Heparin sodium efficient extraction process based on enzymolysis method

Abstract

The invention relates to the technical field of heparin sodium, in particular to a heparin sodium efficient extraction process based on an enzymolysis method. The method comprises the steps of raw material treatment, heating enzymolysis, cooling adsorption, elution, ethanol precipitation, dehydration drying and index determination. In the high-efficiency extraction process of the heparin sodium based on the enzymolysis method, the small intestine mucous membrane is crushed into a minced state to increase the enzyme contact area, and a low-temperature graded heating enzymolysis process is combined, so that the high-efficiency decomposition of a heparin-protein compound is realized, and the problems that the enzymolysis is not thorough and the temperature needs to be increased for multiple times in the traditional enzymolysis method are effectively solved; meanwhile, insoluble impurities are removed through filtration while the solution is hot, protein residues and impurity interference are reduced in cooperation with cooled resin adsorption and gradient elution, the yield of the heparin sodium is increased, production energy consumption and purification difficulty are reduced, and therefore the overall efficiency and economical efficiency of extraction of the heparin sodium are improved.

Description

Technical Field

[0001] This invention relates to the field of heparin sodium technology, and more specifically, to a high-efficiency extraction process for heparin sodium based on enzymatic hydrolysis. Background Technology

[0002] Heparin sodium, as an important anticoagulant, is widely used in the prevention and treatment of cardiovascular diseases and as an adjuvant therapy for tumors. Currently, heparin sodium is mainly derived from animal tissues such as porcine small intestinal mucosa, and its traditional extraction methods include salting out and enzymatic hydrolysis. Salting out is simple to operate and has low cost, but it suffers from incomplete protein removal and insufficient product purity. While enzymatic hydrolysis can improve protein removal, the conventional process suffers from unstable enzymatic hydrolysis conditions, easily leading to incomplete reactions and requiring multiple heating processes, resulting in high energy consumption and affecting the final yield. Furthermore, existing processes often use large amounts of hydrogen peroxide in the decolorization and purification stages, which may damage the bioactivity of heparin sodium, hindering product quality improvement and industry cost control. To overcome the aforementioned limitations, existing technologies improve reaction efficiency by optimizing enzymatic hydrolysis temperature and time parameters. However, they still fall short in achieving sufficient separation of heparin-protein complexes and removing impurities. For example, uneven stirring during traditional enzymatic hydrolysis can lead to insufficient contact between the enzyme and substrate, reducing the release efficiency of heparin sodium and wasting raw materials. Furthermore, residual proteins and enzyme proteins increase the difficulty of subsequent purification. In addition, unstable adsorption and elution steps may damage the resin structure, affecting process stability and reducing the recovery rate and potency of heparin sodium, resulting in product quality fluctuations. Consequently, the overall efficiency and economy of heparin sodium extraction are reduced.

[0003] Therefore, there is an urgent need for a high-efficiency extraction process for heparin sodium based on enzymatic hydrolysis. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient extraction process for heparin sodium based on enzymatic hydrolysis, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, according to Figure 1 As shown, this invention provides a highly efficient extraction process for heparin sodium based on enzymatic hydrolysis, comprising the following extraction steps: S1. Raw material processing: Fresh pig small intestine mucosa is put into a mechanical stirring device and crushed at a speed of 800-1200 r / min for 20-30 min until a uniform porridge is formed. This can effectively destroy cell structure and significantly increase the contact area between enzyme and substrate. S2. Heating and enzymatic hydrolysis: Add 7-9% trypsin by mass to the chyme, adjust the pH of the system to 8-10 using sodium hydroxide solution, and carry out the enzymatic hydrolysis reaction using a low-temperature staged heating method to obtain the enzymatic hydrolysate. S3. Cooling Adsorption: Filter the enzymatic hydrolysate while it is hot to remove insoluble impurities, collect the filtrate, and cool it to 30-40℃. Remove the floating oil on the top layer, add the pretreated D254 resin, and add the resin at 8-10% of the filtrate volume. Use a low-speed stable stirring method to carry out adsorption for 6-7 hours under the conditions of constant temperature 30-40℃ and low-speed stirring speed 60-80rpm. S4. Elution: Transfer the adsorbed resin to an eluent, add 8-12% sodium chloride solution, and elute in steps at 50-55℃ for 3-4 hours each time. Repeat the elution twice, then combine and collect the eluents to form a gradient elution mode. By controlling the ionic strength and temperature of the eluent, the efficient desorption of heparin sodium is increased, while protein impurities weakly bound to the resin and residual enzyme proteins are effectively removed, thus improving the purity and recovery rate of the target product. S5. Ethanol precipitation: After filtering the eluent, transfer it to a precipitation tank, add 80-85% ethanol by mass, stir with a stirrer at a speed of 30-50 r / min, and simultaneously add dilute hydrochloric acid or sodium hydroxide solution dropwise to adjust the pH value to 7-8. Then stop stirring and let it stand in a sealed environment for 10-12 hours to allow heparin sodium to fully coagulate and precipitate from the solution, forming flocculent or granular precipitates that are easy to separate. S6. Dehydration and drying: The precipitate is collected by vacuum filtration or centrifugation, and then transferred to a drying oven for dehydration and drying at 50-60℃ until the moisture content is less than 5%, to obtain white or off-white crude heparin sodium. S7. Index Determination: Multiple quality indexes were determined on the dried crude heparin sodium to obtain the refined heparin sodium product that meets the requirements.

