Heparin sodium product and its extraction method and application

Abstract

The present invention discloses a heparin sodium fine product and its extraction method and application. The extraction method comprises the following steps: (1) pretreatment to obtain precipitate A; (2) acid-base treatment: adjusting the pH value of the precipitate A aqueous solution to 1.5-2.0, filtering to obtain an acidic filtrate; adjusting the pH value of the acidic filtrate to 11-12, stirring at 90-92°C and filtering to obtain an alkaline filtrate; precipitating the alkaline filtrate to obtain precipitate B; (3) oxidation: oxidizing the precipitate B aqueous solution 2-4 times to obtain precipitate C; (4) freeze-drying. The extraction method of the present invention can effectively prevent prions from entering the heparin sodium fine product. The heparin sodium fine product does not contain prions, and the content of other types of protein impurities is also very low, which has great market application value.

Description

Technical Field

[0001] The invention relates to a refined heparin sodium product and an extraction method and application thereof. Background Art

[0002] Heparin sodium is an anticoagulant drug with a wide range of clinical applications. Its basic raw materials are extracted from the visceral tissues of mammals. Common sources are pig small intestinal mucosa, bovine lung and bovine small intestinal mucosa, and sheep small intestinal mucosa, among which pig small intestinal mucosa is the main source of extraction. With the global supply of heparin sodium being tight and the frequent outbreaks of pig diseases, heparin sodium derived from cattle and sheep has the potential to return to the mainstream market.

[0003] In history, cattle and sheep have had Creutzfeldt-Jakob disease (commonly known as mad cow disease, BSE) and sheep pruritus (TSE). The pathogenic source of BSE / TSE is prion (prion protein), which is a protein substance that can replicate itself and mutate normal proteins. Because it has no typical viral structure and does not stimulate immune response, the human body cannot resist the infection of prion (prion protein). When humans are infected with prions (prion proteins), they will cause central nervous system disease and death. Although prions (prion proteins) are not a common impurity and the probability of occurrence may be only one in a few million, in view of its high micro-hazard, it is necessary to remove possible prions (prion proteins) when extracting heparin sodium from cattle and sheep, and minimize the possibility of infection with prions (prion proteins). However, prions (prion proteins) have a special structure and can resist traditional killing methods such as ultraviolet rays, high temperature, and radiation. Therefore, there is an urgent need for a method to effectively remove prions from heparin sodium from cattle and sheep. Summary of the invention

[0004] The present invention solves the defect that the prior art method for extracting bovine or sheep sodium heparin cannot remove prions, and thus provides a sodium heparin product and its extraction method and application. The extraction method of the present invention can effectively prevent prions from entering the sodium heparin product, and the sodium heparin product does not contain prions, and the content of other types of protein impurities is also very low.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for extracting fine heparin sodium, which comprises the following steps:

[0007] (1) Pretreatment: adjusting the pH value of the aqueous solution of crude heparin sodium to 7-9, then mixing it with the resin, filtering it after adsorption, and collecting the adsorbed resin; eluting the adsorbed resin with a sodium chloride aqueous solution, combining the eluates; precipitating the eluates to obtain precipitate A;

[0008] (2) Acid-base treatment: adjusting the pH value of the aqueous solution of precipitate A to 1.5-2.0, filtering to obtain an acidic filtrate; adjusting the pH value of the acidic filtrate to 11-12, stirring at 90-92° C. and filtering to obtain an alkaline filtrate; precipitating the alkaline filtrate to obtain precipitate B;

[0009] (3) Oxidation: The aqueous solution of precipitate B is oxidized 2 to 4 times. After the first oxidation, the oxidized solution is precipitated to obtain precipitate B1. The aqueous solution of precipitate B1 is oxidized for the second time, and so on. After the last oxidation, the oxidized solution is reduced to obtain a reduced solution. The pH value of the reduced solution is adjusted to 6 to 7, and the solution is precipitated to obtain precipitate C.

