CN116731223A - Preparation method of sulodexide bulk drug - Google Patents
Preparation method of sulodexide bulk drug Download PDFInfo
- Publication number
- CN116731223A CN116731223A CN202310547150.3A CN202310547150A CN116731223A CN 116731223 A CN116731223 A CN 116731223A CN 202310547150 A CN202310547150 A CN 202310547150A CN 116731223 A CN116731223 A CN 116731223A
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- stirring
- freeze
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- drying
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- GRYSXUXXBDSYRT-WOUKDFQISA-N (2r,3r,4r,5r)-2-(hydroxymethyl)-4-methoxy-5-[6-(methylamino)purin-9-yl]oxolan-3-ol Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OC GRYSXUXXBDSYRT-WOUKDFQISA-N 0.000 title claims abstract description 73
- 229920000439 Sulodexide Polymers 0.000 title claims abstract description 72
- 229960003491 sulodexide Drugs 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 52
- 239000003814 drug Substances 0.000 title claims abstract description 50
- 229940079593 drug Drugs 0.000 title claims abstract description 42
- 239000012535 impurity Substances 0.000 claims abstract description 77
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- 238000000034 method Methods 0.000 claims abstract description 47
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- 239000002554 heparinoid Substances 0.000 claims abstract description 32
- 238000001556 precipitation Methods 0.000 claims abstract description 30
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- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims abstract description 15
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 6
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- UJMBCXLDXJUMFB-UHFFFAOYSA-K trisodium;5-oxo-1-(4-sulfonatophenyl)-4-[(4-sulfonatophenyl)diazenyl]-4h-pyrazole-3-carboxylate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)C1=NN(C=2C=CC(=CC=2)S([O-])(=O)=O)C(=O)C1N=NC1=CC=C(S([O-])(=O)=O)C=C1 UJMBCXLDXJUMFB-UHFFFAOYSA-K 0.000 description 3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides an industrial preparation method of sulodexide bulk drug, belonging to the technical field of biological medicine. The method takes heparinoids from pig intestine mucosa as an initial raw material, removes pathogenic agents such as viruses and bacteria by alkali treatment (and subsequent potassium permanganate oxidation treatment), denaturizes and filters impurities such as proteins by potassium permanganate oxidation and high-temperature treatment, removes inorganic impurities such as sulfate by strong cation resin exchange and strong anion resin exchange, removes metal ion impurities by chelate resin adsorption, removes pigment impurities by peracetic acid or hydrogen peroxide, removes pigment impurities by preferential organic solvent precipitant, controls polysaccharide components by fractional precipitation, and finally refines and freeze-dries to remove solvent to obtain the sulodexide raw material medicine. The preparation method is obviously different from the prior art, can control impurities and product quality more systematically and effectively, and is suitable for preparing bulk drugs of sulodexide medical products.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a sulodexide bulk drug.
Background
Sulodexide (Sulodexide), trade name of pegin (Vessel Due F), its pharmaceutical active ingredient (Active Pharmaceutical Ingredient, API) is a group of natural glycosaminoglycan and heparinoids compositions. Sulodexide molecules contain about 80% of fast moving heparin (Fast Moving Heparin, FMH) and about 20% Dermatan Sulfate (DS). Wherein, the fast moving heparin is mainly composed of-IdoA 2S-GlcNS 6S-repeating disaccharide units, while dermatan sulfate is mainly composed of-IdoA-GalN 4S-repeating disaccharide units, and dermatan sulfate occasionally has sulfation at the 6-position of GalNAc and the 2-position of IdoA. Their main structural units are schematically shown below:
-IdoA2S-GlcNS6S-；
-IdoA-GalN4S-。
As an antithrombotic drug, sulodide has strong antithrombotic effect on arteries and veins, and can be used for treating various venous diseases. The antithrombotic effect of sulodide is mainly related to the dose-dependent inhibition of some coagulation factors, in particular the inhibition of activated factor x (xa). The effect of the interfering thrombin (i.e. activated factor II, IIa) is next to that, so that the side effects such as bleeding caused by general anticoagulation are substantially avoided. The antithrombotic effect of sulodexide acts not only on free thrombin by Anti-thrombin-III (AT-III) but also on thrombin bound to fibrin by Heparin Cofactor II (HC-II), whereby the antithrombotic effect of sulodexide by inhibition of thrombin is manifested both in the inhibition of thrombosis and in the inhibition of thrombosis growth.
Sulodexide was developed by italy alpha Wei Shiman pharmaceutical company as an antithrombotic agent for the treatment of various cardiovascular diseases and has been used in italy, spanish, eastern europe, south america and asia for nearly 40 years. In China, sulodide was approved for sale in 2014, and its indication is "vascular diseases at risk of thrombosis". Sulodide is one of the most excellent anti-thrombus and anti-coagulant in clinic at present, can be injected intramuscularly, injected intravenously or orally, has higher bioavailability and tolerance, obvious drug effect and large clinical demand.
However, sulodexide has complex components and difficult preparation, is only sold as a raw development agent from Italy in China, is not seen to imitate the pharmacy and bulk drugs to be marketed, and is difficult to meet the increasing market demands of bulk drugs and preparations.
In the prior art, chinese patent publication No. CN 106883321B discloses a method for extracting sulodide bulk drug from heparin sodium byproducts, which oxidizes hydrogen peroxide and carries out fractional alcohol precipitation on heparin through ethanol, but the scheme does not consider the complex impurity type in the heparin byproducts and the pathogenic agent removing process treatment of animal source products, in addition, the method only depends on alcohol precipitation fractional control to quickly move the proportion of heparin and dermatan sulfate, and people in the field are well aware that the fractional efficiency is quite low, the alcohol precipitation time is long, the steps are complicated, the solvent consumption is excessive, and the method is unfavorable for industrial production. Another preparation method of sulodexide bulk drug is disclosed in chinese patent publication No. CN 108484789A, which comprises the steps of grading crude product of heparin sodium by ethanol, eluting and purifying by anion exchange resin with different concentration sodium chloride solution, and obtaining sulodexide bulk drug by ethanol precipitation, but this scheme does not consider the pathogen-removing treatment of raw material animal source, in addition, it is well known to those skilled in the art that crude heparin raw material has a large amount of impurities such as protein and nucleic acid residues, the protein is often combined with heparin, only by one-step ethanol precipitation method, it is difficult to effectively remove slow moving heparin, subsequent anion exchange is performed, 3% sodium chloride solution is used for eluting and 10% sodium chloride solution is used for eluting, this method can retain a large amount of slow moving heparin, thus leading to disqualification of component proportion in sulodexide product, therefore, the preparation method is difficult to ensure quality of sulodexide product.
There are several other proposals in the prior art, including potassium acetate precipitation, ban Shi reagent precipitation, ethanol fractional precipitation, anion exchange, etc. for general purpose of separating slow moving heparin and controlling the ratio of fast moving heparin to dermatan sulfate components, but these proposals are difficult to control the ratio of components by only using partial fractionation method, and on the other hand, basically do not consider the removal of pathogens, anionic and cationic impurities and complex protein impurities, so there is a major disadvantage that it is difficult to meet the demand of pharmaceutical grade products.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a novel preparation method of sulodexide, which meets the industrial production and quality requirements of pharmaceutical grade sulodexide bulk drugs.
