1. A method for separating and purifying heparin by using expanded bed countercurrent chromatography is characterized in that anion exchange resin is used as a solid adsorption medium and is added into an expanded bed countercurrent chromatography separation column, and a heparin solution is loaded to the expanded bed countercurrent chromatography for adsorption, elution, alcohol precipitation, centrifugation and washing to obtain purified heparin.

2. The method for separating and purifying heparin by expanded bed countercurrent chromatography according to claim 1, wherein the anion exchange resin is one of D204, D208, D254 and D301.

3. The method for separating and purifying heparin by expanded bed countercurrent chromatography according to claim 1, wherein the flow rate of loading and elution are both from top to bottom during the process of separating and purifying heparin by expanded bed countercurrent chromatography.

4. The method for separating and purifying heparin by expanded bed countercurrent chromatography as claimed in claim 3, wherein the rotation speed is 200-500 rpm.

5. The method for separating and purifying heparin by expanded bed countercurrent chromatography according to claim 1, wherein the ratio of the addition amount of the anion exchange resin to the effective column volume of the expanded bed countercurrent chromatography separation column is (2.0-5.0) g: 30mL, the concentration of the heparin solution is 50mg/mL, the sample loading flow rate is 0.5-10mL/min, and the ratio of the sample loading amount to the effective column volume of the expanded bed countercurrent chromatography separation column is (50-200) mg: 30 mL.

6. The method for separating and purifying heparin by expanded bed countercurrent chromatography according to claim 1, wherein the mobile phase in the adsorption process is Tris-HCl buffer solution with pH value of 8.5;

the elution specifically comprises: 2-4mol/L NaCl is used as eluent, and the elution flow rate is 1-5 mL/min.

7. The method for separation and purification of heparin by expanded bed countercurrent chromatography according to claim 1, wherein the loading is cyclic loading.

8. The method for separating and purifying heparin by expanded bed countercurrent chromatography according to claim 7, wherein the number of times of sample loading is 4 times.

9. Heparin obtained by the expanded bed countercurrent chromatography method for heparin separation and purification according to any one of claims 1 to 8.

Background

Heparin is a glycosaminoglycan drug with a complex structure, is formed by alternately connecting alpha-D-glucosamine (N-sulfation, O-sulfation or N-acetylation) and O-sulfated uronic acid (alpha-L-iduronic acid or beta-D glucuronic acid) to form a polymer, and has the effect of prolonging the blood coagulation time. The composition is mainly used for resisting blood coagulation and preventing and treating thromboembolic diseases clinically, and is a first choice medicament for preventing and treating deep vein thrombosis, pulmonary thrombosis, disseminated intravascular coagulation and certain thromboembolic complications.

The intestinal mucosa is the waste for producing the sausage casing, the content of heparin is high, and the extraction of heparin from the intestinal mucosa is the main source of heparin in the market at present. The heparin extraction method tends to be mature, and the mainstream extraction method of crude heparin at the present stage is mainly divided into two main methods, namely a salt decomposition-resin adsorption method and an enzymolysis resin adsorption method: (1) salt decomposition-ion exchange process, operating route: fresh pig small intestine mucous membrane → alkaline salt hydrolysis → filtration → resin adsorption filtrate → elution → eluent alcohol precipitation → drying → crude heparin; (2) the enzymolysis-ion exchange method comprises the following operation routes: fresh pig small intestine mucous membrane → enzymolysis → filtration → resin adsorption filtrate → elution → eluent alcohol precipitation → drying → crude heparin; however, both the two methods are general processes for small-scale production, are simple to operate, have low requirements on equipment and industry, and have the defects of low extraction rate and resource waste. Meanwhile, due to the process, the titer of the heparin product extracted from the small intestine of the pig is low, and the original value of the heparin product is greatly reduced. Therefore, the improvement of the extraction process of heparin has important significance for the expanded production of heparin and the improvement of economic benefit.