[0010] (4) Freeze-drying: The pH value of the aqueous solution of precipitate C is adjusted to 5.5-7, filtered, and the obtained filtrate is freeze-dried.

[0011] In step (1), the crude heparin sodium can be commercially available crude bovine or ovine heparin sodium. The potency of the crude heparin sodium is generally 20 to 60 IU / mg. Based on the potency of the crude heparin sodium, the concentration of the crude heparin sodium aqueous solution is preferably 10000 to 15000 IU / mL.

[0012] In step (1), before adjusting the pH value, preferably, sodium chloride is added to the crude sodium heparin aqueous solution. The amount of sodium chloride used is preferably 1.5% to 4%, and the percentage is the mass percentage of sodium chloride in the crude sodium heparin aqueous solution.

[0013] In step (1), the resin can be conventional in the art, preferably an anion exchange resin, more preferably a strong anion exchange resin, such as FPA98 anion exchange resin, WD-1701 anion exchange resin or D-254 anion exchange resin. The amount of the resin can be conventional in the art, preferably 20 to 50 kg of resin for 1 kg of crude heparin sodium.

[0014] In step (1), the adsorption temperature may be conventional in the art, preferably 55 to 65° C., such as 60° C. The adsorption time may be 3 to 6 hours.

[0015] In step (1), the filtration can be performed by conventional methods in the art, such as filtering with a 100-mesh nylon bag.

[0016] In step (1), the concentration of the sodium chloride aqueous solution is preferably 18-23%, where the percentage is the mass percentage of sodium chloride in the sodium chloride aqueous solution. The amount of the sodium chloride aqueous solution is preferably 1.2 L of sodium chloride aqueous solution per 1 kg of the adsorption resin.

[0017] In step (1), the elution temperature may be conventional in the art, preferably 55-65° C., for example 60° C. The elution time may be conventional in the art, preferably 3-6 hours.

[0018] Preferably, the elution is repeated at least twice.

[0019] Preferably, the adsorption resin is rinsed before elution. The preferred operation of the rinsing is: first rinse with tap water, and then rinse with a sodium chloride aqueous solution at room temperature that is twice the weight of the adsorption resin. The concentration of the sodium chloride aqueous solution is preferably 3% to 5%, and the percentage is the mass percentage of sodium chloride in the sodium chloride aqueous solution.

[0020] In step (2), based on the potency of crude heparin sodium, the concentration of the precipitate A aqueous solution is preferably 10000 to 15000 IU / mL.

[0021] In step (2), the filtration can be performed by conventional methods in the art, preferably diatomaceous earth filtration.

[0022] In step (2), the stirring time is preferably 2 to 4 hours.

[0023] In step (3), the concentration of the precipitate B aqueous solution and the precipitate B1 aqueous solution is preferably 10000-15000 IU / mL based on the titer of crude heparin sodium. Similarly, if precipitate B2 and precipitate B3 are present, the concentration of the precipitate B2 aqueous solution and the precipitate B3 aqueous solution is preferably 10000-15000 IU / mL based on the titer of crude heparin sodium. It should be understood that after the second oxidation is completed, the oxidation solution is precipitated to obtain precipitate B2; after the third oxidation is completed, the oxidation solution is precipitated to obtain precipitate B3.

[0024] In step (3), the oxidation operation can be carried out according to conventional methods in the art, generally using hydrogen peroxide and / or peracetic acid.

[0025] When hydrogen peroxide is used for oxidation, the oxidation operation may include: adjusting the pH value of the precipitate B aqueous solution to 10.5-11.5, adding hydrogen peroxide, stirring and filtering to obtain an oxidized solution. Wherein, the amount of hydrogen peroxide used is preferably 0.5%-1%, and the percentage is the mass volume ratio, that is, the mass of hydrogen peroxide used in 100mL solution. For example, the amount of hydrogen peroxide used is 0.5%, which means that the mass of hydrogen peroxide used in 100mL solution is 0.5g. The stirring temperature is preferably 50-60°C, and the stirring time is preferably 3-6 hours. The filtration is preferably performed using diatomaceous earth.