The invention is realized by the following technical scheme:
a process for preparing the raw material medicine of sulodide includes such steps as sequentially alkali treating (and oxidizing with potassium permanganate) to remove virus and bacteria, oxidizing with potassium permanganate, high-temp treating to denature impurities such as protein, filtering, removing sulfate impurities, adsorbing with chelating resin, removing metallic ion impurities, supplementing sodium chloride, removing pigment impurities, depositing with organic solvent, classifying, refining, freeze drying, and removing water and solvent.
The key steps of the preparation method are as follows:
first, the inactivation and removal of pathogens (viruses and microorganisms);
secondly, protein impurities are denatured, solidified and filtered to remove;
third step, column purification: purifying and removing impurities such as free sulfate and the like (if the sulfate content in the raw materials exceeds the standard) by using a (cation and anion) ion exchange column, and removing metal ion impurities by using chelating resin adsorption;
fourth, decoloring and precipitation classification: removing pigment impurities from peracetic acid or hydrogen peroxide, and then precipitating and grading polysaccharide components by using an organic solvent;
and fifthly, refining, freeze-drying to obtain the sulodexide crude drug.
The preparation method of the sulodexide bulk drug is characterized by comprising the following steps:
s1, the pathogen comprises the inactivation and removal of viruses and microorganisms: weighing crude heparinoids, dissolving the crude heparinoids into 15-25% solution by water, adding 3-5% sodium hydroxide solid of the total amount of the feed liquid, and stirring at room temperature to be not less than 0.5 h;
s2, protein impurities are denatured, coagulated and filtered to remove: neutralizing, adding potassium permanganate with the amount of potassium permanganate not exceeding 66 g into each 1/Kg crude heparin raw material, heating to above 70 ℃, maintaining the temperature at high temperature until protein impurities are denatured and solidified, stopping heating, continuously stirring for not less than 10 min to fully separate out protein impurities, adding filter aid with the amount of filter aid added into each 1/Kg crude heparin raw material of 72-132 g, press-filtering, and cooling the filtrate to room temperature;
S3, purifying and removing impurities by a column: when the free sulfate content of the raw material is 0.5% or more, the feed liquid is firstly pumped through a strong cation resin exchange column and a strong anion resin exchange column in sequence for purification, otherwise, the two columns are skipped for purification, the feed liquid is regulated to be in weak alkaline pH, and pumped through a chelating resin column, wherein the dosage of the three resins is generally the same and is 3-5 times of the dosage of the raw material of the crude product heparinoid;
s4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, controlling the room temperature, enabling the mixture to act at least 0.5 h, adjusting pH to be neutral, adding acetone with the volume of 0.45-0.60 times of the feed liquid, stirring, standing at least 4 h, removing a precipitate paste, adding acetone to the clear liquid to be 0.9-1.1 times of the volume of the feed liquid, stirring, standing at least 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding acetone with the volume of 0.7-0.9 times of the purified water, standing at least 4 h after stirring, and discarding supernatant to obtain a precipitate paste;
S5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 0.9-1.1 times of acetone by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating acetone, reserving sediment, adding not less than 1.5 times of acetone of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of acetone washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide drug.
Preferably, the crude heparinoids in step S1 are derived from porcine intestinal mucosa.
In the invention, the room temperature refers to the temperature range of 15-35 ℃.
The reason why the alkali treatment and the potassium permanganate oxidation treatment can remove pathogens such as viruses and bacteria is based on the invention, the treatment can be applied not only in the stage of preparing raw materials of crude heparinoids, but also in other steps or unlimited sequences of preparing raw materials of sulodexide, and the alkali treatment and the potassium permanganate oxidation treatment are used for removing pathogens in other working procedures according to the technical examples of the invention, and the treatment is also considered to be within the technical scope of the invention.
Preferably, the amount of potassium permanganate in the step S2 is adjusted to the concentration of 4 mg/mL solution according to the feed liquid sample 280 Absorbance was added as follows:
| KMnO 4 additive amount (/ raw material feeding amount, g/Kg) | UV A 280 Reading the number |
| 0 | < 0.3 |
| 40 ± 4 | 0.3 – 0.7 |
| 60 ± 6 | > 0.7 |
。
The protein impurity content in the feed liquid can be reflected by the absorption value at the wavelength of 280 nm, the feed liquid sample is adjusted to the solution concentration of 4 mg/mL, if A 280 The absorbance is 0.3, and the corresponding protein impurities are contained in the raw material solids by about 10% assuming that the absorbance is brought by protein impurities, and similarly, A 280 At a absorbance of 0.7, the protein impurity was present in the raw material solids at about 25%.
The reason why the above-mentioned potassium permanganate oxidation and high-temperature treatment of the present invention denature and filter out impurities such as proteins is based on the fact that the treatment may be applied not only in the crude heparinoids raw material stage but also in other steps or not limited sequence of preparation of sulodexide raw material, and the use of potassium permanganate oxidation and high-temperature treatment in other steps to denature and filter out impurities such as proteins according to the technical examples of the present invention is considered to be within the technical scope of the present invention.
Preferably, the temperature heated and maintained in step S2 is 80 ℃ to 90 ℃.
Preferably, the filter aid in the step S2 is diatomaceous earth, which is added as follows:
| KMnO 4 additive amount (/ raw material feeding amount, g/Kg) | Filter aid addition (/ raw material charge, g/Kg) |
| 0 | 80 ± 8 |
| 40 ± 4 | 100 ± 10 |
| 60 ± 6 | 120 ± 12 |
。
Preferably, the press filtration in the step S2 means press filtration by a press filter.
Preferably, the step S3 is characterized in that the pH of the material liquid before the chelating resin column is adjusted to be slightly alkaline, and the pH is adjusted to 7.0-9.0.
The reason why the sulfate impurities are sequentially removed by strong cation resin exchange and strong anion resin exchange and then metal ion impurities are adsorbed and removed by chelating resin is based on the invention, the treatment can be applied not only at the stage after the crude heparinoids raw material is removed from the pathogeny, but also at other steps or unlimited sequences of preparation of sulodite raw material medicines, and the sulfate impurities are sequentially removed by strong cation resin exchange and strong anion resin exchange at other working procedures according to the technical examples of the invention, and then the metal ion impurities are adsorbed and removed by chelating resin, which is also considered to be within the technical scope of the invention.
In one group of examples (example 4) but not limited to this example, sulodexide drug substance can be obtained by first oxidizing heparin raw material with potassium permanganate, then sequentially removing sulfate impurities and metal ions with resin, removing pathogenic impurities with alkali treatment, and bleaching with peracetic acid or hydrogen peroxide to remove pigments.
Preferably, in the step S4, sodium chloride is added to the feed liquid to a final concentration of 1% -10%, including the feed liquid to a final concentration of 4% -6%.
Based on the foregoing removal of pigment impurities by peracetic acid or hydrogen peroxide, the treatment can be applied not only in the stage after the resin adsorption and impurity removal, but also in other steps or unlimited sequences of preparation of sulodexide bulk drug, and the removal of pigment impurities by using peracetic acid or hydrogen peroxide in other working procedure stages according to the technical example transformation or extension of the invention is considered to be within the technical scope of the invention.
In the invention, acetone is used as an organic solvent precipitant of sulodexide bulk drug in the steps S4 and S5.