[0006] Furthermore, in step S2, the enzymatic hydrolysis reaction includes the following steps: First, the reaction is carried out at a constant temperature of 30-40℃ for 2-3 hours to allow trypsin to fully act on the heparin-protein complex. Then, the temperature is raised to 50-60℃ and maintained for 10-20 minutes. Through the step-temperature control mode, the enzymatic hydrolysis efficiency is improved, and the subsequent temperature increase promotes protein denaturation, thereby achieving the complete decomposition of the heparin-protein complex.

[0007] Furthermore, in step S3, the pretreatment of D254 resin includes the following steps: The D254 resin was soaked in a sodium hydroxide solution with a concentration of 0.5-1.0 mol / L at a liquid-to-solid ratio of 3:1-5:1 for 2-4 hours. After soaking, the resin was washed with purified water until the effluent was neutral. Then, it was equilibrated with a sodium chloride solution with a concentration of 0.1-0.3 mol / L for 1-2 hours to obtain an alkaline resin system with stable adsorption properties.

[0008] Furthermore, in S7, the determination of multiple quality indicators includes at least the following: appearance, specific rotation, pH, nucleic acid, total ammonia content, protein content, molecular weight distribution, potency, clarity and color of the solution. Among these, the specific rotation is not less than +50°; the pH value is 5.0-8.0; the absorbance of nucleic acid at a wavelength of 260 nm does not exceed 0.10; the total nitrogen content, calculated on a dried basis, is 1.3%-2.5%; the molecular weight distribution requires a weight-average molecular weight of 15,000-19,000, with the fraction with a molecular weight greater than 24,000 not exceeding 20%, and the ratio of the fraction with a molecular weight of 8,000-16,000 to that of 16,000-24,000 should not be less than 1.0.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In this high-efficiency heparin sodium extraction process based on enzymatic hydrolysis, the small intestinal mucosa is broken into a porridge-like state to increase the enzyme contact area. Combined with a low-temperature fractional heating enzymatic hydrolysis process, the efficient decomposition of the heparin-protein complex is achieved, effectively overcoming the problems of incomplete enzymatic hydrolysis and the need for multiple heating cycles in traditional enzymatic hydrolysis methods. At the same time, hot filtration removes insoluble impurities, and combined with resin adsorption and gradient elution after cooling, protein residue and impurity interference are reduced. While improving the yield of heparin sodium, the production energy consumption and purification difficulty are reduced, thereby improving the overall efficiency and economy of heparin sodium extraction.

[0010] 2. In this high-efficiency extraction process of heparin sodium based on enzymatic hydrolysis, pretreated D254 resin is used, combined with low-speed stable stirring adsorption and stepwise gradient elution technology. By controlling the ionic strength and temperature of the eluent, the efficient desorption of heparin sodium is promoted, and weakly bound protein impurities and residual enzyme proteins are removed. This enhances adsorption selectivity and elution efficiency, reduces resin damage and loss of target product, thereby improving the recovery rate and purity of heparin sodium. Attached Figure Description

[0011] Figure 1 This is a flowchart of the efficient extraction process of heparin sodium according to the present invention. Detailed Implementation

