A preparation method of sulodexide raw material

Abstract

The present invention provides an industrial preparation method of sulodexide raw material, belonging to the field of biomedicine technology. The method uses heparinoids derived from porcine intestinal mucosa as the starting material, removes pathogens such as viruses and bacteria by alkali treatment (and subsequent potassium permanganate oxidation treatment), denatures impurities such as proteins by potassium permanganate oxidation and high temperature treatment and removes them by filtration, removes inorganic impurities such as sulfate by strong cationic resin exchange and strong anionic resin exchange, removes metal ion impurities by chelating resin adsorption, removes pigment impurities by peracetic acid or hydrogen peroxide, and then uses a preferred organic solvent precipitant to grade and precipitate to control the polysaccharide component, and finally purifies and freeze-dries to remove the solvent to obtain the sulodexide raw material. The preparation method is significantly different from the prior art, can more systematically and effectively control impurities and product quality, and is suitable for the preparation of sulodexide pharmaceutical product raw materials.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for preparing a sulodexide raw material drug. Background Art

[0002] Sulodexide, trade name Vessel Due F®, its active pharmaceutical ingredient (API) is a group of natural glycosaminoglycans and heparinoids. The Sulodexide molecule contains about 80% fast moving heparin (FMH) and about 20% dermatan sulfate (DS). Among them, fast moving heparin is mainly composed of -IdoA2S-GlcNS6S-repeating disaccharide units, while dermatan sulfate is mainly composed of -IdoA-GalN4S-repeating disaccharide units. Dermatan sulfate is occasionally sulfated at the 6-position of GalNAc and the 2-position of IdoA. Their main structural units are shown below:

[0003] -IdoA2S-GlcNS6S-;

[0004] -IdoA-GalN4S-.

[0005] As an anticoagulant thrombotic drug, sulodexide has a strong antithrombotic effect on both arteries and veins and can be used to treat various venous diseases. The antithrombotic effect of sulodexide is mainly related to the dose-dependent inhibition of some coagulation factors, especially the inhibition of activated factor X (Xa). Its interference with thrombin (i.e. activated factor II, IIa) is secondary, thus basically avoiding the side effects such as bleeding caused by general anticoagulant effects. The antithrombotic effect of sulodexide is not only through the action of antithrombin (Anti-thrombin-Ⅲ, AT-Ⅲ) on free thrombin, but also through the action of heparin cofactor II (Heparin Cofactor-Ⅱ, HC-Ⅱ) on thrombin bound to fibrin. Therefore, the antithrombotic effect of sulodexide produced by inhibiting thrombin is reflected in preventing thrombus formation and thrombus growth.

[0006] Sulodexide was developed by Alfa Wassermann Pharmaceuticals of Italy as an antithrombotic drug for the treatment of various cardiovascular diseases. It has been used in Italy, Spain, Eastern Europe, South America and Asia for nearly 40 years. In China, Sulodexide was approved for marketing in 2014, and its indication is "vascular diseases with a risk of thrombosis". Sulodexide is one of the best antithrombotic and anticoagulant drugs currently available in clinical practice. It can be injected intramuscularly, intravenously or orally, has high bioavailability and tolerance, significant efficacy, and a large clinical demand.

[0007] However, sulodexide has complex components and is difficult to prepare. Only the original preparation from Italy is sold in China, and no generic drugs or raw materials are on the market, making it difficult to meet the growing market demand for raw materials and preparations.

[0008] In terms of the prior art, a Chinese patent with publication number CN 106883321 B discloses a method for extracting sulodexide API from heparin sodium byproducts, by using hydrogen peroxide to oxidize and ethanol to grade heparin for alcohol precipitation. However, this scheme does not take into account the complex types of impurities in heparin byproducts, nor does it take into account the pathogen removal process of animal-derived products. In addition, it only relies on alcohol precipitation graded control to control the ratio of fast-moving heparin and dermatan sulfate. People in the field are well aware that this graded efficiency is very low, not only the alcohol precipitation time is long, but also the steps are relatively cumbersome, the solvent consumption is too large, and it is not conducive to industrial production. A Chinese patent with publication number CN 108484789 A discloses another method for preparing a sulodexide API, in which crude heparin sodium is graded by ethanol, adsorbed by anion exchange resin and eluted and purified by sodium chloride solutions of different concentrations, and then the sulodexide API is obtained by alcohol precipitation. However, this scheme also does not take into account the pathogen removal treatment of the raw material animal source. In addition, it is well known to technicians in the field that crude heparin raw materials contain a large amount of impurities, such as protein and nucleic acid residues. Proteins are often combined with heparin. It is difficult to effectively remove slow-moving heparin by only one-step alcohol precipitation. The subsequent anion exchange adopts 3% sodium chloride solution washing and 10% sodium chloride solution elution. This method will retain a large amount of slow-moving heparin, resulting in unqualified component ratios in the sulodexide product. Therefore, this preparation method is difficult to ensure the quality of the sulodexide product.

[0009] There are many other public schemes in the prior art. The general methods for separating slow-moving heparin and controlling the ratio of fast-moving heparin and dermatan sulfate components include potassium acetate precipitation, Benedict's reagent precipitation, ethanol graded precipitation and anion exchange. However, these schemes, on the one hand, are difficult to control the component ratio by only using partial graded methods, and on the other hand, basically do not consider the removal of pathogens, anionic and cationic impurities and complex protein impurities. Therefore, there are major defects and it is difficult to meet the requirements of pharmaceutical-grade products. Summary of the invention

[0010] In order to solve the deficiencies of the prior art, the present invention provides a new method for preparing sulodexide, which meets the industrial production and quality requirements of pharmaceutical-grade sulodexide bulk drugs.

[0011] The present invention is achieved through the following technical solutions:

[0012] The preparation method of sulodexide raw material uses heparinoid (or its crude product), which is a byproduct of porcine small intestinal mucosal heparin production, as the starting material, which contains a large amount of dermatan sulfate, fast-moving heparin and slow-moving heparin, etc. The preparation process sequentially removes pathogens such as viruses and bacteria in the crude heparinoid through alkali treatment (and subsequent potassium permanganate oxidation treatment), denatures impurities such as proteins through potassium permanganate oxidation and high-temperature treatment, and removes them by filtration. Then, according to the sulfate content in the crude raw material, if it is excessive, strong cation resin exchange and strong anion resin exchange are sequentially used to remove sulfate impurities, and then chelating resin is used to adsorb and remove metal ion impurities, sodium chloride is supplemented, and pigment impurities are removed by peracetic acid or hydrogen peroxide. Then, selected organic solvent precipitants of different concentrations are used to precipitate and grade to control the proportion of each polysaccharide component, and finally, it is refined and freeze-dried to remove water and solvent to obtain sulodexide raw material.

[0013] The key steps of the preparation method are briefly described as follows:

[0014] The first step is to inactivate and remove pathogens (viruses and microorganisms);

[0015] The second step is to remove protein impurities by denaturation, coagulation and filtration;

[0016] The third step is column purification: (cationic and anionic) ion exchange column purification to remove impurities such as free sulfate (if the sulfate content in the raw material exceeds the standard), and chelating resin adsorption to remove metal ion impurities;

[0017] The fourth step is decolorization and precipitation classification: peracetic acid or hydrogen peroxide is used to remove pigment impurities, followed by precipitation and classification of polysaccharide components with organic solvents;

[0018] The fifth step is refining and freeze-drying to obtain the sulodexide raw material.