Acetone has better effect than ethanol and methanol of the common organic solvent precipitant in the field, and the principle is that the relative dielectric constant of the precipitation mechanism is 20.7, 78.5, 24.5 and 32.7, so that the corresponding acetone is optimal (the dielectric constant is changed the most) and the ethanol is inferior in the precipitation effect of the aqueous solution.
Whereas raw heparan contains a large number of sulfated polysaccharide components, i.e., heparan sulfate, dermatan sulfate, fast-moving heparin and slow-moving heparin, which are similar in nature, the quality standard of target sulodexide requires about 80% fast-moving heparin and 20% dermatan sulfate as main components, and the index range is small, so that fractional precipitation separation of these polysaccharide components requires higher-grade organic solvent precipitants, and as described above, the effect of acetone is optimal according to the relative dielectric constant of the organic solvent precipitants.
For the reasons stated herein, the organic solvent precipitant may be other solvents having relatively low effect, including ethanol and/or methanol, while acetone has a better effect, and thus, the present patent preferably uses acetone as the organic solvent precipitant. However, it is readily understood that the use of other organic solvent precipitants in accordance with the teachings of the present invention are also considered to be within the scope of the present invention.
In addition, the organic solvent precipitant using ethanol as the sulodexide bulk drug is characterized in that in the steps S4 and S5, the ethanol usage amount is 1.1 times of the original acetone usage amount, and the method is specifically as follows:
s4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, controlling the room temperature, enabling the reaction to be not less than 0.5 and h, adjusting pH to be neutral, adding ethanol with the volume of 0.495-0.66 times of the feed liquid, stirring, standing to be not less than 4 h, removing a precipitate paste, adding ethanol to the clear liquid to be 0.99-1.21 times of the volume of the feed liquid, stirring, standing to be not less than 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding ethanol with the volume of 0.77-0.99 times of purified water, standing to be not less than 4 h after stirring, and discarding supernatant to obtain a precipitate paste;
S5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 0.99-1.21 times of ethanol by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating the ethanol, reserving the sediment, adding not less than 1.65 times of ethanol of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of ethanol washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide bulk drug.
In addition, the organic solvent precipitant using methanol as the sulodexide bulk drug is characterized in that in the steps S4 and S5, the use amount of methanol is 1.5 times of that of the original acetone, and the organic solvent precipitant is specifically as follows:
S4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, controlling the room temperature, enabling the reaction to be not less than 0.5 h, adjusting pH to be neutral, adding methanol with the volume of 0.675-0.90 times of the feed liquid, stirring, standing to be not less than 4 h, removing a precipitate paste, adding methanol to the clear liquid to be 1.35-1.65 times of the volume of the feed liquid, stirring, standing to be not less than 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding methanol with the volume of 1.05-1.35 times of purified water, standing to be not less than 4 h after stirring, and discarding supernatant to obtain a precipitate paste;
s5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 1.35-1.65 times of methanol by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating methanol, reserving sediment, adding not less than 2.25 times of methanol of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of methanol washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide bulk drug.
In one set of examples (example 3) but not limited to this example, the solvent acetone in the fractionation and purification of heparin samples was replaced with ethanol or methanol to prepare the sulodexide drug substance.
The time for the precipitation of the organic solvent in the preparation method to stand after stirring is preferably 12 h unless otherwise specified.
Preferably, the step S5 of freeze-drying includes: 3-5 h at-40 ℃ after pre-freezing, starting a cold trap cooling and vacuum pump (50+/-10 Pa), raising the temperature of the inner partition plate to 0+/-10 ℃ for 1-3 hours, keeping 14+/-4 h, keeping the temperature of the inner partition plate at 20+/-5 Pa for 1-3h to 20+/-10 ℃, keeping 14+/-4 h, keeping the temperature of the inner partition plate at 20+/-5 Pa for 1-3h to 40+/-10 ℃, keeping 1-3h, carrying out extreme vacuum, discharging vacuum after the end, and taking out the container.
The freeze drying is an important mode of medicine drying, and compared with the drying modes such as vacuum drying, hot air drying, spray drying and the like, the freeze drying has the advantages that the temperature is low when the product is freeze-dried, the product can be effectively prevented from being damaged by high temperature, and residual solvents can be effectively removed by using sublimation of water, so that the freeze drying is a preferable scheme for ensuring the quality of medicines.
For the reasons stated in the present invention, the drying of the sulodexide drug substance may be vacuum drying, hot air drying, spray drying and freeze drying, but the quality of the drug substance under freeze drying is better, so the present invention preferably uses freeze drying as the drying mode of the sulodexide drug substance finished product. However, it is to be readily understood that the use of vacuum drying and hot air drying, as modified or extended from the technical examples of the present invention, is also considered to be within the technical scope of the present invention.
Preferably, the powdering and sub-packaging in the step S5 comprises crushing the freeze-dried powder by a crusher, sieving, sub-packaging into two layers of plastic bags, respectively plastic packaging, and sealing into an aluminum foil bag or an aluminum barrel.
In one set of examples (example 5) but not limited to this example, the prepared multi-lot sulodexide drug substance products were tested and tested for quality meeting the requirements of the established index.
The preparation method of the sulodexide bulk drug is applied to the production of medicines.
The invention has the outstanding effects that: compared with the prior art, the method is basically different from the prior art, and can remove pathogenic agents such as viruses, bacteria and the like in crude heparinoids by alkali treatment (and subsequent potassium permanganate oxidation treatment), denature impurities such as proteins and remove the impurities by filtration, remove sulfate impurities by strong cation resin exchange and strong anion resin exchange, remove metal ion impurities by chelating resin adsorption, remove pigment impurities by peracetic acid or hydrogen peroxide, remove pigment impurities by organic solvent precipitants with different concentrations, carry out repeated dissolution precipitation fractionation for several times, and finally carry out refining and freeze drying, and the sulodexide bulk drug is prepared.
Drawings
Fig. 1: nuclear magnetic hydrogen spectrum of self-made sulodexide sample and sulodexide reference substance 1 H-NMR) comparison scheme.
Fig. 2: the comparison of the self-made sulodexide sample with the strong anion exchange-high performance liquid chromatography (SAX-HPLC) of the sulodexide control is schematically shown.
Fig. 3: schematic of molecular size exclusion-high performance liquid chromatography (SEC-HPLC) analysis of self-made sulodexide samples.
Fig. 4: and verifying a gel electrophoresis analysis comparison schematic diagram of three batches of self-made sulodexide samples and sulodexide control substances.
Detailed Description
The present invention will be further described in detail with reference to the following specific embodiments, but is not intended to limit the scope of the present invention.
Example 1: preparation of sulodite 1.
The preparation method is a preparation pilot scale of a hundred-gram-grade sulodexide sample, raw materials are commercial crude heparinoids, byproducts from production of porcine intestinal mucosa heparin are materials which are homogenized and powdered after dry materials are mixed, the batch is 215 Kg, and the test data are as follows: the appearance is of off-white powder, and is easy to dissolve in water; the specific rotation is +13°; pH (5%) 7.6; the drying weight loss is 4.4 percent; free sulfate content was 0.69%; in agarose electrophoresis analysis, the percentages of Slow Moving Heparin (SMH), fast Moving Heparin (FMH), dermatan Sulfate (DS) and Chondroitin Sulfate (CS) were 9%, 64%, 25% and <2%, respectively; the anticoagulation activity of sheep plasma method is 64U/mg.