Disclosure of Invention

The invention aims to provide a method for separating and purifying heparin by using an expanded bed countercurrent chromatography, which improves the adsorption rate of resin to heparin, reduces the separation time and obtains a heparin product with high product titer.

The technical scheme provided by the invention is as follows:

a method for separating and purifying heparin by using expanded bed countercurrent chromatography comprises the steps of taking anion exchange resin as a solid adsorption medium, adding the solid adsorption medium into an expanded bed countercurrent chromatography separation column, loading a heparin solution to the expanded bed countercurrent chromatography for adsorption, elution, alcohol precipitation, centrifugation and washing to obtain purified heparin.

Further, the anion exchange resin is one of D204, D208, D254 and D301.

Furthermore, in the process of separating and purifying heparin by the expanded bed countercurrent chromatography, the sampling and elution flow velocity directions are from top to bottom, and the rotating speed direction is clockwise.

Further, the rotation speed is 200-500 rpm.

Further, the ratio of the amount of the anion exchange resin added to the effective column volume of the expanded bed countercurrent chromatography separation column is (2.0-5.0) g: 30mL, the concentration of the heparin solution is 50mg/mL, the sample loading flow rate is 0.5-10mL/min, and the ratio of the sample loading amount to the effective column volume of the expanded bed countercurrent chromatography separation column is (50-200) mg: 30 mL.

Further, the mobile phase in the adsorption process is Tris-HCl buffer solution with the pH value of 8.5;

the elution specifically comprises: 2-4mol/L NaCl is used as eluent, and the elution flow rate is 1-5 mL/min.

Further, the loading is cyclic loading.

Further, the number of times of sample loading was 4 cycles.

The invention also provides heparin obtained by the method for separating and purifying heparin by the countercurrent chromatography of the expanded bed.

Compared with the prior art, the invention has the beneficial effects that:

the invention adds the solid separation medium commonly used in the fixed bed column chromatography into the separation column of the countercurrent chromatography, and forms the state of the expansion and dispersion of the adsorption medium in the separation column based on the countercurrent chromatography fluid dynamics balance principle to construct the expanded bed countercurrent chromatography, which can be used as a novel chromatographic separation method for separating the polar polysaccharide heparin drugs. The method has the advantages of high sample processing amount, remarkably improved utilization rate of the adsorption medium, and good application prospect in separation and purification of polar polysaccharide bioactive macromolecules.

The method is based on comprehensive evaluation of a spiral force field, a gravity field and a centrifugal force field which are applied to liquid in a separation column in the process of countercurrent chromatography operation, and combines the retention state of solid resin in the separation column to determine that the flow velocity directions of sample injection and elution in the countercurrent chromatography operation of an expanded bed are from top to bottom and the rotation speed direction of equipment is clockwise, and respectively determine the proper sample injection flow velocity within the range of 200 plus materials and 500rpm to ensure that the resin is in a stable expansion dispersion state in the separation column. The penetration behavior and elution profile of expanded bed countercurrent chromatography at different heparin concentrations and different eluent flow rates were studied. The result shows that the dynamic adsorption capacity is 275mg/g under the conditions that the addition amount of the D204 resin is 5.0g, the column volume is 30mL, the sample loading concentration is 50mg/mL, the flow rate is 1.5mL/min and the rotating speed is 300rpm, and the dynamic adsorption capacity is gradually reduced along with the increase of the rotating speed. The flow rate has a significant effect on the breakthrough time, reaching the breakthrough point at 176min at a flow rate of 0.5mL/min, with the increase in flow rate to 10mL/min, the breakthrough time shortened to 9.5min, and the dynamic adsorption capacity increased with the increase in flow rate. Compared with fixed bed column chromatography, the adsorption rate of the expanded bed countercurrent chromatography at the flow rate of 1mL/min can reach 91.66% to the maximum, and the expanded bed countercurrent chromatography effectively improves the adsorption rate of the resin to heparin and reduces the separation time.