[0026] When peracetic acid is used for oxidation, the oxidation operation may include: adjusting the pH value of the precipitate B aqueous solution to 9-10, adding peracetic acid, stirring, and obtaining an oxidized solution. The amount of peracetic acid used is preferably 0.8% to 1%, and the percentage is a mass volume ratio, that is, the mass of peracetic acid used in 100 mL of solution. For example, the amount of peracetic acid used is 0.8%, which means that the mass of peracetic acid used in 100 mL of solution is 0.8g. The stirring temperature is preferably 20-30°C, and the stirring time is preferably 1-1.5 hours.

[0027] In step (3), preferably, the aqueous solution of precipitate B is oxidized twice, and first oxidized with hydrogen peroxide and then oxidized with peracetic acid.

[0028] In step (3), preferably, the aqueous solution of precipitate B is oxidized twice using hydrogen peroxide.

[0029] In step (3), the reduction may be carried out by using a conventional reducing agent in the art, and the reducing agent is preferably a sulfite and / or a pyrosulfite. The sulfite is preferably sodium sulfite. The pyrosulfite is preferably sodium pyrosulfite. The amount of the reducing agent is preferably 0.15% to 0.25%, and the percentage is a mass volume ratio, i.e., the mass of the reducing agent used for 100 mL of the oxidizing solution; for example, the amount of the reducing agent is 0.2%, which means that the mass of the reducing agent used for 100 mL of the oxidizing solution is 0.2 g.

[0030] In step (3), after adjusting the pH value of the reducing solution, preferably, sodium chloride is added to the reducing solution. The amount of the sodium chloride is preferably 2% to 4%, where the percentage is the mass percentage of the sodium chloride in the reducing solution.

[0031] In step (4), based on the potency of crude heparin sodium, the concentration of the precipitate C aqueous solution is preferably 10000 to 15000 IU / mL.

[0032] In step (4), the filtration can be performed using conventional methods in the art, preferably using a 0.2 μm filter.

[0033] In step (4), the freeze-drying can be carried out by conventional methods in the art, and the preferred operation includes: first freezing at -35 to -40°C, then sublimating at 3 to 5°C, and finally drying at 60 to 70°C.

[0034] In the present invention, the general operation of the wine precipitation includes: mixing the liquid to be precipitated with ethanol, and obtaining a precipitate after standing. The amount of the ethanol is preferably 40% to 60%, and the percentage is the volume percentage of the ethanol in the mixed liquid of the ethanol and the liquid to be precipitated. The liquid to be precipitated refers to the object for wine precipitation, for example, in step (1) it refers to the eluent, in step (2) it refers to the alkaline filtrate, and in step (3) it refers to the oxidizing solution or the reducing solution.

[0035] In the present invention, the method for adjusting the pH value may be conventional in the art, preferably using sodium hydroxide or hydrochloric acid. When sodium hydroxide is used to adjust the pH value, a sodium hydroxide aqueous solution is generally used, and the concentration of the sodium hydroxide aqueous solution is preferably 20% to 50%, and the percentage is the mass percentage of sodium hydroxide in the sodium hydroxide aqueous solution. When hydrochloric acid is used to adjust the pH value, the hydrochloric acid may be conventionally defined in the art, i.e., an HCl aqueous solution. The concentration of the hydrochloric acid may be conventional in the art, preferably 5% to 10%, and the percentage is a mass volume ratio. For example, a hydrochloric acid concentration of 5% means that 100 mL of hydrochloric acid contains 5 g of HCl.