The preparation process and the result of the sulodexide bulk drug are as follows:
s1, the pathogen comprises the inactivation and removal of viruses and microorganisms: raw heparinoids 500, g are weighed into a beaker, added with water approximately 2.5, L, mechanically stirred until substantially clear, then added with 125, g sodium hydroxide solids, and stirred for 1, h.
S2, protein impurities are denatured, coagulated and filtered to remove: adjusting pH to 8.2 with 6 mol/L hydrochloric acid solution, sampling (accurately sucking 2.4. 2.4 mL feed liquid according to the feeding concentration, diluting with water to constant volume to 100 mL to obtain 4 mg/mL solution to be tested), and determining A 280 0.798, according to a comparison table, adding potassium permanganate 33 g, heating to 70deg.C, maintaining high temperature (heating is stopped as soon as the feed liquid is boiled) until protein impurity is denatured and coagulated, stopping heating, stirring for 1 h to precipitate protein impurity sufficiently, adding diatomite 62 g, press-filtering with pump, and cooling filtrate to 26deg.C.
S3, purifying and removing impurities by a column: because the free sulfate content of the raw materials exceeds 0.5%, the feed liquid is firstly pumped through a 2.5L strong cation resin exchange column and a 2.5L strong anion resin exchange column in sequence, the feed liquid is subjected to top washing by 1L water after passing through the columns, filtrate is collected, the pH of the feed liquid is regulated to 8.2, the feed liquid is pumped through a 2.5L chelating resin column, and the filtrate is obtained by washing 1L by water, so that the filtrate is about 4.8L.
S4, decoloring and precipitation classification: adding 480, 480 g sodium chloride into the feed liquid, adding 25, 25 g peracetic acid, adjusting the pH to 9.8, stirring, and standing for decoloring 2 h; adjusting pH to 7.0, adding 2.6L acetone, stirring, standing for 12 h, removing precipitate paste, adding 2.2L acetone into the clear liquid, stirring, standing for 12 h, discarding supernatant, adding 3L purified water into the precipitate paste, stirring until the solution is clear, wherein the volume of the feed liquid is about 3.4L, adding 2.8L acetone, stirring, standing for 12 h, discarding supernatant to obtain precipitate paste about 600 g.
S5, refining and drying: adding 1.5. 1.5L purified water to the above precipitate paste, stirring until the solution is clear, wherein the volume of the feed liquid is about 2.0L, adding 2.0L acetone, standing for 12. 12 h after stirring, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating acetone, leaving the precipitate, adding 1.5. 1.5L acetone, stirring for 10 min, standing, removing the supernatant, repeating the operations of washing and dehydrating the same amount of acetone for 3 times, wherein the precipitate is in a particulate matter dispersion state, suction-filtering to obtain a solid, drying at 60 ℃ for 12 h, transferring the solid powder to obtain 329 g, dissolving with 1.2. 1.2L water for injection, filtering at 0.22 μm, loading the filtrate into a stainless steel freeze-drying plate of a freeze-drying device, and carrying out the solution height of about 12 mm, and freeze-drying the procedures: prefreezing 5 h at-40deg.C; starting a cold trap cooling and vacuum pump (60 Pa); 3 h inner partition plate is raised to 10 ℃, 18 h is kept, and vacuum is 25 Pa;3 h inner partition plate is raised to 30 ℃, 18 h is kept, and vacuum is 25 Pa; raising the temperature of the partition plate in the chamber 3 h to 45 ℃, and maintaining the temperature at 3 h and the extreme vacuum; and discharging the vacuum after the completion of the process, and taking out the container. Collecting lyophilized product, pulverizing with pulverizer, sieving with 80 mesh sieve, packaging into double-layer PE bag, and coating with aluminum foil bag to obtain sulodexide product 306 g with yield of 61.2%.
Test results: white freeze-dried powder, easy to dissolve in water; electrophoretic identification meets the regulations; specific rotation (measured as 4% aqueous solution) was +22.6° on a dry basis; the color of the solution is lighter than the standard color of EP yellow No. 5; the pH (1%) was 7.1; a as 0.4% solution of nucleic acid impurity 260nm The absorbance was 0.112; residual solvent(acetone) 176 ppm; protein impurity (Lowry method) 0.12%; the organic sulfur content was 9.0%; sulfonate/carboxylate ratio 1.8; the percentages of Fast Moving Heparin (FMH), dermatan Sulfate (DS) and Slow Moving Heparin (SMH) in agarose electrophoresis analysis were 79%, 20% and, respectively<2%; the anti-Xa activity was 80 IU/mg; the anticoagulation activity of the sheep plasma method is 58U/mg; the esterase activity was 13 LSU/mg.
Example 2: preparation of sulodite 2.
This is another pilot run of a sample of hundred gram grade sulodexide from a byproduct heparinoids of the purification refining stage of the fine heparin production, as tested in the following data: white powder in appearance and easy to dissolve in water; the specific rotation is +38°; pH (5%) 7.1; drying weight loss is 5.4%; free sulfate content was 0.34%; in agarose electrophoresis analysis, the percentages of Slow Moving Heparin (SMH), fast Moving Heparin (FMH), dermatan Sulfate (DS) and Chondroitin Sulfate (CS) were 22%, 64%, 14% and 0%, respectively; the anticoagulation activity of sheep plasma method is 103U/mg.
The process and preparation results of sulodite are as follows:
s1, the pathogen comprises the inactivation and removal of viruses and microorganisms: the crude heparinoids 1 Kg was weighed and added to a 50L reactor, water was added at about 15L, 600 g NaOH powder was added with stirring, and 2 h was stirred with a mechanical stirrer.
S2, protein impurities are denatured, coagulated and filtered to remove: adjusting pH to 8.0 with 4 mol/L hydrochloric acid, sampling (accurately sucking 6.0 mL feed liquid according to the feeding concentration, diluting with water to constant volume to 100 mL to obtain 4 mg/mL solution to be tested), and determining A 280 0.316, KMnO was added according to a comparison table 4 126 And g, starting a heater for heating, starting heat preservation and controlling at 85 ℃ plus or minus 5 ℃ when the temperature reaches 85 ℃ until the protein impurities are denatured and solidified, stopping heating, continuing stirring for 1 to h, fully precipitating the protein impurities, adding diatomite for 300 g, performing filter pressing by using a test type filter press, and pouring the filtrate into another reaction tank for cooling to 24 ℃.
S3, purifying and removing impurities by a column: since the free sulfate content of the raw material is less than the limit value of 0.5%, the feed solution is adjusted to ph=8.0, and the feed solution is directly pumped through a 12L chelating resin column, and washed with water for 3L, to obtain filtrate 18L.
S4, decoloring and precipitation classification: adding solid NaCl powder 900 g, adding hydrogen peroxide acid 120 g, removing residual pigment in the solution, controlling pH=9.0, reacting at room temperature to 1.5 h, adjusting pH=7.0, adding 9L acetone, stirring, standing for 4 h, removing precipitate paste, adding 18L of acetone into the clear liquid, stirring, standing for 6 h, discarding supernatant, adding 7L purified water into the precipitate paste, stirring until the solution is clear, adding 6.5L acetone, stirring, standing for 6 h, discarding supernatant, and obtaining precipitate paste about 1.5 Kg.
