0-5.0. This pH range is close to the isoelectric point of tiduglutide, making its net charge approach zero and minimizing intermolecular electrostatic repulsion, which is conducive to crystal nucleation and crystal growth. If the pH deviates from this range (such as pH 6.0 in Example 6), the yield will drop sharply to 56.1%, demonstrating the importance of this pH window.

[0045] Parameter 3: Temperature. Allowing the product to stand at a low temperature of 2℃-8℃ further reduces its solubility and increases the crystallization yield.

[0046] Finally, the crystals collected by centrifugation were reconstituted with an appropriate amount of sodium hydroxide solution and then freeze-dried. During this process, teduglutide was reconstituted into a salt, yielding the clinically usable teduglutide sodium salt form in one step, completely avoiding the cumbersome and wasteful process of traditional dialysis or re-chromatography.

[0047] In each example and comparative example, the purity and yield of tiduglutide were determined by the following high-performance liquid chromatography (HPLC) method: Column: Venusil XBP C8, 5μm, 4.6×250mm (or other equivalent octylsilane-bonded silica column); Mobile phase A: 25 mM sodium dihydrogen phosphate aqueous solution (pH adjusted to 2.5 with phosphoric acid) / acetonitrile = 90 / 10 (v / v); Mobile phase B: 25 mM sodium dihydrogen phosphate aqueous solution (pH adjusted to 2.5 with phosphoric acid) / acetonitrile = 20 / 80 (v / v); Gradient program: 0 min (100% A) → 5 min (100% A) → 35 min (0% A, 100% B) → 40 min (0% A, 100% B) → 40.1 min (100% A) → 50 min (100% A); Column temperature: 30℃; flow rate: 1.0 mL / min; detection wavelength: 214 nm; injection volume: 10 μL.

[0048] Yield calculation method: Yield = (Weight of obtained product × Purity of product in this step) / (Weight of starting material × Purity of starting material) × 100%. The total yield is the product of the yields of each step.

[0049] The present invention is described in detail below. Example 1: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0050] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0051] Step 2: Using a single loading volume of approximately 5 mg / mL, the crude tiduglutide solution was subjected to gradient elution using RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography) for 30 min. The tiduglutide fraction was collected from the appearance of the main peak to its end, and the fraction with a purity of over 95% was separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octadecylsilane-bonded silica gel was used as the stationary phase.

[0052] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 6.0 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The acetonitrile concentration of teduglutide with a purity of over 99% is separated from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0053] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 14% and the pH value to 5, then allow it to stand to crystallize. After centrifugation, dissolve the solid in a suitable sodium hydroxide solution and freeze-dry it to obtain pure teduglutide.

[0054] Table 1 shows the detection chromatogram data of crude tiduglutide, which indicates that it contains many impurities and has a relatively complex composition.

[0055] Table 1. Spectral data of crude tiduglutide

[0056]

[0057]

[0058] Figure 1 Table 2 shows the detection spectral data of the teduglutide product, indicating that it has few impurities and high purity.

[0059] Table 2. Spectral data of teduglutide finished product

[0060] Example 2: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0061] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0062] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The fraction with a purity of tiduglutide of ≥95% was separated for further purification. The gradient elution used trifluoroacetic acid aqueous solution (0.2% by volume) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0063] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate-triethylamine with a pH of 7.0 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The acetonitrile concentration of teduglutide with a purity of over 99% is separated from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0064] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 10% and the pH value to 5, then allow it to stand to crystallize. After centrifugation, dissolve the solid in a suitable sodium hydroxide solution and freeze-dry it to obtain pure teduglutide.

[0065] The purification process is basically the same as in Example 1, except that the mobile phase A system in step 2 is a 0.2% trifluoroacetic acid aqueous solution.

[0066] Example 3: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0067] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage, ammonia 0.5-1.0%) to obtain a crude teduglutide solution.

[0068] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The fraction with a purity of tiduglutide of ≥95% was separated for further purification. The gradient elution used a phosphoric acid aqueous solution (0.1% by volume) at pH 2.0 as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0069] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 6.5 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The acetonitrile concentration of teduglutide with a purity of over 99% is separated from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0070] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 12% and the pH value to 4.5, allow it to stand to crystallize, centrifuge the resulting solid, dissolve it in a suitable sodium hydroxide solution, freeze-dry it, and the resulting product is pure teduglutide.

[0071] The purification process is basically the same as in Example 1, except that the mobile phase A system in step 2 is a 0.1% aqueous phosphoric acid solution.