[0019] A method for preparing a sulodexide bulk drug, characterized in that it comprises the following steps:

[0020] S1. Inactivation and removal of pathogens including viruses and microorganisms: Weigh the crude heparin raw material, dissolve it in water to form a 15% - 25% solution, add 3% - 5% sodium hydroxide solid of the total amount of the liquid, and stir at room temperature for not less than 0.5 h;

[0021] S2. Denaturation and coagulation of protein impurities and filtration removal: neutralize, add potassium permanganate, the amount of potassium permanganate added per 1 kg of crude heparin raw material should not exceed 66 g, heat to above 70°C, and maintain high temperature until the protein impurities are denatured and coagulated, then stop heating, continue stirring for not less than 10 min to allow the protein impurities to be fully precipitated, add filter aid, the amount of filter aid added per 1 kg of crude heparin raw material should be 72-132 g, filter, and cool the filtrate to room temperature;

[0022] S3, column purification and impurity removal: When the free sulfate content of the raw material is 0.5% or above, the feed liquid is first pumped through a strong cation resin exchange column and a strong anion resin exchange column in sequence for purification. Otherwise, the two column purifications are skipped, the feed liquid is adjusted to a weakly alkaline pH, and pumped through a chelating resin column. The dosage of the above three resins is generally the same, which is 3-5 times the amount of crude heparin raw material input;

[0023] S4, decolorization and precipitation classification: sodium chloride is added to the feed solution to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the crude heparin raw material feed amount, and the pH is controlled to be around 8 - 10. The reaction is carried out at room temperature for at least 0.5 h, and the pH is adjusted to be neutral. Acetone is added in an amount of 0.45 - 0.60 times the volume of the feed solution, and the mixture is stirred and allowed to stand for at least 4 h, and the precipitated paste is removed. Acetone is added to the clear liquid to a volume of 0.9 - 1.1 times the volume of the feed solution, and the mixture is stirred and allowed to stand for at least 4 h, and the supernatant is discarded. Purified water in an amount of 4 - 10 times the volume of the crude heparin raw material feed amount is added to the precipitated paste, and the mixture is stirred until dissolved. Acetone is added in an amount of 0.7 - 0.9 times the volume of the purified water, and the mixture is stirred and allowed to stand for at least 4 h, and the supernatant is discarded to obtain a precipitated paste;

[0024] S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 0.9-1.1 times the volume of acetone of the feed liquid, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with acetone, retain the precipitate, add 1.5 times the amount of acetone of the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of acetone 1-5 times until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the feed liquid in each freeze-drying plate is not more than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

[0025] Preferably, the crude heparinoid raw material in step S1 is derived from porcine intestinal mucosa.

[0026] In the present invention, the room temperature refers to a temperature range of 15°C - 35°C.

[0027] Based on the reason that the aforementioned alkaline treatment and potassium permanganate oxidation treatment of the present invention can remove pathogens such as viruses and bacteria, the treatment can be applied not only at the crude heparin raw material stage, but also in other steps of the preparation of sulodexide raw materials or in any order. According to the technical paradigm transformation or extension of the present invention, the use of alkaline treatment and potassium permanganate oxidation treatment in other process stages to remove pathogens should also be regarded as within the technical scope of the present invention.

[0028] Preferably, the amount of potassium permanganate in step S2 is adjusted according to the A when the feed liquid sample is adjusted to a solution concentration of 4 mg / mL. 280 Absorbance values ​​are added as follows:

[0029] <![CDATA[KMnO 4 Addition amount ( / raw material feeding amount, g / Kg)]]> <![CDATA[UV A 280 Reading]]> 0 < 0.3 40 ± 4 0.3 – 0.7 60 ± 6 > 0.7 .

[0030] The protein impurity content in the feed solution can be reflected by the absorbance value at a wavelength of 280 nm. The feed solution sample is adjusted to a solution concentration of 4 mg / mL. If A 280 The absorbance value is 0.3. Assuming that the absorbance value is caused by protein impurities, the corresponding protein impurity content in the raw material solid is about 10%. Similarly, A 280 When the absorbance value is 0.7, the content of protein impurities in the raw material solid is about 25%.

[0031] Based on the reason that the potassium permanganate oxidation and high temperature treatment mentioned above denature and filter out impurities such as proteins, the treatment can be applied not only in the crude heparin raw material stage, but also in other steps of the preparation of sulodexide raw materials or in any order. According to the transformation or extension of the technical example of the present invention, the use of potassium permanganate oxidation and high temperature treatment in other process stages to denature and filter out impurities such as proteins should also be regarded as within the technical scope of the present invention.

[0032] Preferably, the temperature for heating and maintaining in step S2 is 80°C - 90°C.

[0033] Preferably, the filter aid in step S2 is diatomaceous earth, which is added as follows:

[0034] <![CDATA[KMnO 4 Addition amount ( / raw material feeding amount, g / Kg)]]> Amount of filter aid added ( / raw material feed amount, g / Kg) 0 80 ± 8 40 ± 4 100 ± 10 60 ± 6 120 ± 12 .

[0035] Preferably, the filtration in step S2 refers to pressure filtration using a filter press.

[0036] Preferably, the step S3 of adjusting the pH of the feed solution before the chelating resin column to a weakly alkaline pH includes adjusting the pH to 7.0-9.0.

[0037] Based on the reason for the above-mentioned sequential removal of sulfate impurities by strong cation resin exchange and strong anion resin exchange, followed by removal of metal ion impurities by adsorption with a chelating resin, the treatment can be applied not only in the stage after the pathogens are removed from the crude heparin raw material, but also in other steps of the preparation of sulodexide raw materials or in any order. According to the technical paradigm transformation or extension of the present invention, the sequential removal of sulfate impurities by strong cation resin exchange and strong anion resin exchange in other process stages, followed by removal of metal ion impurities by adsorption with a chelating resin, should also be regarded as within the technical scope of the present invention.

[0038] In one group of embodiments (Example 4) but not limited to this embodiment, the heparin raw material is first oxidized with potassium permanganate, then subjected to resin to remove sulfate impurities and metal ions, and then subjected to alkali treatment to remove pathogenic impurities, and then bleached and depigmented with peracetic acid or hydrogen peroxide to obtain the sulodexide raw material.

[0039] Preferably, in step S4, sodium chloride is added to the feed solution to a final concentration of 1% - 10%, including adding sodium chloride to the feed solution to a final concentration of 4% - 6%.

[0040] Based on the aforementioned removal of pigment impurities by peracetic acid or hydrogen peroxide in the present invention, the treatment can be applied not only in the stage after resin adsorption and impurity removal, but also in other steps of the preparation of sulodexide raw materials or in any order. According to the transformation or extension of the technical examples of the present invention, the use of peracetic acid or hydrogen peroxide to remove pigment impurities in other process stages should also be regarded as within the technical scope of the present invention.

[0041] In the present invention, acetone is used as an organic solvent precipitant for the sulodexide raw material drug in steps S4 and S5.

[0042] Acetone has a better effect than ethanol and methanol, which are commonly used organic solvent precipitants in the field. The principle lies in the relative dielectric constant of the precipitation mechanism, which is 20.7 for acetone, 78.5 for water, 24.5 for ethanol, and 32.7 for methanol. Therefore, correspondingly, in terms of the precipitation effect of aqueous solution, acetone is the best (the dielectric constant changed the most), followed by ethanol, and methanol is relatively worse.