S5, refining and drying: adding 4.5L purified water, stirring to dissolve, adding 6.0L acetone, stirring, standing 12. 12 h, discarding supernatant, repeating the operations of dissolving the same amount of purified water and precipitating acetone, collecting precipitate, adding 3.0L acetone, stirring for 10 min, standing, removing supernatant, repeating the operation of washing and dehydrating the same amount of acetone for 2 times, taking precipitate as particulate matter dispersed state, taking solid, drying 12 h at 60 ℃, transferring solid powder to obtain 0.75 Kg, dissolving with 4 times amount of water for injection, filtering 0.22 μm, loading filtrate into stainless steel freeze-drying plate of freeze drying equipment, regulating solution height to about 24 mm, pre-freezing to-40 ℃ for 3 h, starting cold trap cooling and vacuum pump (40 Pa), heating 1 h inner partition plate to-5 ℃, maintaining 12 h, heating 1 h inner partition plate to 10 ℃, maintaining 12 h, heating 1 Pa inner partition plate to 40 ℃ in vacuum 15, maintaining 1 Pa, maintaining h, and discharging out of vacuum tank after the end. And (3) collecting freeze-dried products, crushing by a crusher, sieving by a 80-mesh sieve, packaging by a double-layer PE bag, sealing an aluminum foil bag on the outer layer, and sub-packaging to obtain a sulodexide raw material medicine final product 672 g, wherein the yield is 67.2%.
Test results: white freeze-dried powder, easy to dissolve in water; electrophoretic identification meets the regulations; specific rotation (measured as 4% aqueous solution) was +24.3° on a dry basis; the color of the solution is lighter than the standard color of EP yellow No. 6; pH (1%) was 7.0; a as 0.4% solution of nucleic acid impurity 260nm The absorbance was 0.082; residual solvent (acetone) was 93 ppm; protein impurity (Lowry method) 0.05%; the organic sulfur content was 9.1%; sulfonate/carboxylate ratio of1.8; the percentages of Fast Moving Heparin (FMH), dermatan Sulfate (DS) and Slow Moving Heparin (SMH) in agarose electrophoresis analysis were 80%, 19% and, respectively<1%; the anti-Xa activity was 94 IU/mg; the anticoagulation activity of the sheep plasma method is 64U/mg; the esterase activity was 13 LSU/mg.
The small knot: the product yield of this example is improved over example 1, probably because fewer impurities are used in the starting material, e.g. lower free sulfate content, and thus less loss is possible in the column purification step.
Example 3: preparation of sulodite 3.
In this example, the organic solvent precipitant using ethanol as the fractional precipitation step was examined, the raw materials (and the amounts of the materials) were the same as in example 1, and the S1 causative agent in the preparation process included inactivation and removal of viruses and microorganisms, denaturation and solidification of S2 protein impurities, filtration removal, and purification by S3 column and removal of impurities were the same as in example 1, and the remaining preparation processes and results were as follows:
s4, decoloring and precipitation classification: adding sodium chloride 480 g into the filtrate of about 4.8 and L, adding peracetic acid 25 g, adjusting the pH to 9.2, stirring at room temperature, and decoloring by 2 h; adjusting pH to 7.0, adding 2.9L ethanol, stirring, standing for 12 h, removing precipitate paste, adding ethanol 2.6L into the clear liquid, stirring, standing for 12 h, discarding supernatant, adding 3L purified water into the precipitate paste, stirring until the solution is clear, wherein the volume of the feed liquid is about 3.4L, adding 3.0L ethanol, stirring, standing for 12 h, discarding supernatant to obtain precipitate paste about 570 g.
S5, refining and drying: adding 1.55L purified water to the above precipitate paste, stirring until the solution is clear, wherein the volume of the solution is about 2.0L, adding 2.2L ethanol, stirring, standing for 12. 12 h, discarding the supernatant, repeating the above operation of dissolving the same amount of purified water and precipitating ethanol, collecting the precipitate, adding 1.7L ethanol, stirring for 10 min, standing, removing the supernatant, repeating the above operation of washing and dehydrating the same amount of ethanol for 3 times, wherein the precipitate is in a particulate matter dispersion state, suction-filtering to obtain solid, drying at 60deg.C for 14 h, transferring solid powder to obtain 288 g, dissolving with 1.7L water for injection, filtering with 0.22 μm, loading the filtrate into a stainless steel freeze-drying plate of a freeze-drying device, and the liquid medicine height is about 17 mm, and freeze-drying the steps: prefreezing at-40deg.C to 4 h; starting a cold trap cooling and vacuum pump (50 Pa); 2 h, raising the temperature of the separator to 10 ℃, maintaining the temperature at 12 h and vacuum at 20 Pa;2 h, raising the temperature of the separator to 25 ℃, maintaining the temperature at 12 h and vacuum at 20 Pa; raising the temperature of the separator in the chamber 2 h to 40 ℃, and maintaining the temperature at 2 h and the extreme vacuum; and discharging the vacuum after the completion of the process, and taking out the container. Collecting lyophilized product, pulverizing with pulverizer, sieving with 80 mesh sieve, packaging into double-layer PE bag, and coating with aluminum foil bag to obtain sulodexide product 262 g with yield of 52.4%.
Test results: white freeze-dried powder, easy to dissolve in water; electrophoretic identification meets the regulations; specific rotation (measured as 4% aqueous solution) was +22.5° on a dry basis; the color of the solution is lighter than the standard color of EP yellow No. 5; the pH (1%) was 7.1; a as 0.4% solution of nucleic acid impurity 260nm The absorbance was 0.122; residual solvent (acetone) was 137 ppm; protein impurity (Lowry method) 0.11%; the organic sulfur content was 8.4%; sulfonate/carboxylate ratio 1.6; the percentages of Fast Moving Heparin (FMH), dermatan Sulfate (DS) and Slow Moving Heparin (SMH) in agarose electrophoresis analysis were 78%, 21% and, respectively<2%; the anti-Xa activity was 85 IU/mg; the anticoagulation activity of the sheep plasma method is 56U/mg; the esterase activity was 11 LSU/mg.
The small knot: the ethanol is used as an organic solvent precipitator for grading, and the product meeting the quality standard requirement of the established sulodexide bulk drug can be obtained, and the yield is slightly lower than that of the preparation method using acetone as the organic solvent precipitator described in the embodiment 1.
Example 4: preparation 4 of sulodite.
The preparation method for examining different step sequences is to adjust the core step sequences of alkali treatment for pathogen removal, protein impurity removal and the like, and the examined sulodexide raw material medicine is prepared, and the raw materials (and the feeding amount) are the same as those in the embodiment 1, and the process and the result are as follows:
(1) Protein impurities are denatured, coagulated and filtered to remove: (in this step, the pathogen and protein impurities were removed by treatment with potassium permanganate first) crude heparinoid starting material 1 Kg from example 1 was weighed, water was used to determine the volume to 5L, and samples were taken (and diluted with water to 4 mg/mL depending on the feed concentration) for determination of A 280 Adding potassium permanganate 65 g according to a comparison table, heating to 85deg.C, maintaining the temperature at 85deg.C+ -5deg.C until protein impurity is denatured and coagulated, stopping heating, stirring for 1 h to precipitate protein impurity, adding diatomite 120 g, press-filtering with a tester, and cooling the filtrate to room temperature.