Meanwhile, the analysis shows that the titer of the purified heparin separated and purified by column chromatography is 205.51 +/-7.90 IU/mg, and the titer of the heparin separated and purified by the expanded bed countercurrent chromatography is 216.09 +/-11.89 IU/mg, which all reach the standard of 180IU/mg specified by the pharmacopoeia. And high performance liquid chromatography (HPLC-ELSD) is used for detecting the purity of the heparin, and the result shows that the heparin after chromatographic purification is a mixture with different molecular weights, and compared with the heparin separated and purified by column chromatography, the heparin separated and purified by the expanded bed countercurrent chromatography has smaller molecular weight and higher Fxa/FIIa value, and is suitable for being used as an exogenous anticoagulant.

Drawings

FIG. 1 is a schematic diagram of expanded bed countercurrent chromatography (a) and a schematic diagram of experimental procedure (b) in example 2 of the present invention;

FIG. 2 is an expanded bed countercurrent chromatography experimental apparatus in example 2 of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

In the following examples of the present invention, the resins D204, D254, D208 and D301 used were purchased from Shanghai-base industries, Ltd., and the physical parameters are shown in Table 1;

TABLE 1

Example 1: expanded bed countercurrent chromatography adsorbent media screening

(1) Pretreatment and regeneration of resins

Putting the four resins in a 500mL beaker respectively, adding distilled water with the volume about 5 times of that of the four resins, soaking for 24 hours, stirring for multiple times during the period, pouring an upper solution after settling, draining the upper solution by using a suction filter funnel, adding absolute ethyl alcohol, soaking for 2 hours, stirring at any time, draining and washing until the absolute ethyl alcohol smell is removed, then carrying out acid-base acid treatment on the resins, firstly soaking the resins in 2mol/L HCl for 2 hours, continuously stirring during the period, carrying out suction filtration, draining, washing with distilled water until the resins are neutral, soaking the resins in 2mol/L HCl for 2 hours, carrying out suction filtration, draining, washing with distilled water until the resins are neutral, detecting the pumped liquids are washed with distilled water until the liquids are neutral, and then soaking the resins in distilled water for standby.

After the resin is used for a period of time, the adsorbed impurities are close to a saturated state, the adsorption performance is reduced, and regeneration treatment is carried out to recover the original composition and performance. The regeneration of the resin is soaked for 2h with 2moL/L HCl solution and then washed to neutrality with water. Soaking the mixture for 2 hours by using 2moL/L NaOH, soaking the mixture for 2 hours by using 2moL/L HCl solution again, and then washing the mixture to be neutral by using water for reuse.

(2) The heparin concentration is measured by adopting a concentrated sulfuric acid oxidation method;

(3) determining an adsorption kinetics curve, wherein the adsorption kinetics curve fitting adopts a quasi-first-stage kinetics model, a quasi-second-stage kinetics model and an internal diffusion kinetics model to research the micro process of resin for adsorbing heparin, and the adsorption isotherm fitting adopts a Langmuir model and a Freundlich model to fit the isothermal adsorption behavior;

taking 2.0g of each of the four resins, placing the resins into a 50mL conical flask, adding 10mL of 20mg/mL heparin, placing the resins into a 150rpm shaking table, shaking the resins at 25 ℃, and sampling and detecting the concentration of the solution after 0, 0.5, 1, 2, 4, 6 and 8 hours respectively. And drawing an adsorption kinetic curve by taking the time as an abscissa and the heparin adsorption amount as an ordinate.