[0043] Considering that the raw material heparin contains a large amount of sulfated polysaccharide components with similar properties, such as heparan sulfate, dermatan sulfate, fast-moving heparin and slow-moving heparin, and the quality standard of the target sulodexide requires that the main components are about 80% fast-moving heparin and 20% dermatan sulfate, and the indicator range is very small, therefore, the graded precipitation separation of these polysaccharide components requires an organic solvent precipitant with higher discrimination. As mentioned above, according to the relative dielectric constant of the organic solvent precipitant, acetone has the best effect.

[0044] Based on the reasons described in the present invention, the organic solvent precipitant may also be other solvents with relatively low effects, including ethanol and / or methanol, while acetone has a better effect. Accordingly, the present invention preferably uses acetone as the organic solvent precipitant. However, it is easy to understand that according to the transformation or extension of the technical examples of the present invention, the use of other organic solvent precipitants should also be regarded as within the technical scope of the present invention.

[0045] In addition, the use of ethanol as an organic solvent precipitant for the sulodexide bulk drug is characterized in that, in the steps S4 and S5, the amount of ethanol used is 1.1 times the original amount of acetone used, as follows:

[0046] S4, decolorization and precipitation classification: sodium chloride is added to the feed liquid to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the crude heparin raw material feed amount, and the pH is controlled to be around 8 - 10. The reaction is carried out at room temperature for not less than 0.5 h, and the pH is adjusted to be neutral. Ethanol in an amount of 0.495 - 0.66 times the volume of the feed liquid is added, and the mixture is stirred and allowed to stand for not less than 4 h, and the precipitated paste is removed. Ethanol is added to the clear liquid to a volume of 0.99 - 1.21 times the volume of the feed liquid, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded. Purified water in an amount of 4 - 10 times the volume of the crude heparin raw material is added to the precipitated paste, and the mixture is stirred until dissolved. Ethanol in an amount of 0.77 - 0.99 times the volume of the purified water is added, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded to obtain a precipitated paste;

[0047] S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 0.99-1.21 times the volume of ethanol, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above-mentioned operation of dissolving with the same amount of purified water and precipitating with ethanol, retain the precipitate, add 1.65 times the amount of ethanol as the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above-mentioned operation of washing and dehydrating with the same amount of ethanol 1-5 times, until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the feed liquid in each freeze-drying plate is not higher than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

[0048] In addition, the use of methanol as an organic solvent precipitant for the sulodexide bulk drug is characterized in that, in the steps S4 and S5, the amount of methanol used is 1.5 times the original amount of acetone used, as follows:

[0049] S4, decolorization and precipitation classification: sodium chloride is added to the feed solution to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the crude heparin raw material feed amount, and the pH is controlled to be around 8 - 10. The reaction is carried out at room temperature for at least 0.5 h, and the pH is adjusted to be neutral. Methanol is added in an amount of 0.675 - 0.90 times the volume of the feed solution, and the mixture is stirred and allowed to stand for at least 4 h, and the precipitated paste is removed. Methanol is added to the clear liquid to a volume of 1.35 - 1.65 times the volume of the feed solution, and the mixture is stirred and allowed to stand for at least 4 h, and the supernatant is discarded. Purified water in an amount of 4 - 10 times the volume of the crude heparin raw material feed amount is added to the precipitated paste, and the mixture is stirred until dissolved. Methanol is added in an amount of 1.05 - 1.35 times the volume of the purified water, and the mixture is stirred and allowed to stand for at least 4 h, and the supernatant is discarded to obtain a precipitated paste;

[0050] S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 1.35-1.65 times the volume of the feed liquid of methanol, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with methanol, retain the precipitate, add no less than 2.25 times the amount of methanol of the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of methanol 1-5 times, until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying tray of the freeze-drying equipment, the height of the feed liquid in each freeze-drying tray is not more than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

[0051] In one group of embodiments (Example 3) but not limited to this embodiment, the solvent acetone in the process of heparin sample classification and purification is replaced with ethanol or methanol to produce sulodexide raw material in the same way.

[0052] Unless otherwise specified, the standing time after the organic solvent precipitation and stirring in the preparation method is preferably 12 h.

[0053] Preferably, the freeze-drying procedure in step S5 includes: pre-freezing at -40°C for 3-5 h, starting the cold trap to cool down and the vacuum pump (50±10 Pa), raising the internal partition to 0±10°C within 1-3 h, maintaining for 14±4 h, vacuum at 20±5Pa, raising the internal partition to 20±10°C within 1-3 h, maintaining for 14±4 h, vacuum at 20±5Pa, raising the internal partition to 40±10°C within 1-3 h, maintaining for 1-3 h, ultimate vacuum, releasing the vacuum after completion, and unpacking.

[0054] Freeze drying is an important method of drug drying. Compared with other drying methods such as vacuum drying, hot air drying and spray drying, the advantage of freeze drying is that the temperature of the product is low during freeze drying, which can effectively protect the product from being damaged by high temperature. The sublimation of water can also more effectively remove residual solvents. Therefore, freeze drying is the preferred option for ensuring the quality of drugs.

[0055] Based on the reasons described in the present invention, the drying of the sulodexide API can also be vacuum drying, hot air drying, spray drying and freeze drying, and the quality of the drug under freeze drying is better. Accordingly, the present invention preferably uses freeze drying as the drying method for the sulodexide API finished product. However, it is easy to understand that according to the transformation or extension of the technical examples of the present invention, the use of vacuum drying and hot air drying should also be regarded as within the technical scope of the present invention.

[0056] Preferably, the powdering and packaging in step S5 includes crushing the freeze-dried powder with a pulverizer, sieving, packaging into two layers of plastic bags, plastic-sealing each, and then sealing into aluminum foil bags or aluminum barrels.

[0057] In one set of embodiments (Example 5) but not limited to this embodiment, multiple batches of prepared sulodexetide raw material products were inspected and tested, and the quality met the requirements of the established indicators.

[0058] The invention discloses a method for preparing the sulodexide raw material medicine and its application in medicine production.

[0059] The outstanding effects of the present invention are: providing a method for preparing high-quality sulodexide raw materials suitable for the pharmaceutical industry, the method using heparinoids derived from porcine intestinal mucosa as the starting material, and compared with the prior art, the method uses alkali treatment (and subsequent potassium permanganate oxidation treatment) to remove pathogens such as viruses and bacteria in crude heparinoids, uses potassium permanganate oxidation and high-temperature treatment to denature and filter out impurities such as proteins, uses strong cation resin exchange and strong anion resin exchange to remove sulfate impurities, uses chelating resin adsorption to remove metal ion impurities, and uses peracetic acid or hydrogen peroxide to remove impurities such as proteins. The pigment impurities are removed, and then the precipitation is repeated with different concentrations of organic solvent precipitants for several times, and finally refined, freeze-dried to remove water and solvent to obtain the sulodexide raw material. These processes are basically different from the existing technology, and can more systematically and effectively remove pathogens, inorganic impurities (sulfate and metal ion impurities), pigment impurities and residual solvents. In addition, the organic solvent acetone, which is more suitable for fast-moving heparin and dermatan sulfate graded precipitation, is preferably selected, which can fully guarantee the quality of the sugar component of the product sulodexide. Based on this, the present invention is significantly superior to the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 :NMR 1H spectra of self-made sulodexide samples and sulodexide reference substances ( 1 Schematic diagram of H-NMR comparison.

[0061] Figure 2 : Schematic diagram of the strong anion exchange-high performance liquid chromatography (SAX-HPLC) comparison of the homemade sulodexide sample and the sulodexide reference substance.