[0036] In the present invention, the heparin sodium fine product extraction method cannot be carried out continuously, and the mother liquor cannot be recycled and reused. Between different batches of production, strict cleaning must be carried out, and the inner wall of the reaction container must be circulated and cleaned with a sodium hydroxide solution with a pH>11 and a temperature>90°C for more than 4 hours. Only after the inner wall is tested to have no protein residue, can the next batch of production be carried out.

[0037] The present invention also provides a heparin sodium fine product, which is prepared by the heparin sodium fine product extraction method.

[0038] The present invention also provides the application of the heparin sodium fine product in the preparation of anticoagulant blood products.

[0039] In the present invention, the anticoagulant product can be any product with anticoagulant function, such as anticoagulant drugs, cosmetics with anticoagulant function, anticoagulant coatings of medical devices, anticoagulant solutions in indwelling needles, or anticoagulant additives in blood products.

[0040] The present invention preferably provides the use of the heparin sodium fine product in the preparation of anticoagulant drugs.

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive and progressive effects of the present invention are:

[0044] Compared with the traditional process, the present invention can reduce the content of total protein, thereby greatly reducing the probability of prions causing disease, and can also reduce the probability of impurity proteins in heparin causing adverse reactions in the human body. The method for extracting heparin sodium fine product described in the present invention can be oxidatively inactivated in multiple steps, effectively preventing possible prions (prion proteins) from entering the heparin sodium fine product; the obtained bovine and sheep heparin sodium fine product does not contain prions, and the content of other types of protein impurities is also very low, which has great market application value. DETAILED DESCRIPTION

[0045] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0046] Example 1

[0047] A method for extracting fine heparin sodium comprises the following steps:

[0048] (1) Pretreatment: Take 5000MU (about 100kg, potency 50IU / mg) of crude bovine heparin sodium, dissolve it in 500L pure water at 10000IU / mL, add 10kg of sodium chloride, and adjust the pH to 8 with 5% hydrochloric acid or 20% sodium hydroxide aqueous solution; then add 2000kg of FPA98 anion exchange resin, adsorb at 60°C for 3 hours, filter and collect the adsorbed resin; first rinse with tap water, then rinse with 4% sodium chloride aqueous solution at room temperature twice the weight of the adsorbed resin, and then elute with 20% sodium chloride aqueous solution, elute twice, and combine the eluates; add ethanol to 50% for alcohol precipitation to obtain precipitate A;

[0049] (2) Acid-base treatment: Precipitate A was dissolved in 500 L of pure water, the pH was adjusted to 1.8 with 5% hydrochloric acid and maintained for 0.5 hours, and filtered through diatomaceous earth to obtain an acidic filtrate; the pH was adjusted to 11.5 with 20% sodium hydroxide aqueous solution, and stirred at 90°C for 3 hours, and filtered through diatomaceous earth to obtain an alkaline filtrate; ethanol was added to 50% for alcohol precipitation to obtain Precipitate B;

[0050] (3) Oxidation: Precipitate B was dissolved in 500 L of pure water, the pH was adjusted to 11 with 20% sodium hydroxide aqueous solution, hydrogen peroxide was added to 1%, stirred at 55°C for 5 hours, filtered with diatomaceous earth to obtain an oxidation solution, ethanol was added to 50% for alcohol precipitation, and Precipitate B1 was obtained;

[0051] The precipitate B1 was dissolved in 500L pure water, the pH was adjusted to 8.5 with 20% sodium hydroxide aqueous solution, peracetic acid was added to 1%, and stirred at 25°C for 1 hour, sodium bisulfite was added to 0.2%, and stirred for 1 hour to obtain a reduction solution, the pH was adjusted to 6.5 with 20% sodium hydroxide aqueous solution or 5% hydrochloric acid, and then 2% sodium chloride was added; ethanol was added to 40% for alcohol precipitation, and the alcohol precipitation operation was repeated 3 times to obtain precipitate C;

[0052] (4) Freeze drying: The precipitate C is dissolved in 100 L of pure water, and the pH is adjusted to 6 with 20% sodium hydroxide aqueous solution or 5% hydrochloric acid. After filtering with a 0.2 μm filter, the filtrate is first frozen at -40°C, then sublimated at 5°C, and finally dried at 70°C to obtain the heparin sodium concentrate. The obtained heparin sodium concentrate is stored in a double-layer PE bag or aluminum foil bag.