(2) Purifying and removing impurities by a column: pumping the feed liquid through a 4L strong cation resin exchange column and a strong anion resin exchange column in sequence, flushing with 1L water after each column passage, collecting filtrate, adjusting the pH value of the feed liquid to 8.0, pumping through a 4L chelating resin column, flushing with water for 1L, and obtaining filtrate 7.3L.
(3) Alkali treatment: sodium hydroxide solid 290, g, was added to the feed solution and mechanically stirred 2 h.
(4) Decoloring, precipitation and classification: adjusting pH to 10.2 with hydrochloric acid solution, adding hydrogen peroxide 42 g, checking that the pH is 9.5, reacting at room temperature to 1.5 h, adjusting pH to 7.0, adding 3.7L acetone, stirring, standing 12 h, removing precipitate paste, adding 7.3L acetone into the clear liquid, stirring, standing 12 h, discarding supernatant, adding 6L purified water into the precipitate paste, stirring until the solution is clear, adding 4.8L acetone, stirring, standing 12 h, discarding supernatant to obtain precipitate paste 1.2L.
(5) Refining and drying: the paste was precipitated by 1.2. 1.2L, 3. 3L of purified water was added, stirred until it was dissolved, 3. 3L of acetone was added, stirred and allowed to stand for 12. 12 h, the supernatant was discarded, the same amount of purified water was repeated for dissolution and acetone precipitation, the precipitate was left, 3.7 of L of acetone was added, stirred for 10 minutes, and allowed to stand for 10 minutes, the same amount of acetone was repeated for 4 times for washing and dehydration, acetone was 3.7 of L each time, and stirred for 10 minutes each time, at this time the precipitate was in a particulate dispersion form, and was allowed to stand, a solid was taken, dried at 60℃for 12. 12 h, and the solid powder was transferred to obtain 0.66. 0.66 Kg, dissolved with 5 times of water for injection, 0.22 μm was filtered, the filtrate was equally divided into three portions, and loaded into a stainless steel freeze-drying plate of a freeze-drying apparatus, the liquid medicine was about 11 mm in height, and the freeze-drying method was the same as in example 3, and taken out of the case. Collecting the freeze-dried product, pulverizing into powder, and packaging to obtain the final product 611 and g of the sulodexide crude drug, wherein the yield is 61.1%.
Test results: white jellyDry powder, readily soluble in water; electrophoretic identification meets the regulations; specific rotation (measured as 4% aqueous solution) was +27.1° calculated on a dry basis; the color of the solution is lighter than the standard color of EP yellow No. 5; pH (1%) 7.3; a as 0.4% solution of nucleic acid impurity 260nm The absorbance was 0.078; residual solvent (acetone) was 126 ppm; protein impurity (Lowry method) 0.09%; the organic sulfur content was 8.7%; sulfonate/carboxylate ratio 1.7; the percentages of Fast Moving Heparin (FMH), dermatan Sulfate (DS) and Slow Moving Heparin (SMH) in agarose electrophoresis analysis were 82%, 17% and, respectively<2%; the anti-Xa activity was 91 IU/mg; the anticoagulation activity of the sheep plasma method is 60U/mg; the esterase activity was 12 LSU/mg.
The small knot: the preparation method adopts potassium permanganate to remove pathogens, carries out oxidation filtration, then carries out alkali treatment, decolorization, alcohol precipitation classification and final refining after column purification and impurity removal, has smooth and controllable preparation process, and the product meets the index requirements of sulodexide bulk drug through inspection, so that the core steps of the preparation method can be adjusted, and the preparation method is suitable for process flow layout and industrial amplification.
Example 5: and (3) verifying the production and preparation of the sulodexide bulk drug in three batches continuously.
The production of three batches of sulodexide raw medicines is verified, the raw materials are the same as in example 1, the feeding amount of each batch is 35 Kg, the preparation methods of the three batches are the same, only the process control, the intermediate weight, the material consumption and the like are slightly different according to the process control value change, and the preparation process is taking the first batch as an example, and the preparation method comprises the following steps:
S1, the pathogen comprises the inactivation and removal of viruses and microorganisms: weighing crude heparinoids 35.0. 35.0 Kg, adding into a stainless steel tank T1, adding water about 157.5L, stirring for 30 min, and dissolving the feed liquid until the feed liquid is clear, wherein the volume is about 175L; 7.0. 7.0 Kg sodium hydroxide solids were added and stirring was continued for 1 h.
S2, protein impurities are denatured, coagulated and filtered to remove: slowly adding 6 mol/L hydrochloric acid solution of about 25L, adjusting pH to 8.2, and measuring the total amount of the materials at about 200L, and weighing 2.29 g materials (according to the volumes of the materials and the materials, diluting the materials with water to constant volume to 100 mL, which is 4 mg/mL of the sample solution to be measured), to obtain a sampleDecide A 280 0.724, adding potassium permanganate 2.1 Kg according to the comparison table, heating to 85deg.C, maintaining for 15 min, stopping heating, continuing stirring for 0.5 h, adding diatomite 3.5 Kg, and maintaining stirring; preparing a plate-frame filter, press-filtering the feed liquid, flushing with 30L hot water, collecting filtrate to a stainless steel tank T2, and cooling to room temperature.
S3, purifying and removing impurities by a column: pumping the cooled feed liquid through a 140L strong cation resin exchange column 1 and a 140L strong anion resin exchange column 2 which are connected in series in sequence, performing top washing with 30L water after passing through the columns, and collecting filtrate to a stainless steel tank T3; the pH of the feed solution was adjusted to 8.2, the feed solution was continuously pumped through a 140L chelating resin column, water was flushed through 30L, and the filtrate was collected into stainless steel tank T4 to give a filtrate of about 300L.
S4, decoloring and precipitation classification: adding sodium chloride 15-Kg into the feed liquid, and stirring until the solution is clear; adding peracetic acid 1.05 and Kg, regulating the pH to 9.0 with dilute alkali solution, stirring, and standing at room temperature for decoloration 2 h; adjusting pH to 7.0, adding 150L acetone, stirring, standing for 12 h, transferring supernatant to a precipitation tank T5, and discarding precipitate paste; adding acetone 150L into the clear liquid, stirring, standing for 12 h, discarding supernatant, adding 210L purified water into the precipitate paste, stirring until the solution is clear, wherein the volume of the solution is about 260L, adding 208L acetone, stirring, standing for 12 h, discarding supernatant to obtain precipitate paste about 44 Kg.