[0062] Figure 3: Schematic diagram of size exclusion-high performance liquid chromatography (SEC-HPLC) analysis of homemade sulodexide samples.

[0063] Figure 4 : Schematic diagram of gel electrophoresis analysis comparison between three batches of homemade sulodexide samples and sulodexide reference substance. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods, but it is not intended to limit the protection scope of the present invention.

[0065] Example 1: Preparation of Sulodexide 1.

[0066] This is a small test for the preparation of 100-gram-level sulodexide samples. The raw material is commercially available crude heparin, a by-product of porcine intestinal mucosal heparin production, which is a material obtained by homogenizing and powdering after mixing dry materials. The batch size is 215 Kg, and the test data are as follows: it appears as an off-white powder and is easily soluble in water; the specific rotation is + 13°; the pH (5%) is 7.6; the loss on drying is 4.4%; the free sulfate content is 0.69%; in agarose electrophoresis analysis, the percentages of slow-moving heparin (SMH), fast-moving heparin (FMH), dermatan sulfate (DS) and chondroitin sulfate (CS) are 9%, 64%, 25% and <2%, respectively; the anticoagulant activity of the sheep plasma method is 64 U / mg.

[0067] The preparation process and results of Sulodexide API are as follows:

[0068] S1. Inactivation and removal of pathogens including viruses and microorganisms: Weigh 500 g of crude heparin raw material, add it into a beaker, add about 2.5 L of water, stir mechanically until it is basically dissolved, then add 125 g of sodium hydroxide solid, and stir for 1 hour.

[0069] S2. Denaturation, coagulation and filtration removal of protein impurities: Adjust the pH to 8.2 with 6 mol / L hydrochloric acid solution, take a sample (accurately take 2.4 mL of the feed solution according to the feed concentration, dilute it to 100 mL with water, and obtain a 4 mg / mL test solution), and determine A 280 The pH value was 0.798. According to the reference table, 33 g potassium permanganate was added and the mixture was heated to 70°C and kept at high temperature (heating was stopped as soon as the liquid started to boil) until the protein impurities were denatured and solidified. Then the heating was stopped and stirring was continued for 1 h to allow the protein impurities to be fully separated. 62 g diatomaceous earth was added and filtered with a pump. The filtrate was cooled to 26°C.

[0070] S3, column purification and impurity removal: Since the free sulfate content of the raw material exceeds 0.5%, the feed liquid is first pumped through a 2.5 L strong cation resin exchange column and a 2.5 L strong anion resin exchange column in sequence, and then top washed with 1 L of water, and the filtrate is collected. Then the pH of the feed liquid is adjusted to 8.2, and pumped through a 2.5 L chelating resin column, and rinsed with 1 L of water to obtain about 4.8 L of filtrate.

[0071] S4, decolorization and precipitation classification: add 480 g of sodium chloride and 25 g of peracetic acid to the feed solution, adjust the pH to 9.8, stir and let stand for 2 h to decolorize; adjust the pH to 7.0, add 2.6 L of acetone, stir and let stand for 12 h, remove the precipitated paste, add 2.2 L of acetone to the clear liquid, stir and let stand for 12 h, discard the supernatant, add 3 L of purified water to the precipitated paste, stir until dissolved, at this time the volume of the feed solution is about 3.4 L, add 2.8 L of acetone, stir and let stand for 12 h, discard the supernatant, and obtain about 600 g of precipitated paste.

[0072] S5, purification and drying: add 1.5 L purified water to the above precipitate paste, stir until dissolved, the volume of the feed liquid is about 2.0 L, add 2.0 L acetone, stir and let stand for 12 h, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with acetone, keep the precipitate, add 1.5 L acetone, stir for 10 min, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of acetone 3 times, the precipitate is now in the form of dispersed particles, filter out the solid, dry at 60°C for 12 h, transfer the solid powder to obtain 329 g, dissolve it in 1.2 L water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the solution height is about 12 mm, freeze-drying procedure: pre-freeze at -40°C for 5 h; start the cold trap to cool down and vacuum pump (60Pa); within 3 h, raise the partition to 10°C, keep it for 18 h, vacuum 25 Pa; within 3 h, raise the partition to 30°C, keep it for 18 h, vacuum 25Pa; within 3 h, the partition temperature rises to 45℃, maintained for 3 h, ultimate vacuum; after completion, the vacuum is released and the product is taken out of the box. The freeze-dried product is collected, ground into powder by a grinder, passed through an 80-mesh sieve, and then packed into a double-layer PE bag, covered with an aluminum foil bag, and 306 g of sulodexide product is obtained, with a yield of 61.2%.

[0073] Test results: White lyophilized powder, easily soluble in water; electrophoresis identification meets the requirements; specific rotation (measured with 4% aqueous solution) calculated with dry product is +22.6°; the color of the solution is lighter than EP yellow No. 5 standard color; pH (1%) is 7.1; nucleic acid impurities are measured with A of 0.4% solution 260nmThe absorbance was 0.112; the residual solvent (acetone) was 176 ppm; the protein impurity (Lowry method) was 0.12%; the organic sulfur content was 9.0%; the sulfonate / carboxylate ratio was 1.8; in agarose electrophoresis analysis, the percentages of fast-moving heparin (FMH), dermatan sulfate (DS), and slow-moving heparin (SMH) were 79%, 20%, and <2%, respectively; the anti-Xa activity was 80 IU / mg; the anticoagulant activity by sheep plasma method was 58 U / mg; and the esterase activity was 13 LSU / mg.

[0074] Example 2: Preparation of Sulodexide 2.

[0075] This is another preparation test of 100-gram level sulodexide sample. The raw material comes from the by-product heparin in the purification and refining stage of fine heparin production. The test data are as follows: it appears as a white powder and is easily soluble in water; the specific rotation is +38°; the pH (5%) is 7.1; the loss on drying is 5.4%; the free sulfate content is 0.34%; in agarose electrophoresis analysis, the percentages of slow-moving heparin (SMH), fast-moving heparin (FMH), dermatan sulfate (DS) and chondroitin sulfate (CS) are 22%, 64%, 14% and 0%, respectively; the anticoagulant activity of the sheep plasma method is 103 U / mg.

[0076] The process and preparation results of Sulodexide are as follows:

[0077] S1. Inactivation and removal of pathogens including viruses and microorganisms: Weigh 1 kg of the crude heparin raw material mentioned above, add it into a 50 L reactor, add about 15 L of water, stir and add 600 g of NaOH powder, and stir with a mechanical stirrer for 2 h.

[0078] S2. Denaturation, coagulation and filtration removal of protein impurities: Adjust the pH to 8.0 with 4 mol / L hydrochloric acid, take a sample (according to the feed concentration, accurately take 6.0 mL of the feed solution, dilute it to 100 mL with water, and obtain a 4 mg / mL test solution), and determine A 280 is 0.316. According to the reference table, add 126 g of KMnO4, turn on the heater for heating, and when the temperature reaches 85°C, start the insulation control at 85°C±5°C until the protein impurities denature and coagulate. Then stop heating and continue stirring for 1 h to allow the protein impurities to be fully precipitated. Add 300 g of diatomaceous earth, filter with a test filter press, and pour the filtrate into another reaction tank and cool to 24°C.

[0079] S3. Column purification and impurity removal: Since the free sulfate content of the raw material is less than the limit value of 0.5%, the feed liquid is adjusted to pH = 8.0, and directly passed through a 12 L chelating resin column with a pressure pump, and washed with 3 L of water to obtain 18 L of filtrate.