[0072] Example 4: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0073] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage: 1.0% ammonia) to obtain a crude teduglutide solution.

[0074] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The tiduglutide fraction with a purity of over 95% was separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0075] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 7.8 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The acetonitrile concentration of teduglutide with a purity of over 99% is separated from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0076] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 14% and the pH value to 4.2, then allow it to stand to crystallize. After centrifugation, dissolve the solid in a suitable sodium hydroxide solution and freeze-dry it to obtain pure teduglutide.

[0077] The purification process is basically the same as in Example 1, except that the mobile phase A system in step 3 is an aqueous solution of phosphate-triethylamine with pH=7.8.

[0078] Example 5: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0079] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0080] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The tiduglutide fraction with a purity of over 95% was separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0081] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 7.4 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The acetonitrile concentration of teduglutide with a purity of over 99% is separated from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0082] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 18% and the pH value to 5, then allow it to stand to crystallize. After centrifugation, dissolve the solid in a suitable sodium hydroxide solution and freeze-dry it to obtain pure teduglutide.

[0083] The purification process is basically the same as in Example 1, except that in step 4, the acetonitrile ratio for diluting the solution is less than 20%.

[0084] Example 6: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0085] Step 1: Dissolve 50% pure crude teduglutide in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0086] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The tiduglutide fraction with a purity of over 95% was separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0087] Step 3: The collected teduglutide fraction with a purity of over 95% was subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 7.4 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) was used as mobile phase A, and acetonitrile was used as mobile phase B. The teduglutide with a purity of over 99% was separated, and the acetonitrile concentration was increased from 33% to 35% by volume percentage. Octylsilane-bonded silica gel was used as the stationary phase.

[0088] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 8% and the pH value to 6, then allow it to stand to crystallize. After centrifugation, dissolve the solid in a suitable sodium hydroxide solution and freeze-dry it to obtain pure teduglutide.

[0089] The purification process is basically the same as in Example 1, except that the pH is adjusted to 6 in step 4.

[0090] Example 7: A method for purifying crude tiduglutide is provided, and the specific steps are as follows.

[0091] Step 1: Dissolve crude teduglutide with a purity of 22.5% in an aqueous ammonia solution (volume percentage: 1.0% ammonia) to obtain a crude teduglutide solution.

[0092] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 5 mg / mL. The tiduglutide fraction was collected from the appearance of the main peak to the end of the main peak. The tiduglutide fraction with a purity of over 95% was separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) as the mobile phase A and acetonitrile as the mobile phase B. The acetonitrile concentration was increased from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase.

[0093] Step 3: The collected teduglutide fraction with a purity of over 95% is subjected to gradient elution again. A mixed aqueous solution of phosphate and triethylamine with a pH of 7.0 (volume percentage: 0.35% phosphate, 0.35% triethylamine, pH adjusted with ammonia) is used as mobile phase A, and acetonitrile is used as mobile phase B. The teduglutide with a purity of over 99% is separated, and the acetonitrile concentration is increased from 33% to 35% by volume percentage. Octylsilane-bonded silica gel is used as the stationary phase.

[0094] Step 4: Adjust the volume percentage of acetonitrile in the separated teduglutide fraction (purity above 99%) to 12.5% ​​and the pH value to 4.5, allow it to stand to crystallize, centrifuge the solid obtained, dissolve it in a suitable sodium hydroxide solution, freeze-dry it to obtain pure teduglutide.

[0095] The purification process is basically the same as in Example 1, except that the purity of the crude product in step 1 is 20%-30%.

[0096] Experimental Example 1: The yields and purities of steps 2, 3, and 4 in each of the statistical examples 1-7 are shown in Table 3.

[0097] Table 3. Yields and purity of each step in each example.