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0013] Take 100 kg of fresh pig small intestine mucosa and put it into a mechanical stirring device. Grind it at 1000 r / min for 25 min to form a uniform porridge. Add 8% (w / w) trypsin to the porridge, adjust the pH to 9.0 using sodium hydroxide solution, and react at a constant temperature of 35℃ for 2.5 h. Then raise the temperature to 55℃ and maintain it for 15 min to complete the enzymatic hydrolysis reaction. The enzymatic hydrolysate was filtered while hot to remove insoluble impurities. The filtrate was collected, cooled to 35°C, and the supernatant oil was removed. 8 L of pretreated D254 resin was added (pretreatment method: soaking in 0.8 mol / L sodium hydroxide solution at a liquid-to-solid ratio of 4:1 for 3 h, washing with purified water until neutral, and equilibrating in 0.2 mol / L sodium chloride solution for 1.5 h), and adsorption was carried out at 35°C and 70 rpm under low-speed stirring for 6.5 h. The resin after adsorption was completed was transferred to an eluent, and a 10% sodium chloride solution was added. Elution was carried out stepwise at 52°C, with stirring for 3.5 hours each time. The elution was repeated twice, and the eluents were combined and collected. After filtration, the eluent was transferred to a sedimentation tank, and 82% ethanol (by mass) was added. The mixture was stirred at 40 rpm while adjusting the pH to 7.5. After stirring was stopped, the mixture was sealed and allowed to stand for 11 hours. The precipitate was collected and dehydrated at 55°C until the moisture content was 4.5%, yielding crude heparin sodium.

[0014] Quality indicators showed the following: specific rotation +52°, pH 6.8, nucleic acid absorbance 0.08, total nitrogen 1.8%, protein content met the requirements, weight-average molecular weight 17,000, molecular weight greater than 24,000 accounted for 18%, molecular weight 8,000-16,000 to 16,000-24,000 molecular weight fraction ratio was 1.2, and potency was 8.5 billion units / mg. Example 2

[0015] 150 kg of fresh pig small intestine mucosa was taken and put into a mechanical stirring device for high-speed crushing at 900 r / min for 28 min to form a uniform porridge. Trypsin at 8.5% by mass was added to the porridge, and the pH was adjusted to 8.5 with sodium hydroxide solution. The reaction was first carried out at a constant temperature of 32℃ for 3 h, and then the temperature was raised to 58℃ and maintained for 12 min to complete the enzymatic hydrolysis reaction. The enzymatic hydrolysate was filtered while hot to remove insoluble impurities. The filtrate was collected, cooled to 38°C, and the supernatant oil was removed. 14 L of pretreated D254 resin was added (pretreatment method: soaking in 0.6 mol / L sodium hydroxide solution at a liquid-to-solid ratio of 4.5:1 for 2.5 h, washing with purified water until neutral, and equilibrating in 0.15 mol / L sodium chloride solution for 2 h), and adsorption was carried out at 38°C and 65 rpm for 7 h under low-speed stirring. The resin after adsorption was completed was transferred to an eluent, and a 9% sodium chloride solution was added. Elution was carried out stepwise at 54°C, with stirring for 4 hours each time. The elution was repeated twice, and the eluents were combined and collected. After filtration, the eluent was transferred to a sedimentation tank, and 83% ethanol (by mass) was added. The mixture was stirred at 35 rpm while adjusting the pH to 7.3. After stirring was stopped, the mixture was sealed and allowed to stand for 12 hours. The precipitate was collected and dehydrated at 58°C until the moisture content was 4.2%, yielding crude heparin sodium.

[0016] Quality indicators showed the following: specific rotation +55°, pH 6.5, nucleic acid absorbance 0.07, total nitrogen 1.6%, protein content met the requirements, weight-average molecular weight 16,500, molecular weight greater than 24,000 accounted for 16%, molecular weight 8,000-16,000 to 16,000-24,000 ratio was 1.4, and potency was 8.8 billion units / mg. Example 3