[0080] S4, decolorization and precipitation classification: add 900 g of NaCl solid powder to the feed liquid, add 120 g of hydrogen peroxide acid, this step is used to remove the residual pigment in the solution, control pH = 9.0, act at room temperature for 1.5 h, adjust pH = 7.0, add 9 L of acetone, stir and let stand for 4 h, remove the precipitated paste, add 18 L of acetone to the clear liquid, stir and let stand for 6 h, discard the supernatant, add 7 L of purified water to the precipitated paste, stir until dissolved, add 6.5 L of acetone, stir and let stand for 6 h, discard the supernatant, and obtain about 1.5 Kg of precipitated paste.

[0081] S5, purification and drying: add 4.5 L purified water, stir until clear, add 6.0 L acetone, stir and let stand for 12 h, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with acetone, retain the precipitate, add 3.0 L acetone, stir for 10 min, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of acetone twice, at this time the precipitate is dispersed in particles, take the solid, dry it at 60°C for 12 h, transfer the solid powder to obtain 0.75 Kg, dissolve it with 4 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the solution height is about 24 mm, pre-freeze at -40°C for 3 h, start the cold trap to cool down and the vacuum pump (40 Pa), raise the partition to -5°C within 1 h, keep it for 12 h, vacuum 15 Pa, raise the partition to 10°C within 1 h, keep it for 12 h, vacuum 15 Pa, raise the partition to 40°C within 1 h, keep it for 1 h, ultimate vacuum, release the vacuum after the end, and take it out of the box. Collect the freeze-dried product, crush it with a grinder, pass it through an 80-mesh sieve, package it with a double-layer PE bag, seal it with an aluminum foil bag on the outer layer, and package it to obtain 672 g of the final product of sulodexide raw material, with a yield of 67.2%.

[0082] Test results: White lyophilized powder, easily soluble in water; electrophoresis identification meets the requirements; specific rotation (measured with 4% aqueous solution) calculated with dry product is +24.3°; the color of the solution is lighter than EP yellow No. 6 standard color; pH (1%) is 7.0; nucleic acid impurities are measured with A of 0.4% solution 260nm The absorbance was 0.082; the residual solvent (acetone) was 93 ppm; the protein impurity (Lowry method) was 0.05%; the organic sulfur content was 9.1%; the sulfonate / carboxylate ratio was 1.8; in agarose electrophoresis analysis, the percentages of fast-moving heparin (FMH), dermatan sulfate (DS), and slow-moving heparin (SMH) were 80%, 19%, and <1%, respectively; the anti-Xa activity was 94 IU / mg; the anticoagulant activity by sheep plasma method was 64 U / mg; and the esterase activity was 13 LSU / mg.

[0083] Summary: The product yield of this example is higher than that of Example 1, which may be due to the fact that the raw materials used have fewer impurities, such as a lower free sulfate content, and thus the column purification step may have less loss.

[0084] Example 3: Preparation of Sulodexide 3.

[0085] This example investigates the use of ethanol as an organic solvent precipitant in the graded precipitation step. The raw materials (and feed amounts) are the same as those in Example 1. The preparation process includes the inactivation and removal of pathogens including viruses and microorganisms, the denaturation, coagulation and filtration removal of protein impurities in S1, and the removal of impurities by column purification in S3. The rest of the preparation process and results are as follows:

[0086] S4, decolorization and precipitation classification: the filtrate is about 4.8 L, add 480 g of sodium chloride, add 25 g of peracetic acid, adjust the pH to 9.2, and decolorize for 2 h after stirring at room temperature; adjust the pH to 7.0, add 2.9 L of ethanol, stir and let stand for 12 h, remove the precipitated paste, add 2.6 L of ethanol to the clear liquid, stir and let stand for 12 h, discard the supernatant, add 3 L of purified water to the precipitated paste, stir until dissolved, at this time the volume of the feed liquid is about 3.4 L, add 3.0 L of ethanol, stir and let stand for 12 h, discard the supernatant, and obtain about 570 g of precipitated paste.

[0087] S5, purification and drying: add 1.55 L purified water to the above precipitate paste, stir until dissolved, the volume of the feed liquid is about 2.0 L, add 2.2 L ethanol, stir and let stand for 12 h, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and ethanol precipitation, keep the precipitate, add 1.7 L ethanol, stir for 10 min, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of ethanol 3 times, the precipitate is now in the form of dispersed particles, filter out the solid, dry at 60 ° C for 14 h, transfer the solid powder, get 288 g, dissolve with 1.7 L water for injection, filter at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the drug liquid is about 17 mm, freeze-drying procedure: pre-freeze at -40 ° C for 4 h; start the cold trap to cool down and vacuum pump (50Pa); within 2 h, the partition is raised to 10 ° C, maintained for 12 h, vacuum 20 Pa; within 2 h, the partition is raised to 25 ° C, maintained for 12 h, vacuum 20 Pa; The temperature of the partition was raised to 40℃ for 2 hours and maintained at the ultimate vacuum. After the vacuum was released, the product was taken out of the box. The freeze-dried product was collected, ground into powder by a grinder, passed through an 80-mesh sieve, and then packed into a double-layer PE bag and covered with an aluminum foil bag to obtain 262 g of sulodexide with a yield of 52.4%.

[0088] Test results: White lyophilized powder, easily soluble in water; electrophoresis identification meets the requirements; specific rotation (measured with 4% aqueous solution) calculated with dry product is +22.5°; the color of the solution is lighter than EP yellow No. 5 standard color; pH (1%) is 7.1; nucleic acid impurities are measured with A of 0.4% solution 260nm The absorbance was 0.122; the residual solvent (acetone) was 137 ppm; the protein impurity (Lowry method) was 0.11%; the organic sulfur content was 8.4%; the sulfonate / carboxylate ratio was 1.6; in agarose electrophoresis analysis, the percentages of fast-moving heparin (FMH), dermatan sulfate (DS), and slow-moving heparin (SMH) were 78%, 21%, and <2%, respectively; the anti-Xa activity was 85 IU / mg; the anticoagulant activity by sheep plasma method was 56 U / mg; and the esterase activity was 11 LSU / mg.

[0089] Summary: By using ethanol as the organic solvent precipitant for classification, a product that meets the established quality standard requirements of sulodexide raw material can also be obtained, and the yield is slightly lower than the preparation method using acetone as the organic solvent precipitant described in Example 1.

[0090] Example 4: Preparation of Sulodexide 4.

[0091] This example is a preparation method for investigating different step sequences, specifically adjusting the sequence of core steps such as alkali treatment to remove pathogens and protein impurities, and investigating the preparation of sulodexide API. The raw materials (and feed amounts) are the same as in Example 1, and the process and results are as follows:

[0092] (1) Denaturation, coagulation and filtration removal of protein impurities: (In this step, potassium permanganate is first used to remove pathogens and oxidation to remove protein impurities) Weigh 1 kg of the crude heparin raw material of Example 1, dilute to 5 L with water, take a sample (and dilute to 4 mg / mL with water according to the feed concentration) and measure the A 280 is 0.804. According to the reference table, add 65 g of potassium permanganate and heat to 85°C. Maintain the high temperature of 85°C±5°C until the protein impurities denature and coagulate. Then stop heating and continue stirring for 1 h to allow the protein impurities to be fully precipitated. Add 120 g of diatomaceous earth and filter with a testing machine. Cool the filtrate to room temperature.