[0098] Table 3 shows that the first chromatographic purification, the second chromatographic purification, and the crystallization-salt conversion of the present invention constitute an interlocking, mutually restrictive, and synergistically progressive process data chain. These steps collectively demonstrate that the "three-in-one" design of this technical solution is not an arbitrary superposition of steps, but an organic whole with inherent logic and synergistic effects. The "three-in-one" steps of the present invention are a step-by-step purification process. The purity of the product at each step directly determines the feed quality of the next step, thus affecting the separation difficulty and efficiency of the next step. As can be seen from Table 3, the fraction obtained from the first chromatographic purification is not less than 95%, indicating that it contains relatively few impurities with properties similar to the target peptide. This high-purity fraction can significantly improve the purification effect under neutral pH conditions when entering the second chromatographic purification. The reason why the second chromatographic purification can achieve the ultimate purification "from 95% to 99%" within an extremely narrow pH window (6.0-7.0) is that the first chromatographic purification has already removed most of the impurities with large differences in hydrophobicity. What is "handed over" to the second chromatographic purification is a "pure raw material" with controllable impurity levels and a relatively concentrated impurity spectrum. The purity of the second-step chromatographic purification product is ≥99%, which means that the total amount of impurities has been reduced to below 1%. This "extremely clean" solution environment is a necessary prerequisite for the third-step crystallization and salt conversion to crystallize smoothly with an ultra-high purity of no less than 99.3%. If the purity of the second-step product is only 98% or lower, the purity and yield of the third-step crystallization will decrease significantly, and it may even be impossible to obtain qualified crystals.

[0099] Example 8: Based on the aforementioned Example 1, an optimized method for purifying crude tiduglutide is provided, the specific steps of which are as follows.

[0100] Step 1: Dissolve crude teduglutide with a purity of 52.3% in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0101] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 8 mg / mL. Tiduglutide fractions were collected from the appearance of the main peak to its cessation. The fractions with a purity of 95% or higher were separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, and 1.8 mM hydroxybenzaldehyde, pH adjusted with ammonia) as mobile phase A, and acetonitrile as mobile phase B, with the acetonitrile concentration increasing from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase. The yield was 83.6%, and the purity was 96.5%.

[0102] Step 3: The collected tiduglutide fraction with a purity of over 95% was subjected to gradient elution again. A mixed aqueous solution of phosphate-triethylamine at pH 6.0 (obtained by adjusting the pH of the phosphate-triethylamine mixed aqueous solution from Step 2 with ammonia) was used as mobile phase A, and acetonitrile was used as mobile phase B. The acetonitrile concentration was increased from 33% to 35% by volume to separate tiduglutide with a purity of over 99%. Octylsilane-bonded silica gel was used as the stationary phase. The yield was 90.4%, and the purity was 99.3%.

[0103] Step 4: The tiduglutide fraction with a purity of over 99% was separated above. The acetonitrile volume percentage in the fraction was adjusted to 14%, and the pH value was adjusted to 5. After standing and crystallization, the resulting solid was dissolved in a suitable sodium hydroxide solution and freeze-dried to obtain pure tiduglutide. The yield was 97.8%, and the purity was 99.3%.

[0104] The purification process was basically the same as in Example 1, except that the single loading amount for chromatographic purification in step 2 was 8 mg / mL, and 1.8 mM hydroxybenzaldehyde was added to the mixed aqueous solution of phosphate-triethylamine.

[0105] Example 9: Based on the aforementioned Example 6, a method for purifying crude tiduglutide is provided, the specific steps of which are as follows.

[0106] Step 1: Dissolve 50% pure crude teduglutide in an aqueous ammonia solution (volume percentage: 0.8% ammonia) to obtain a crude teduglutide solution.

[0107] Step 2: The crude tiduglutide solution was subjected to gradient elution by RP-HPLC for 30 min at a single loading volume of approximately 10 mg / mL. Tiduglutide fractions were collected from the appearance of the main peak to its cessation. The fractions with a purity of 95% or higher were separated for further purification. The gradient elution used a mixed aqueous solution of phosphate and triethylamine at pH 2.3 (volume percentage: 0.35% phosphate, 0.35% triethylamine, 2.0 mM hydroxybenzaldehyde, pH adjusted with ammonia) as mobile phase A, and acetonitrile as mobile phase B, with the acetonitrile concentration increasing from 30% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase. The yield was 84.3%, and the purity was 95.5%.

[0108] Step 3: The collected tiduglutide fraction with a purity of over 95% was subjected to gradient elution again. A mixed aqueous solution of phosphate-triethylamine at pH 7 (obtained by adjusting the pH of the phosphate-triethylamine mixed aqueous solution from Step 2 with ammonia) was used as mobile phase A, and acetonitrile was used as mobile phase B. Tiduglutide with a purity of over 99% was separated, with the acetonitrile concentration increasing from 33% to 35% by volume. Octylsilane-bonded silica gel was used as the stationary phase. The yield was 89.8%, and the purity was 99.6%.