S5, refining and drying: adding 110L purified water into the precipitate paste, stirring until the solution is clear, transferring the feed liquid to a precipitation tank T6 (a little purified water washing tank T5 is combined), wherein the volume of the feed liquid is about 150L, adding 150L acetone, stirring, standing for 12 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating acetone, collecting precipitate, adding 100L acetone, stirring for 10 min, standing, removing the supernatant, repeating the same amount of acetone washing and dehydrating operations for 2 times, and dispersing the precipitate into particles; unloading, suction filtering to obtain solid, transferring to a drying disc, vacuum drying at 60 ℃ to obtain 14. 14 h, transferring solid powder to obtain 22.75 Kg; adding the powder into a 200L liquid preparation tank in a C-level clean area liquid preparation room, adding about 110L of injection water precooled to room temperature, and stirring for about 30 min, wherein the liquid is dissolved; the feed solution was filtered in series through 0.45 μm and then 22 μm filters, and the filtrate was transferred to stainless steel lyophilization trays with a clean beaker at about 2 per tray L and a drug solution height of about 20 mm, the procedure set for the lyophilization machine was as follows: prefreezing at-40deg.C to 4 h; starting a cold trap cooling and vacuum pump (50 Pa); 2 h inner partition plate is raised to 0 ℃, kept at 14 h and vacuum 20 Pa;2 h inner partition plate is raised to 20 ℃, kept at 14 h and vacuum is 20 Pa; raising the temperature of the separator in the chamber 2 h to 40 ℃, and maintaining the temperature at 2 h and the extreme vacuum; and discharging the vacuum after the completion of the process, and taking out the container. Collecting the freeze-dried product blocks, transferring to a stainless steel barrel with a cover, pulverizing, sieving, weighing and bagging by a double-layer PE bag, respectively packaging in plastic bags, and then packaging in plastic bags by an aluminum foil bag to obtain the finished product of the sulodexide bulk drug.
Results: the product weights 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 test results are shown in Table 1 below.
Table 1 shows the results of the test for continuously verifying three batches of sulodexide drug substance
。
The data show that the quality inspection results of the three continuous batches of verification batches are highly consistent, and reflect the high controllability of the preparation process and quality.
Example 6: nuclear magnetic hydrogen spectrum @ 1 H-NMR) analysis compares the homemade sulodexide drug substance with the control.
Test article: the sulodexide drug substance was self-made from examples 1 and 2.
Control: freeze-dried powder of original preparation injection.
Sample preparation: 35 mg test or control samples were dissolved in 0.6 mL deuterium water (0.002% TSP was added as an internal standard calibration zero), the solution was transferred to a nuclear magnetic tube of 5 mm, and sonicated for 2 min.
Instrument: NMR Bruker AC500 HD;
software: bruker Top Spin 3.2 (Bruker Biospin GmbH).
Nuclear magnetic analysis was performed on samples of examples 1 and 2 and as-ground sulodexide, 1 H-NMRthe graph results and the comparison graph are shown in figure 1.
The result shows that the peak (response) and the peak position of the sulodexide crude drug prepared by the invention are almost consistent with those of the original ground reference substance, so that the functional group composition and the structure of the sulodexide crude drug can be judged to be the same.
Example 7: and (3) analyzing and comparing the self-made sulodexide crude drug with a control substance by strong anion exchange-high performance liquid chromatography (SAX-HPLC).
Test article: the self-made sulodexide drug substance was from example 1.
Control: freeze-dried powder of original preparation injection.
Sample treatment: preparing a sample into a 20 mg/mL solution, taking 20 mu L, adding 70 mu L of a pH 7.0 calcium acetate solution and 100 mu L of a heparinase solution, uniformly mixing, standing at least for 48 h in a water bath at 25 ℃, and carrying out heparinase enzymolysis to obtain a sample degradation solution. The control is prepared by the same method.
SAX-HPLC chromatographic conditions:
chromatographic column: waters spherisorb SAX, (4.0X1250 mm, 5 μm); high performance liquid chromatography mobile phase: 0.1 M NH 4 Ac + 0.02% NaN 3 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 0.8 mL/min; column temperature: room temperature; detector wavelength: 232 nm; sample injection volume: 18. 2, L; run time: 79 min; sample injection amount: 20. mu L.
The chromatographic analysis results and the spectrogram comparison are shown in figure 2.
The results show that the disaccharide composition of the sample prepared by the invention is consistent with the type and proportion composition of the sulodexide reference substance through heparinase enzymolysis and SAX-HPLC separation analysis.
Example 8: size exclusion-high performance liquid chromatography (SEC-HPLC) analysis.
Test article: the self-made sulodexide drug substance was from example 1.
Molecular weight calculation calibrator: USP heparin sodium molecular weight calibration standard.
The sample treatment method comprises the following steps: the test sample and the calculation standard sample are diluted by mobile phase to prepare 5 mg/mL water solution, and the water solution are filtered by a 0.45 mu m filter membrane.
HPLC detection conditions:
chromatographic column: 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 NH 4 Ac + 0.02% NaN 3 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 0.6 mL/min; column temperature: 30 ℃; a detector: differential; sample injection volume: 20. 2, L; run time: 50 And (5) min.
The molecular weight distribution results and SEC-HPLC chromatograms of the test samples are shown in FIG. 3.
The results show that in the samples prepared by the invention, the fast moving heparin accounts for 79.5%, the average molecular weight is about 11000 and Da, the dermatan sulfate accounts for about 19.5%, and the average molecular weight is about 36000 and Da. The composition ratio of the sample is consistent with the index of the original ground product.
Example 9: agarose gel electrophoresis analysis compares the homemade sulodexide drug substance with the control.
Test article: self-made sulodexide drug substance example 5 three batches of samples were validated.
Control: freeze-dried powder of original preparation injection.
Standard reference substance: mixing standard solution of Slow Moving Heparin (SMH), fast Moving Heparin (FMH), dermatan Sulfate (DS) and Chondroitin Sulfate (CS), and positioning electrophoresis strip.
Sample treatment: the test sample and the control sample were diluted and prepared into 20. 20 mg/mL aqueous solutions, respectively, and diluted 10-fold with 0.02% cresol red solution (20% glycerol was added) to give a solution concentration of 2.0 mg/mL. Standard substance reference substance solution is prepared by the same method.
Agarose gel: the preparation method comprises the steps of preparing a 20 mM barium acetate solution, heating to boil until the agarose concentration is 0.6%, preparing the gel, and cooling.
Electrophoresis system: 20 mM barium acetate solution, current 160-400 mA, voltage 140-200V, time 90 min.
The results of the electrophoresis analysis are shown in FIG. 4, and the percentage data are shown in Table 1 in example 5.
The results demonstrate that three batches of self-made sulodexide samples were validated to be consistent with the original sulodexide control, with lower control of the slow moving heparin component.
All the data reveal that the preparation method is practical and efficient, the product sulodexide is consistent with the original ground reference substance, and meets the requirements of the established drug indexes, and the preparation method can systematically and effectively control the impurities and the product quality, thereby being suitable for the industrialized preparation of sulodexide medical products.
There are, of course, many specific embodiments of the invention, not set forth herein. All technical solutions formed by equivalent substitution or equivalent transformation fall within the scope of the invention claimed.