[0093] (2) Column purification and impurity removal: The feed liquid was pumped through a 4 L strong cation resin exchange column and a strong anion resin exchange column in turn. After each column pass, it was rinsed with 1 L of water and the filtrate was collected. The feed liquid was then adjusted to pH = 8.0 and pumped through a 4 L chelating resin column. 1 L of water was used to rinse the column to obtain 7.3 L of filtrate.

[0094] (3) Alkali treatment: Add 290 g of sodium hydroxide solid to the liquid and stir mechanically for 2 h.

[0095] (4) Decolorization and precipitation classification: Adjust the pH to 10.2 with hydrochloric acid solution, add 42 g of hydrogen peroxide, check that the pH is 9.5, and let it act at room temperature for 1.5 h. Adjust the pH to 7.0, add 3.7 L of acetone, stir and let stand for 12 h, remove the precipitate paste, add 7.3 L of acetone to the clear liquid, stir and let stand for 12 h, discard the supernatant, add 6 L of purified water to the precipitate paste, stir until dissolved, add 4.8 L of acetone, stir and let stand for 12 h, discard the supernatant, and obtain 1.2 L of precipitate paste.

[0096] (5) Purification and drying: 1.2 L of the precipitate paste was added with 3 L of purified water, stirred until dissolved, 3 L of acetone was added, stirred and allowed to stand for 12 h, the supernatant was discarded, the same amount of purified water dissolution and acetone precipitation were repeated, the precipitate was retained, 3.7 L of acetone was added, stirred for 10 min, allowed to stand, the supernatant was discarded, the same amount of acetone washing and dehydration was repeated 4 times, 3.7 L of acetone was added each time, and the stirring time was 10 min each time. At this time, the precipitate was dispersed in particles, allowed to stand, solid was taken, dried at 60°C for 12 h, and solid powder was transferred to obtain 0.66 kg, which was dissolved with 5 times the amount of water for injection, filtered at 0.22 μm, and the filtrate was divided into three portions and loaded into the stainless steel freeze-drying tray of the freeze-drying equipment. The height of the drug solution was about 11 mm. The freeze-drying method was the same as that in Example 3, and the product was taken out of the box. The freeze-dried product was collected, powdered, and packaged to obtain 611 g of the final product of sulodexide raw material drug with a yield of 61.1%.

[0097] Test results: White lyophilized powder, easily soluble in water; electrophoresis identification meets the requirements; specific rotation (measured with 4% aqueous solution) calculated with dry product is +27.1°; the color of the solution is lighter than EP yellow No. 5 standard color; pH (1%) is 7.3; nucleic acid impurities are measured with A of 0.4% solution 260nm The absorbance was 0.078; the residual solvent (acetone) was 126 ppm; the protein impurity (Lowry method) was 0.09%; the organic sulfur content was 8.7%; the sulfonate / carboxylate ratio was 1.7; in agarose electrophoresis analysis, the percentages of fast-moving heparin (FMH), dermatan sulfate (DS), and slow-moving heparin (SMH) were 82%, 17%, and <2%, respectively; the anti-Xa activity was 91 IU / mg; the anticoagulant activity by sheep plasma method was 60 U / mg; and the esterase activity was 12 LSU / mg.

[0098] Summary: This embodiment adopts the method of first adding potassium permanganate to remove pathogens and oxidative filtration, followed by column purification to remove impurities, and then alkali treatment, decolorization, alcohol precipitation classification and final refining. The preparation process is smooth and controllable, and the product meets the index requirements of sulodexide raw materials after inspection, indicating that the order of the core steps of the preparation method of the present invention can be adjusted, which is suitable for process flow layout and industrial scale-up.

[0099] Example 5: Production and preparation of three consecutive validation batches of sulodexide API.

[0100] The production of three batches of sulodexide API was verified. The raw materials were the same as in Example 1, and the feed amount of each batch was 35 Kg. The preparation methods of the three batches were the same, except that the process control, the weight of the intermediates, the amount of materials, etc. were slightly different according to the process control values. The preparation process was taken as an example of the first batch, as follows:

[0101] S1. Inactivation and removal of pathogens including viruses and microorganisms: Weigh 35.0 kg of crude heparin raw material, add it to stainless steel tank T1, add about 157.5 L of water, stir for 30 min, at which time the liquid has dissolved and the volume is about 175 L; add 7.0 kg of sodium hydroxide solid and continue stirring for 1 h.

[0102] S2. Denaturation, coagulation and filtration of protein impurities: Slowly add about 25 L of 6 mol / L hydrochloric acid solution and adjust the pH to 8.2. At this time, the total volume of the feed liquid is about 200 L. Take a sample (based on the volume of the feed and the current feed liquid, weigh 2.29 g of the feed liquid and dilute it to 100 mL with water. This is a 4 mg / mL sample solution to be tested). Determine A 280 is 0.724. According to the reference table, add 2.1 kg of potassium permanganate, heat to 85°C, and keep for 15 min. Then stop heating, continue stirring for 0.5 h, add 3.5 kg of diatomaceous earth, and keep stirring. Prepare a plate and frame filter, filter the material liquid, rinse with 30 L of hot water, collect the filtrate into stainless steel tank T2, and cool to room temperature.

[0103] S3, column purification and impurity removal: the cooled feed liquid is pumped through the 140 L strong cation resin exchange column 1 and the 140 L strong anion resin exchange column 2 connected in series in sequence, and then top washed with 30 L of water, and the filtrate is collected into a stainless steel tank T3; the feed liquid pH is adjusted to 8.2, and then pumped through the 140 L chelating resin column, and rinsed with 30 L of water, and the filtrate is collected into a stainless steel tank T4, and about 300 L of filtrate is obtained.

[0104] S4, decolorization and precipitation classification: add 15 kg of sodium chloride to the feed liquid, stir until it is dissolved; add 1.05 kg of peracetic acid, adjust the pH to 9.0 with a dilute alkaline solution, stir and stand at room temperature for 2 hours to decolorize; adjust the pH to 7.0, add 150 L of acetone, stir and stand for 12 hours, transfer the supernatant to the precipitation tank T5, and discard the precipitated paste; add 150 L of acetone to the clear liquid, stir and stand for 12 hours, discard the supernatant, add 210 L of purified water to the precipitated paste, stir until it is dissolved, at this time the volume of the feed liquid is about 260 L, add 208 L of acetone, stir and stand for 12 hours, discard the supernatant, and obtain about 44 kg of precipitated paste.

[0105] S5, refining and drying: add 110 L purified water to the above precipitate paste, stir until dissolved, transfer the feed liquid to the precipitation tank T6 (a little purified water rinse tank T5 is combined), the feed liquid volume is about 150 L at this time, add 150 L acetone, stir and let stand for 12 h, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with acetone, keep the precipitate, add 100 L acetone, stir for 10 min, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of acetone twice, at this time the precipitate is dispersed in particles; unload, filter out the solid, transfer to the drying plate, vacuum dry at 60 ° C for 14 h, transfer the solid powder, and obtain 22.75 Kg; in the liquid preparation room of the Class C clean area, add the powder to the 200 L liquid preparation tank, add about 110 L injection water precooled to room temperature, stir for about 30 min, at this time the feed liquid has dissolved; the feed liquid is sieved at 0.45 μm and then 22 μm filter was used for serial filtration, and the filtrate was transferred to a stainless steel freeze-drying plate with a clean beaker. Each plate was about 2 L, and the height of the drug solution was about 20 mm. The setting program of the freeze dryer was as follows: pre-freeze at -40℃ for 4 h; start the cold trap to cool down and vacuum pump (50 Pa); within 2 h, the partition was raised to 0℃, maintained for 14 h, and vacuum was 20 Pa; within 2 h, the partition was raised to 20℃, maintained for 14 h, and vacuum was 20 Pa; within 2 h, the partition was raised to 40℃, maintained for 2 h, and ultimate vacuum; after the end, the vacuum was released and the product was taken out of the box. The freeze-dried block was collected into a stainless steel barrel with a lid, transferred to the pulverizer room, powdered, sieved, weighed and bagged with double-layer PE bags, the inner bags were plastic-sealed separately, and then the outer aluminum foil bags were plastic-sealed to obtain the finished drug substance of Sulodexide.