[0109] Step 4: The tiduglutide fraction with a purity of over 99% was separated above. The acetonitrile volume percentage in the fraction was adjusted to 8%, and the pH value to 6. After standing and crystallization, the resulting solid was dissolved in a suitable sodium hydroxide solution and freeze-dried to obtain pure tiduglutide. The yield was 97.7%, and the purity was 99.3%.

[0110] The purification process is basically the same as in Example 6, except that the single loading amount for chromatographic purification in step 2 is 10 mg / mL, 1.8 mM hydroxybenzaldehyde is added to the phosphate-triethylamine mixed aqueous solution, and the pH of mobile phase A in step 3 is 7.

[0111] The inventors discovered that adding trace amounts (1.7-2.0 mM) of hydroxybenzaldehyde to a mixed aqueous solution of phosphate-triethylamine significantly increases the single chromatographic injection volume to 8-10 mg / mL. Tiduglutide exhibits slight differences in its three-dimensional conformation, surface charge distribution, and the microenvironment surrounding the N-terminal amino group compared to impurities. Therefore, the following mechanism is hypothesized: hydroxybenzaldehyde acts as a dynamic selectivity modifier in chromatographic purification. Its benzene ring provides hydrophobic interaction sites, while its aldehyde and phenolic hydroxyl groups provide hydrogen bond donors / acceptors. This enables reversible non-covalent interactions between the peptide molecule, mobile phase, and stationary phase. The synergistic effect of hydrophobic-hydrogen bonds selectively alters the apparent hydrophobicity difference between tiduglutide and structurally similar impurities, increasing the separation factor of key impurity pairs. Ultimately, baseline separation is achieved even with higher loading volumes. Macroscopically, this significantly increases the loading volume while maintaining efficiency and yield, ultimately achieving high loading separation that is impossible under traditional physical partitioning mechanisms. This provides a strong guarantee for higher loading volumes through chemical-chromatographic coupling.

[0112] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0113] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0114] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0115] While the foregoing detailed descriptions have shown, described, and pointed out novel features applicable to various embodiments, it should be understood that various omissions, substitutions, and changes may be made to the form and details of the described apparatus or methods without departing from the spirit of this disclosure. Furthermore, the various features and methods described above may be used independently of each other or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. Many of the foregoing embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or applications, as well as their obvious modifications and equivalents. Therefore, the invention is not intended to be limited to the specific disclosure of the preferred embodiments herein.

[0116] All matters not covered in this invention are common knowledge.

Claims

1. A method for purifying crude tiduglutide, characterized in that... Includes the following steps: First chromatographic purification: The crude tiduglutide solution was subjected to gradient elution on a reversed-phase column, and the first fraction with a purity ≥95% was collected; wherein, mobile phase A was phosphate-triethylamine buffer at pH 2.0-3.0, and mobile phase B was acetonitrile, and during the gradient elution process, the volume percentage concentration of mobile phase B increased from 30% to 35%; Second chromatographic purification: The first fraction was subjected to gradient elution on a reversed-phase column, and the second fraction with a purity ≥99% was collected; wherein, mobile phase A was phosphate-triethylamine buffer at pH 6.0-7.0, and mobile phase B was acetonitrile, and during the gradient elution process, the volume percentage concentration of mobile phase B increased from 33% to 35%; Crystallization and salt conversion: Adjust the acetonitrile volume percentage concentration of the second fraction to 12.5-14% and adjust its pH value to 4.5-5.0, allow it to stand to crystallize, and collect the solid by centrifugation; dissolve the obtained solid in sodium hydroxide solution and freeze-dry to obtain pure teduglutide sodium salt.

2. The method according to claim 1, characterized in that: The phosphate-triethylamine buffer solution contains 0.2-0.5% phosphate and 0.2-0.5% triethylamine.

3. The method according to claim 1, characterized in that: Adjusting the acetonitrile volume percentage concentration of the second fraction to 12.5-14% is achieved by diluting the second fraction with purified water.

4. The method according to claim 1, characterized in that: The mobile phase A of the first and / or second chromatographic purification contains 1.7-2.0 mM hydroxybenzaldehyde.

5. The method according to claim 1, characterized in that: The freeze-drying conditions are as follows: pre-freezing at -40°C to -50°C for 2-4 hours, followed by sublimation drying under a vacuum of 10-30 Pa.

6. The method according to claim 1, characterized in that: The reversed-phase column in the first chromatographic purification step and the reversed-phase column in the second chromatographic purification step are the same or different columns, and their column temperature is controlled at 25℃-35℃.

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