[0053] Effect Example 1

[0054] The Thermo Fisher qPCR equipment was used to measure the nucleic acid content in the bovine heparin sodium product prepared in Example 1 by real-time fluorescence quantitative PCR. Real-time fluorescence quantitative PCR (Quantitative Real-time PCR) is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. The animal DNA remaining in the heparin sodium is multiplied and then measured to obtain a quantitative result. The results show that the residual nucleic acid content of the bovine heparin sodium product obtained in Example 1 cannot be detected.

[0055] Effect Example 2

[0056] The protein content of the crude bovine heparin sodium obtained in Example 1 was less than 0.2% as measured in the United States Pharmacopoeia, while the protein content of the refined heparin sodium extracted by the traditional method was about 2%.

[0057] Effect Example 3

[0058] The activity of the bovine heparin sodium fine product prepared in Example 1 was measured according to the method for measuring the potency of heparin sodium recorded in the Chinese Pharmacopoeia (2015 edition), and the result was 120 IU / mg.

Claims

1. A method for extracting fine heparin sodium, comprising the following steps: (1) Pretreatment: adjusting the pH value of the aqueous solution of crude bovine and sheep heparin sodium to 7-9, then mixing it with the resin, filtering it after adsorption, and collecting the adsorbed resin; eluting the adsorbed resin with a sodium chloride aqueous solution, combining the eluates; precipitating the eluates to obtain precipitate A; (2) Acid-base treatment: adjusting the pH value of the aqueous solution of precipitate A to 1.5-2.0, filtering to obtain an acidic filtrate; adjusting the pH value of the acidic filtrate to 11-12, stirring at 90-92° C. and filtering to obtain an alkaline filtrate; precipitating the alkaline filtrate to obtain precipitate B; (3) Oxidation: The aqueous solution of precipitate B is oxidized 2 to 4 times. After the first oxidation, the oxidized solution is precipitated to obtain precipitate B1. The aqueous solution of precipitate B1 is oxidized for the second time, and so on. After the last oxidation, the oxidized solution is reduced to obtain a reduced solution. The pH value of the reduced solution is adjusted to 6 to 7, and the solution is precipitated to obtain precipitate C. (4) Freeze-drying: adjusting the pH value of the precipitate C solution to 5.5-7, filtering, and freeze-drying the obtained filtrate; In step (2), the filtration is performed using diatomaceous earth; In step (1), the potency of the crude bovine and ovine heparin sodium is 20 to 60 IU / mg; The concentration of the aqueous solution of crude bovine and ovine heparin sodium is 10000 to 15000 IU / mL based on crude heparin sodium; Before adjusting the pH value, sodium chloride is added to the crude bovine and sheep heparin sodium aqueous solution; the amount of sodium chloride used is 1.5% to 4%, and the percentage is the mass percentage of sodium chloride in the crude bovine and sheep heparin sodium aqueous solution; The resin is FPA98 anion exchange resin; the amount of the resin used is 20-50 kg of resin for 1 kg of crude heparin sodium; The adsorption temperature is 55 to 65°C; the adsorption time is 3 to 6 hours; The filtration is carried out using a 100-mesh nylon bag; In step (2), the concentration of the precipitate A aqueous solution is 10000-15000 IU / mL based on the crude bovine and sheep heparin sodium; The stirring time is 2 to 4 hours; In step (3), the reducing agent is sulfite and / or pyrosulfite; the sulfite is sodium sulfite; the pyrosulfite is sodium pyrosulfite; the amount of the reducing agent is 0.15% to 0.25%, where the percentage is the mass volume ratio, i.e., the mass of the reducing agent used for 100 mL of the oxidizing solution; After adjusting the pH value of the reducing solution, sodium chloride is added to the reducing solution; the amount of the sodium chloride is 2% to 4%, where the percentage is the mass percentage of the sodium chloride in the reducing solution.