[0106] Results: The weights of the finished products of the three validation batches were 21.4 Kg, 20.7 Kg and 22.3 Kg, respectively, with corresponding yields of 61.1%, 59.1% and 63.7%; the inspection results are shown in Table 1 below.

[0107] Table 1 Continuous verification of the test results of three batches of sulodexide API

[0108] .

[0109] The above data show that the quality inspection results of three consecutive validation batches are highly consistent, reflecting the high controllability of this preparation process and quality.

[0110] Example 6: H NMR spectroscopy ( 1 H-NMR) analysis and comparison of homemade sulodexide API and reference substance.

[0111] Test sample: Homemade sulodexide API from Example 1 and Example 2.

[0112] Reference substance: lyophilized powder of original preparation injection.

[0113] Sample preparation: 35 mg of the test or control sample was dissolved in 0.6 mL of deuterated water (0.002% TSP was added as an internal standard to correct the zero point), the solution was transferred to a 5 mm NMR tube, and ultrasonicated for 2 min.

[0114] Instrument: NMR Bruker AC500 HD;

[0115] Software: Bruker Top Spin 3.2 (Bruker Biospin GmbH).

[0116] The samples of Examples 1 and 2 and the original research sulodexide were subjected to nuclear magnetic resonance analysis. 1 H-NMR spectrum results and comparison chart are shown in Figure 1 .

[0117] The results showed that the peak type (response) and peak position of the sulodexide raw material prepared by the present invention and the original reference substance were almost the same, so it can be judged that their functional group composition and structure are the same.

[0118] Example 7: Strong anion exchange-high performance liquid chromatography (SAX-HPLC) analysis and comparison of homemade sulodexide bulk drug and reference substance.

[0119] Test sample: Homemade sulodexide API from Example 1.

[0120] Reference substance: lyophilized powder of original preparation injection.

[0121] Sample treatment: Prepare a 20 mg / mL solution of the test sample, take 20 μL, add 70 μL pH 7.0 calcium acetate solution and 100 μL heparinase solution, mix well, and place in a 25°C water bath for at least 48 h for heparinase enzymolysis to obtain sample degradation solution. Prepare the reference substance in the same way.

[0122] SAX-HPLC chromatographic conditions:

[0123] Chromatographic column: Waters spherisorb SAX, (4.0×250 mm, 5 μm); HPLC mobile phase: 0.1 M NH4Ac + 0.02% NaN3; flow rate: 0.8 mL / min; column temperature: room temperature; detector wavelength: 232 nm; injection volume: 18 μL; run time: 79 min; injection volume: 20 μL.

[0124] Chromatographic analysis results and spectra comparison, see Figure 2 .

[0125] The results show that the disaccharide composition of the sample prepared by the present invention is consistent with the type and proportion composition of the sulodexide reference substance after heparinase hydrolysis and SAX-HPLC separation and analysis.

[0126] Example 8: Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC) analysis.

[0127] Test sample: Homemade sulodexide API from Example 1.

[0128] Molecular weight calculation calibrator: USP heparin sodium molecular weight calibration standard.

[0129] Sample processing method: The test sample and calculation standard were diluted with mobile phase to prepare 5 mg / mL aqueous solution and filtered through 0.45 μm filter membrane.

[0130] HPLC detection conditions:

[0131] Chromatographic columns: Analytical Column BioCore SEC-300 (7.5 mm × 30 cm, 5 µm) and BioCore SEC-500 (7.5 mm × 30 cm, 5 µm); mobile phase: 0.1 mol / mL NH4Ac + 0.02% NaN3; flow rate: 0.6 mL / min; column temperature: 30°C; detector: differential; injection volume: 20 µL; run time: 50 min.

[0132] The molecular weight distribution results and SEC-HPLC chromatograms of the test samples are shown in Figure 3 .

[0133] The results show that in the sample prepared by the present invention, fast-moving heparin accounts for 79.5%, with an average molecular weight of about 11,000 Da, and dermatan sulfate accounts for about 19.5%, with an average molecular weight of about 36,000 Da. The composition ratio of the sample is consistent with the indicators of the original product.

[0134] Example 9: Agarose gel electrophoresis analysis and comparison of homemade sulodexide bulk drug and reference substance.

[0135] Test sample: Homemade sulodexide API Example 5 Verification of three batches of samples.

[0136] Reference substance: lyophilized powder of original preparation injection.

[0137] Standard reference: Take the slow-moving heparin (SMH), fast-moving heparin (FMH), dermatan sulfate (DS) and chondroitin sulfate (CS) of the standard solution, mix them, and use them for electrophoresis band positioning.

[0138] Sample treatment: Dilute the test sample and reference sample to a 20 mg / mL aqueous solution, and then dilute them 10 times with 0.02% cresol red solution (add 20% glycerol) to make the solution concentration 2.0 mg / mL. Prepare the standard and reference solutions in the same way.

[0139] Agarose gel: Prepare with 20 mM barium acetate solution, agarose concentration of 0.6%, heat to boiling, make gel, and cool.

[0140] Electrophoresis system: 20 mM barium acetate solution, current 160 - 400 mA, voltage 140 - 200 V, time 90 min.

[0141] The electrophoresis analysis results are shown in Figure 4 , the percentage data are shown in Table 1 in Example 5.

[0142] The results showed that the three batches of homemade sulodexide samples were verified to be consistent with the original sulodexide reference substance, with lower control of the slow-moving heparin component.

[0143] All the above data reveal that the preparation method of the present invention is practical and efficient, the product Sulodexide is consistent with the original reference substance and meets the requirements of established drug indicators, and the preparation method can systematically and effectively control impurities and product quality, and is suitable for the industrial preparation of Sulodexide pharmaceutical products.