Claims (12)
1. The preparation method of the sulodexide bulk drug is characterized by comprising the following steps:
s1, the pathogen comprises the inactivation and removal of viruses and microorganisms: weighing crude heparinoids, dissolving the crude heparinoids into 15-25% solution by water, adding 3-5% sodium hydroxide solid of the total amount of the feed liquid, and stirring at room temperature to be not less than 0.5 h;
s2, protein impurities are denatured, coagulated and filtered to remove: neutralizing, adding potassium permanganate with the amount of potassium permanganate not exceeding 66 g into each 1/Kg crude heparin raw material, heating to above 70 ℃, maintaining the temperature at high temperature until protein impurities are denatured and solidified, stopping heating, continuously stirring for not less than 10 min to fully separate out protein impurities, adding filter aid with the amount of filter aid added into each 1/Kg crude heparin raw material of 72-132 g, press-filtering, and cooling the filtrate to room temperature;
s3, purifying and removing impurities by a column: when the free sulfate content of the raw material is 0.5% or more, the feed liquid is firstly pumped through a strong cation resin exchange column and a strong anion resin exchange column in sequence for purification, otherwise, the two columns are skipped for purification, the feed liquid is regulated to be in weak alkaline pH, and pumped through a chelating resin column, wherein the dosage of the three resins is generally the same and is 3-5 times of the dosage of the raw material of the crude product heparinoid;
S4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, reacting at room temperature, not less than 0.5 and h, adjusting pH to be neutral, adding acetone with the volume of 0.45-0.60 times of the feed liquid, stirring, standing for not less than 4 h, removing a precipitate paste, adding acetone to the clear liquid to be 0.9-1.1 times of the volume of the feed liquid, stirring, standing for not less than 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding acetone with the volume of 0.7-0.9 times of purified water, stirring, standing for not less than 4 h, and discarding supernatant to obtain a precipitate paste;
s5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 0.9-1.1 times of acetone by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating acetone, reserving sediment, adding not less than 1.5 times of acetone of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of acetone washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide drug.
2. The method of claim 1, wherein the crude heparinoids in step S1 are derived from porcine intestinal mucosa.
3. The method according to claim 1, wherein the amount of potassium permanganate in the step S2 is A when the solution concentration is adjusted to 4 mg/mL according to the sample of the feed solution 280 Values were added as follows: a is that 280 Reading a value<0.3 without adding potassium permanganate, A 280 The addition amount of potassium permanganate is 40+/-4 g/Kg of the raw material feeding amount when the reading value is 0.3-0.7, A 280 Reading a value>The addition amount of potassium permanganate at 0.7 is used as raw material60+/-6 g/Kg of the feeding amount.
4. The method of claim 1, wherein the temperature heated and maintained in step S2 is 80 ℃ to 90 ℃.
5. The process according to claim 1 and claim 3, wherein the filter aid in step S2 is diatomaceous earth, added as follows: when the potassium permanganate is not added, the addition amount of the filter aid is 80+/-8 g/Kg of the raw material feeding amount; when the addition amount of the potassium permanganate is 40+/-4 g/Kg of the raw material addition amount, the addition amount of the filter aid is 100+/-10 g/Kg of the raw material addition amount; when the addition amount of potassium permanganate is 60+/-6 g/Kg of the raw material, the addition amount of the filter aid is 120+/-12 g/Kg of the raw material.
6. The method according to claim 1, wherein the step S3 of adjusting the pH of the feed solution to a slightly alkaline pH comprises adjusting the pH to 7.0 to 9.0.
7. The method of claim 1, wherein the feed solution is added with sodium chloride to a final concentration of 1% -10% in step S4, including the feed solution being added with sodium chloride to a final concentration of 4% -6%.
8. The method according to claim 1, wherein the step S5 of freeze-drying comprises: 3-5 h at-40 ℃, starting a cold trap cooling and vacuum pump (50+/-10 Pa), raising the temperature of a 1-3h inner partition plate to 0+/-10 ℃, keeping 14+/-4 h, raising the temperature of a 1-3h inner partition plate to 20+/-10 ℃, keeping 14+/-4 h, keeping the temperature of a 20+/-5 Pa, raising the temperature of a 1-3h inner partition plate to 40+/-10 ℃, keeping 1-3h, carrying out extreme vacuum, discharging vacuum after the end, and discharging from a box.
9. The preparation method of claim 1, wherein the powdering and sub-packaging in the step S5 comprises crushing the freeze-dried powder by a crusher, sieving with a 80-mesh sieve, sub-packaging into two layers of plastic bags, respectively plastic packaging, and sealing into an aluminum foil bag or an aluminum barrel.
10. The method of claim 1, wherein the following process steps are used in any order: the method comprises the steps of S1 application steps of alkali treatment inactivation and pathogen removal, S2 application steps of potassium permanganate oxidation and heat treatment to denature, solidify and filter protein impurities, S3 application steps of cation exchange column adsorption removal of free sulfate radical and chelating resin column adsorption removal of metal ions, and S4 application steps of peracetic acid or hydrogen peroxide decoloration and organic solvent precipitant fractional precipitation.
11. The preparation method according to claim 1, wherein in the steps S4 and S5, ethanol is used as an organic solvent precipitator instead of acetone, the ethanol is used in an amount 1.1 times that of the original acetone, and the specific steps are as follows:
s4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, controlling the room temperature, enabling the reaction to be not less than 0.5 and h, adjusting pH to be neutral, adding ethanol with the volume of 0.495-0.66 times of the feed liquid, stirring, standing to be not less than 4 h, removing a precipitate paste, adding ethanol to the clear liquid to be 0.99-1.21 times of the volume of the feed liquid, stirring, standing to be not less than 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding ethanol with the volume of 0.77-0.99 times of purified water, standing to be not less than 4 h after stirring, and discarding supernatant to obtain a precipitate paste;
s5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 0.99-1.21 times of ethanol by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating the ethanol, reserving the sediment, adding not less than 1.65 times of ethanol of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of ethanol washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide bulk drug.
12. The preparation method according to claim 1, wherein in the steps S4 and S5, methanol is used as an organic solvent precipitant instead of acetone, the amount of methanol being 1.5 times that of the original acetone, and the specific steps are as follows:
s4, decoloring and precipitation classification: adding sodium chloride to a final concentration of 1% -10%, adding peracetic acid or hydrogen peroxide for decolorization, the quantity of which is 2% -5% of the feeding quantity of crude heparinoids raw materials, controlling pH to be about 8-10, controlling the room temperature, enabling the reaction to be not less than 0.5 h, adjusting pH to be neutral, adding methanol with the volume of 0.675-0.90 times of the feed liquid, stirring, standing to be not less than 4 h, removing a precipitate paste, adding methanol to the clear liquid to be 1.35-1.65 times of the volume of the feed liquid, stirring, standing to be not less than 4 h, discarding supernatant, adding purified water with the feeding quantity of 4-10 times of crude heparinoids raw materials into the precipitate paste, stirring until the solution is clear, adding methanol with the volume of 1.05-1.35 times of purified water, standing to be not less than 4 h after stirring, and discarding supernatant to obtain a precipitate paste;
s5, refining and drying: measuring the volume or weight of the sediment paste, adding 2-3 times of purified water, stirring until the sediment is dissolved, adding 1.35-1.65 times of methanol by volume of the liquid material, standing after stirring for not less than 4 h, discarding the supernatant, repeating the operations of dissolving the same amount of purified water and precipitating methanol, reserving sediment, adding not less than 2.25 times of methanol of the sediment paste in the step, stirring for not less than 5 min, standing, removing the supernatant, repeating the same amount of methanol washing and dehydrating operations for 1-5 times until the sediment is in a particle dispersion state, taking solid, drying at not more than 70 ℃, not less than 6 h, transferring solid powder, dissolving with 4-6 times of water for injection, filtering at 0.22 mu m, loading the filtrate into a stainless steel freeze-drying disc of freeze-drying equipment, keeping the height of the liquid material in each freeze-drying disc to be not more than 25 mm, starting a freeze-dryer, freeze-drying, collecting freeze-dried products, powdering, and obtaining the final product of the sulodide bulk drug.
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