[0017] 120 kg of fresh pig small intestine mucosa was taken and put into a mechanical stirring device for high-speed crushing at 1100 r / min for 22 min to form a uniform porridge. 7.5% trypsin by mass was added to the porridge, and the pH was adjusted to 9.5 with sodium hydroxide solution. The reaction was first carried out at a constant temperature of 38℃ for 2 h, and then the temperature was raised to 52℃ and maintained for 18 min to complete the enzymatic hydrolysis reaction. The enzymatic hydrolysate was filtered while hot to remove insoluble impurities. The filtrate was collected, cooled to 32°C, and the supernatant oil was removed. 11 L of pretreated D254 resin was added (pretreatment method: soaking in 0.9 mol / L sodium hydroxide solution at a liquid-to-solid ratio of 3.5:1 for 3.5 h, washing with purified water until neutral, and equilibrating in 0.25 mol / L sodium chloride solution for 1 h), and adsorption was carried out at 32°C and 75 rpm for 6 h under low-speed stirring. The resin that has completed adsorption is transferred to an eluent, and an 11% sodium chloride solution is added. The eluent is eluted stepwise at 51°C, with stirring for 3 hours each time. The elution is repeated twice, and the eluents are combined and collected. After filtration, the eluent was transferred to a sedimentation tank, and 81% ethanol (by mass) was added. The mixture was stirred at 45 rpm while adjusting the pH to 7.8. After stirring was stopped, the tank was sealed and allowed to stand for 10 hours. The precipitate was collected and dehydrated at 52°C until the moisture content was 4.8%, yielding crude heparin sodium.

[0018] Quality indicators showed the following: specific rotation +58°, pH 7.2, nucleic acid absorbance 0.06, total nitrogen 1.9%, protein content met the requirements, weight-average molecular weight 17,500, molecular weight greater than 24,000 fractions accounted for 15%, molecular weight 8,000-16,000 to 16,000-24,000 fractions ratio 1.3, and potency 9 billion units / mg.

[0019] To verify that the efficient extraction of heparin sodium in the embodiments of the present invention has good extraction efficiency and product quality stability, the following experimental examples are used to illustrate the efficient extraction of heparin sodium provided in the embodiments of the present invention.

[0020] Test case The purpose of this experimental group is to investigate the effect of different component ratios on the efficient extraction of heparin sodium, and to detect the heparin sodium yield, potency, protein content, nucleic acid absorbance and molecular weight distribution of the heparin sodium of this invention.

[0021] Experimental Objective: Experimental groups A, B, and C used the proportions of components obtained from the highly efficient heparin sodium extraction provided in Examples 1-3, respectively; the control group consisted of control groups A, B, C, and D, wherein: Control group A 100 kg of fresh pig small intestine mucosa was taken, cut by ordinary mechanical means (not high-speed crushing), and 8% trypsin was added. The pH was adjusted to 9.0, and the mixture was enzymatically hydrolyzed at 40℃ for 3 h, omitting the step of staged heating. After filtration of the enzymatic hydrolysate, it was stirred at 150 rpm at room temperature for 6 h (using untreated D254 resin). The mixture was eluted once with a single concentration (10%) sodium chloride solution at 50℃ and stirred for 2 h. The eluent was precipitated with 82% ethanol, the pH was adjusted to 7.5, and the precipitate was collected after standing for 8 h and dried at 55℃ to obtain the crude product.

[0022] Control group B Take 100 kg of fresh pig small intestine mucosa, cut it, add 5% sodium chloride solution to extract for 2 h, then add 5% trypsin by mass and enzymatically hydrolyze at 45℃ for 2 h; after filtering the extract, purify it by a combination of salting out (adding 25% sodium chloride) and ethanol precipitation (80% ethanol), let it stand for 10 h, collect the precipitate and dry it.

[0023] Control group C Take 100 kg of fresh pig small intestinal mucosa, crush it normally, add 9% trypsin by mass, adjust the pH to 8.5, and directly enzymatically hydrolyze it at 55℃ for 2.5 h (without a low temperature stage); the subsequent adsorption, elution and precipitation steps are the same as control group A.

[0024] Control group D The raw material processing and enzymatic hydrolysis steps were the same as those in experimental group A. After filtration, the enzymatic hydrolysate was not adsorbed by resin, but directly precipitated with 82% ethanol. The pH was adjusted to 7.5, and after standing for 12 hours, the precipitate was collected and dried at 55°C to obtain crude heparin sodium.