[0144] Of course, there are many specific implementations of the present invention, which are not listed here one by one. All technical solutions formed by equivalent replacement or equivalent transformation fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a sulodexide bulk drug, characterized in that: The following steps are involved: S1. Inactivation and removal of pathogens including viruses and microorganisms: Weigh the crude heparin raw material, dissolve it in water to form a 15% -25% solution, add 3% - 5% sodium hydroxide solid of the total amount of the liquid, and stir at room temperature for not less than 0.5 h; S2. Denaturation and coagulation of protein impurities and filtration removal: neutralize, add potassium permanganate, the amount of potassium permanganate added per 1 kg of crude heparin raw material should not exceed 66 g, heat to above 70°C, and maintain high temperature until the protein impurities are denatured and coagulated, then stop heating, continue stirring for not less than 10 min to allow the protein impurities to be fully precipitated, add filter aid, the amount of filter aid added per 1 kg of crude heparin raw material should be 72-132 g, filter, and cool the filtrate to room temperature; S3, column purification and impurity removal: when the free sulfate content of the raw material is 0.5% or above, the feed liquid is first pumped through a strong cation resin exchange column and a strong anion resin exchange column in sequence for purification, otherwise, the two column purifications are skipped, the feed liquid is adjusted to a weakly alkaline pH, and pumped through a chelating resin column, the dosage of the above three resins is the same, which is 3-5 times the amount of crude heparin raw material; S4, decolorization and precipitation classification: sodium chloride is added to the feed solution to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the crude heparin raw material feed amount, and the pH is controlled at 8 - 10, and the reaction is carried out at room temperature for not less than 0.5 h, and the pH is adjusted to neutral, and acetone is added in an amount of 0.45 - 0.60 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the precipitated paste is removed, and acetone is added to the clear liquid to a volume of 0.9 - 1.1 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded, and purified water in an amount of 4 - 10 times the volume of the crude heparin raw material is added to the precipitated paste, and the mixture is stirred until dissolved, and acetone is added in an amount of 0.7 - 0.9 times the volume of the purified water, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded to obtain a precipitated paste; S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 0.9-1.1 times the volume of acetone of the feed liquid, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above operation of dissolving with the same amount of purified water and precipitating with acetone, retain the precipitate, add 1.5 times the amount of acetone of the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above operation of washing and dehydrating with the same amount of acetone 1-5 times, until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the feed liquid in each freeze-drying plate is not more than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

2. The preparation method according to claim 1, characterized in that The crude heparinoid raw material in step S1 is derived from porcine intestinal mucosa.

3. The preparation method according to claim 1, characterized in that: The amount of potassium permanganate in step S2 is adjusted to a concentration of 4 mg / mL according to the feed sample. 280 Values, add as follows: A 280 When the reading value is < 0.3, potassium permanganate is not added. 280 When the reading value is 0.3 – 0.7, the amount of potassium permanganate added is 40 ± 4 g / Kg of the raw material feed amount, A 280 When the reading value is >0.7, the amount of potassium permanganate added is 60 ± 6 g / Kg of the raw material feed amount.

4. The preparation method according to claim 1, characterized in that: The temperature of heating and maintaining in step S2 is 80°C - 90°C.

5. The preparation method according to claim 1 or claim 3, characterized in that: The filter aid in step S2 is diatomaceous earth, which is added as follows: when potassium permanganate is not added, the amount of filter aid added is 80±8 g / Kg of the raw material feed amount; when the amount of potassium permanganate added is 40±4 g / Kg of the raw material feed amount, the amount of filter aid added is 100±10 g / Kg of the raw material feed amount; when the amount of potassium permanganate added is 60±6 g / Kg of the raw material feed amount, the amount of filter aid added is 120±12 g / Kg of the raw material feed amount.

6. The preparation method according to claim 1, characterized in that: The step S3 of adjusting the pH of the feed solution before the chelating resin column to a weakly alkaline pH includes adjusting the pH to 7.0-9.

0.

7. The preparation method according to claim 1, characterized in that: In step S4, sodium chloride is added to the feed solution to a final concentration of 1% - 10%, including adding sodium chloride to the feed solution to a final concentration of 4% - 6%.

8. The preparation method according to claim 1, characterized in that: The freeze-drying procedure in step S5 includes: pre-freezing at -40°C for 3-5 h, starting the cold trap to cool down and the vacuum pump (50±10 Pa), raising the internal partition to 0±10°C within 1-3 h, maintaining for 14±4 h, vacuum at 20±5 Pa, raising the internal partition to 20±10°C within 1-3 h, maintaining for 14±4 h, vacuum at 20±5 Pa, raising the internal partition to 40±10°C within 1-3 h, maintaining for 1-3 h, ultimate vacuum, releasing the vacuum after completion, and unpacking.

9. The preparation method according to claim 1, characterized in that: The powdering and packaging in step S5 includes crushing the freeze-dried powder with a pulverizer, passing it through an 80-mesh sieve, packaging it into two layers of plastic bags, sealing them separately, and then sealing them into aluminum foil bags or aluminum barrels.

10. The preparation method according to claim 1, characterized in that: In the steps S4 and S5, ethanol is used instead of acetone as the organic solvent precipitant, and the amount of ethanol used is 1.1 times the original amount of acetone. The specific steps are as follows: S4, decolorization and precipitation classification: sodium chloride is added to the feed solution to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the crude heparin raw material feed amount, and the pH is controlled to 8 - 10, and the reaction is carried out at room temperature for not less than 0.5 h, and the pH is adjusted to neutral, and ethanol is added in an amount of 0.495 - 0.66 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the precipitated paste is removed, and ethanol is added to the clear liquid to a volume of 0.99 - 1.21 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded, and purified water in an amount of 4 - 10 times the volume of the crude heparin raw material is added to the precipitated paste, and the mixture is stirred until dissolved, and ethanol is added in an amount of 0.77 - 0.99 times the volume of the purified water, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded to obtain a precipitated paste; S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 0.99-1.21 times the volume of ethanol in the amount of the feed liquid, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above-mentioned operation of dissolving with the same amount of purified water and precipitating with ethanol, retain the precipitate, add 1.65 times the amount of ethanol in the amount of the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above-mentioned operation of washing and dehydrating with the same amount of ethanol 1-5 times, until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the feed liquid in each freeze-drying plate is not higher than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

11. The preparation method according to claim 1, characterized in that: In the steps S4 and S5, methanol is used instead of acetone as the organic solvent precipitant, and the amount of methanol used is 1.5 times the original amount of acetone. The specific steps are as follows: S4, decolorization and precipitation classification: sodium chloride is added to the feed solution to a final concentration of 1% - 10%, and peracetic acid or hydrogen peroxide for decolorization is added in an amount of 2% - 5% of the amount of crude heparin raw material fed, and the pH is controlled at 8 - 10, and the reaction is carried out at room temperature for not less than 0.5 h, and the pH is adjusted to neutral, and methanol is added in an amount of 0.675 - 0.90 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the precipitated paste is removed, and methanol is added to the clear liquid to a volume of 1.35 - 1.65 times the volume of the feed solution, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded, and purified water in an amount of 4 - 10 times the amount of crude heparin raw material fed is added to the precipitated paste, and the mixture is stirred until dissolved, and methanol is added in an amount of 1.05 - 1.35 times the volume of purified water, and the mixture is stirred and allowed to stand for not less than 4 h, and the supernatant is discarded to obtain a precipitated paste; S5. Purification and drying: Measure the volume or weight of the precipitated paste, add 2-3 times the amount of purified water, stir until dissolved, add 1.35-1.65 times the volume of the feed liquid of methanol, stir and let stand for not less than 4 hours, discard the supernatant, repeat the above-mentioned operation of dissolving with the same amount of purified water and precipitating with methanol, retain the precipitate, add no less than 2.25 times the amount of methanol of the precipitated paste in this step, stir for not less than 5 minutes, let stand, remove the supernatant, repeat the above-mentioned operation of washing and dehydrating with the same amount of methanol 1-5 times, until the precipitate is dispersed in particles, take the solid, dry it at not more than 70°C for not less than 6 hours, transfer the solid powder, dissolve it with 4-6 times the amount of water for injection, filter it at 0.22 μm, load the filtrate into the stainless steel freeze-drying plate of the freeze-drying equipment, the height of the feed liquid in each freeze-drying plate is not higher than 25 mm, start the freeze dryer, freeze-dry, collect the freeze-dried product, grind it into powder, and package it to obtain the final product of sulodexide raw material.

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