2. The method for extracting heparin sodium according to claim 1, characterized in that: In step (1), the concentration of the sodium chloride aqueous solution is 18-23%, where the percentage is the mass percentage of sodium chloride in the sodium chloride aqueous solution; the amount of the sodium chloride aqueous solution used is 1.2 L of sodium chloride aqueous solution for 1 kg of the adsorption resin; The elution is repeated at least twice; And / or, the adsorption resin is rinsed before elution; the preferred operation of the rinsing is: first rinse with tap water, and then rinse with a sodium chloride aqueous solution at room temperature that is twice the weight of the adsorption resin, wherein the concentration of the sodium chloride aqueous solution is 3% to 5%, and the percentage is the mass percentage of sodium chloride in the sodium chloride aqueous solution.

3. The method for extracting heparin sodium according to claim 1, characterized in that: In step (3), the concentration of the precipitate B aqueous solution and the precipitate B1 aqueous solution is 10000-15000 IU / mL based on the potency of crude heparin sodium; And / or, the oxidation is carried out using hydrogen peroxide and / or peracetic acid; When hydrogen peroxide is used for oxidation, the oxidation operation includes: adjusting the pH value of the precipitate B aqueous solution to 10.5-11.5, adding hydrogen peroxide, stirring and filtering to obtain an oxidized solution; wherein the amount of hydrogen peroxide used is 0.5%-1%, and the percentage is a mass volume ratio, that is, the mass of hydrogen peroxide used in 100 mL of the solution; the stirring temperature is 50-60° C.; the stirring time is 3-6 hours; and the filtration is performed using diatomaceous earth; When peracetic acid is used for oxidation, the oxidation operation includes: adjusting the pH value of the precipitate B aqueous solution to 9-10, adding peracetic acid, stirring, and obtaining an oxidation solution; wherein the amount of the peracetic acid used is 0.8%-1%, and the percentage is a mass volume ratio, that is, the mass of the peracetic acid used in 100 mL of the solution; the stirring temperature is 20-30° C.; and the stirring time is 1-1.5 hours; The aqueous solution of Precipitate B is oxidized twice, and firstly oxidized with hydrogen peroxide and then oxidized with peracetic acid; the aqueous solution of Precipitate B is oxidized twice with hydrogen peroxide.

4. The method for extracting heparin sodium according to claim 1, characterized in that: In step (4), the concentration of the precipitate C aqueous solution is 10000 to 15000 IU / mL based on the potency of crude heparin sodium; And / or, the filtering uses a 0.2 μm filter; And / or, the freeze-drying comprises: first freezing at -35 to -40°C, then sublimating at 3 to 5°C, and finally drying at 60 to 70°C.

5. The method for extracting heparin sodium concentrate according to any one of claims 1 to 4, characterized in that: The wine precipitation operation includes: mixing the liquid to be precipitated with ethanol, and letting it stand to obtain precipitation; wherein the amount of the ethanol used is 40% to 60%, and the percentage is the volume percentage of the ethanol in the mixed liquid of the ethanol and the liquid to be precipitated; And / or, the pH value is adjusted using sodium hydroxide or hydrochloric acid; when sodium hydroxide is used to adjust the pH value, a sodium hydroxide aqueous solution is used, and the concentration of the sodium hydroxide aqueous solution is 20% to 50%, and the percentage is the mass percentage of sodium hydroxide in the sodium hydroxide aqueous solution; when hydrochloric acid is used to adjust the pH value, the concentration of the hydrochloric acid is 5% to 10%, and the percentage is the mass volume ratio.