[0025] Experimental methods: The yield, potency, protein content, nucleic acid absorbance, and molecular weight distribution of heparin sodium according to the present invention were tested separately, and the specific experimental methods are as follows: Heparin sodium yield: Weigh the dried crude heparin sodium and record its mass. Calculate the percentage yield of heparin sodium based on the initial mass of the raw material, porcine small intestinal mucosa. The specific operation is as follows: accurately weigh the mass of the dried crude product, divide it by the total mass of the fresh porcine small intestinal mucosa used, and then multiply by 100% to obtain the yield result. This process is carried out at room temperature to ensure that the data accurately reflects the extraction efficiency. Potency: The anti-factor IIa assay was used, with heparin standard as a reference. The biopotency of the sample was calculated using the principle of parallel linear reaction. The specific steps included: diluting the heparin sodium sample and the standard to a series of concentrations, adding antithrombin solution and thrombin solution, reacting at 37°C, adding a chromogenic substrate and measuring the absorbance at 405 nm wavelength. The biopotency was obtained through linear regression and statistical processing, and the unit was expressed in international units per milligram. Protein content: The protein content is determined by using appropriate analytical methods. Specifically, after dissolving the heparin sodium sample, the residual amount of protein in the sample is determined using specific reagents or instrument conditions to ensure that it meets the prescribed limits. During the operation, pH and temperature conditions are strictly controlled to ensure the repeatability and accuracy of the results. Nucleic acid absorbance: Take the heparin sodium sample, dissolve it in water and dilute it to the specified concentration, and use a UV-Vis spectrophotometer to measure its absorbance at a wavelength of 260 nm; the specific steps are as follows: place the sample solution in a quartz cuvette, use water as a blank control, and read the absorbance value. This value does not exceed the limit specified in the standard to assess the residual status of nucleic acid impurities; Molecular weight distribution: High-performance gel permeation chromatography (HPLC) was used with hydrophilic modified bonded silica gel as the packing material (TSK pre-column connected in series with TSK gel G4000SWXL and TSK gel G3000SWXL columns). The mobile phase was 0.1 mol / L ammonium acetate solution, the flow rate was 0.6 mL / min, the column temperature was 30℃, and detection was performed using a differential refractive index detector. The injection volume was 25 μL. First, a heparin molecular weight system suitability reference solution was injected to verify system suitability, ensuring complete separation of the main peak and solvent peak, and that the weight-average molecular weight was within ±500 of the labeled value. Within the specified range, a cubic equation calibration curve (correlation coefficient not less than 0.990) is established using heparin molecular weight reference standard with retention time as the x-axis and the logarithm of molecular weight as the y-axis. Finally, the test solution is injected, and the weight-average molecular weight is calculated according to the formula Mw=∑(RIiMi) / ∑RI based on the chromatogram. The proportion of each fraction is analyzed, requiring a weight-average molecular weight of 15,000-19,000, with the fraction with a molecular weight greater than 24,000 not exceeding 20%, and the ratio of the molecular weight of 8,000-16,000 to the fraction with a molecular weight of 16,000-24,000 not less than 1.0.

[0026] Specific testing indicators are shown in Table 1.

[0027]

[0028] Based on the comparative data shown in Table 1, the technical solution of the present invention demonstrates a comprehensive and significant improvement in the overall performance of heparin sodium extraction compared to traditional processes and incomplete improvement schemes: In terms of extraction efficiency, the heparin sodium yield of the process of this invention (experimental groups A, B, and C) remained stable at a relatively high level of 2.78% to 2.92%, which was better than all control groups. This indicates that the present invention achieves a more complete release of heparin by increasing the enzyme contact area and optimizing the enzymatic hydrolysis kinetics. In particular, compared with the process that only uses a single enzymatic hydrolysis temperature (control group C) or omits the resin adsorption purification step (control group D), the yield advantage of the present invention is more prominent, and it can release heparin more thoroughly, thereby greatly improving the utilization rate of raw materials. Regarding the core quality indicators of the product, this invention has significant effects: First, the potency of the obtained heparin sodium is stably maintained at 8.5 to 9 billion units / mg, which is not only high in absolute value but also has small batch-to-batch differences, meeting the stringent requirements for bioactivity of high-quality heparin sodium. In contrast, the potency of each control group product decreased to varying degrees, reflecting that the mild and efficient purification process of this invention can better protect the natural structure and activity of heparin molecules. Second, in terms of purity, the nucleic acid absorbance value (0.06-0.08) of the product of this invention is much lower than that of the control group, and the protein content meets the requirements, indicating that the adsorption and gradient elution process can effectively remove nucleic acid and protein impurities. Furthermore, this invention achieves precise control over the molecular weight distribution of heparin sodium, with its weight-average molecular weight concentrated in the ideal range (16,500-17,500), a lower proportion of high molecular weight fractions (>24,000), and a more reasonable proportion of low molecular weight fractions (fraction ratio ≥1.2). This molecular weight characteristic is closely related to the high potency and good uniformity of the product.

[0029] In summary, this invention solves the problems of low enzymatic hydrolysis efficiency, difficulty in removing impurities, and easy damage to product activity in traditional methods by using the processes of "high-speed crushing of raw materials", "stepwise temperature-controlled enzymatic hydrolysis", "resin stable adsorption", and "gradient elution", thus achieving high yield, high quality, and high efficiency extraction of heparin sodium.

[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high-efficiency extraction process of heparin sodium based on enzymatic hydrolysis, characterized in that, The extraction process comprises the following steps: S1, put fresh pig small intestine mucosa into a mechanical stirring device for crushing treatment until a uniform paste is formed; S2, add trypsin to the paste, adjust the pH value of the system to 8-10 using sodium hydroxide solution, and perform enzymatic reaction by low-temperature staged heating to obtain an enzymatic hydrolysate; S3, filter the enzymatic hydrolysate while hot, collect the filtrate, cool it to 30-40℃, remove the upper floating oil, add pretreated D254 resin, and adsorb by low-speed stable stirring; S4, transfer the adsorbed resin to an elution device, add sodium chloride solution, elute by stepwise elution, and then collect the eluate; S5, filter the eluate and move it into a precipitation tank, stir it using a stirrer, drop in dilute hydrochloric acid or sodium hydroxide solution, adjust the pH value to 7-8, and then stand still in a sealed environment for 10-12 hours to form a precipitate; S6, separate and collect the precipitate, and then transfer it to a drying oven to obtain crude heparin sodium; S7, measure multiple quality indicators of the dried crude heparin sodium to obtain refined heparin sodium product.

2. The high efficiency extraction process of heparin sodium based on enzymatic method according to claim 1, characterized in that, In S1, the mechanical stirring device is crushed at a high speed of 800-1200 r / min for 20-30 min.

3. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S2, the trypsin is added at 7-9% of the mass of the paste. In S3, the D254 resin is added at 8-10% of the volume of the filtrate. In S4, the sodium chloride solution is used at a mass fraction of 8-12%. In S5, the ethanol is used at a mass fraction of 80-85%.

4. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S2, the enzymatic reaction comprises the following steps: First, react at a constant temperature of 30-40℃ for 2-3 hours to allow the trypsin to fully act on the heparin-protein complex; then, raise the temperature to 50-60℃ and maintain it for 10-20 min.

5. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S3, the pretreatment of D254 resin comprises the following steps: Soak the D254 resin in a 0.5-1.0 mol / L sodium hydroxide solution with a liquid-solid ratio of 3:1-5:1 for 2-4 hours, then wash it with purified water until the effluent is neutral, and then balance it with a 0.1-0.3 mol / L sodium chloride solution for 1-2 hours to obtain an alkaline resin system with stable adsorption performance.

6. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S3, the low-speed stable stirring is performed at a constant temperature of 30-40℃ and a low-speed stirring speed of 60-80 rpm for 6-7 hours.

7. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S4, the stepwise elution is repeated twice, each time for 3-4 hours.

8. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S6, the drying oven is used for dehydration drying at 50-60℃ until the water content is less than 5%.

9. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 1, characterized in that, In S7, the multiple quality indicator measurements include at least the following: appearance, specific rotation, pH, nucleic acid, total ammonia content, protein content, molecular weight distribution, potency, solution clarity, and color.

10. The process for high efficient extraction of heparin sodium based on enzymatic hydrolysis according to claim 9, characterized in that, The specific rotation is not less than +50°; the pH value of the acid-base degree is 5.0-8.0; the absorbance of the nucleic acid at 260 nm wavelength is not more than 0.10; the total nitrogen content calculated by dry product is 1.3%-2.5%; the molecular weight distribution requires that the weight average molecular weight is 15000-19000, the fraction with molecular weight greater than 24000 is not more than 20%, and the fraction ratio of 8000-16000 to 16000-24000 should be not less than 1.0.

Images