The results show that the quasi-second order kinetic correlation coefficient R of the four resins²Quasi first order kinetic correlation coefficient R²Closer to 1, and fitted q_eAnd the measured value is closer to the value measured by the experiment. Therefore, the quasi-secondary dynamics better accords with the adsorption process of resin to heparin, and the straight line fitted by the internal diffusion dynamic model shows that the particle internal diffusion plays a certain role in adsorption. At the beginning of adsorption, the adsorption rate was broad and the adsorption amount increased quite rapidly because a large number of adsorption sites were present in the resin. With increasing time, the adsorption sites on the adsorbent become fewer and fewer until the adsorption reaction reaches an adsorption equilibrium state. Wherein the time for adsorbing heparin by the D204 resin to reach saturation adsorption is short, the adsorption capacity is high, and the adsorption capacity reaches 87.34% after 2h, so that the resin can be used as an alternative resin of an adsorption medium.

Respectively preparing 50mg/mL heparin solutions with NaCl concentrations of 0, 1, 2, 3 and 4mol/L, taking 2.0g of each of the four pretreated resins, putting the resins into a 50mL conical flask, adding 10mL of 50mg/mL heparin with different salt concentrations, putting the resins into a 150rpm shaking table, fully shaking the bottles at 25 ℃, and respectively sampling and detecting the concentration of the solutions after 0, 2, 4, 6 and 8 hours. And drawing adsorption kinetic curves under different salt concentrations by taking the time as an abscissa and the heparin adsorption amount as an ordinate.

The results show that: the adsorption capacity of the D204 resin is increased rapidly in the first 1h or so, the adsorption capacity is close to the saturated adsorption capacity in 1h or so and hardly changes, the adsorption capacity of the resin to heparin is gradually reduced along with the increase of the salt concentration, and the heparin is hardly adsorbed under the conditions that the salt concentration is 2mol/L, 3mol/L and 4 mol/L. The adsorption kinetics of heparin under different salt concentrations in 8h are measured, adsorption research is carried out through quasi-first-order kinetics, quasi-second-order kinetics and internal diffusion kinetics, linear fitting is carried out, and the result shows that the quasi-second-order kinetics correlation coefficient R of the D204 resin is obtained when the NaCl concentration is 0-4mol/L²Quasi first order kinetic correlation coefficient R²Closer to 1, and fitted q_eAnd the measured value is closer to the value measured by the experiment. Thus, the quasi-secondary kinetics is more consistent with the adsorption process of resin to heparin. When the NaCl concentration is 4mol/L, the correlation coefficient R is obtained by an internal diffusion dynamic model²0.9334 and intercept C is near the origin, where intraparticle diffusion plays a major role in adsorption.

The adsorption capacity of the D208 resin is increased very rapidly in the first 2h or so, the adsorption capacity is increased slowly in 2-6h, the adsorption capacity is close to the saturated adsorption capacity in 8h or so and hardly changes, the adsorption capacity of the resin to heparin is gradually reduced obviously along with the increase of the salt concentration, and the heparin is hardly adsorbed when the salt concentration is 4 mol/L. Determining the adsorption kinetics of heparin under different salt concentrations within 8h, performing adsorption research through quasi-first-order kinetics, quasi-second-order kinetics and internal diffusion kinetics, and performing linear fitting; the quasi-second order kinetic correlation coefficient R of the D204 resin at the NaCl concentration of 0-4mol/L²Quasi-first order dynamics related coefficient R²Closer to 1, and fitted q_eThe value is closer to the value measured by the experiment, thereby showing that the quasi-secondary dynamics is more in line with the adsorption process of resin to heparin. The straight line fitted by the internal diffusion kinetic model is high in fitting degree under low concentration, indicates that particles are diffused in the particles and plays a certain role in adsorption;

the adsorption amount of the D254 resin is gradually increased along with the time, but the adsorption process is very slow, the saturated adsorption amount is reached within about 6 hours, the adsorption amount of the resin to the heparin is obviously gradually reduced along with the increase of the salt concentration, and the heparin is hardly adsorbed when the salt concentration is 3mol/L and 4 mol/L. Determining adsorption kinetics of heparin under different salt concentrations within 8h, performing adsorption research through quasi-first-order kinetics, quasi-second-order kinetics and internal diffusion kinetics, and performing linear fitting; quasi-first order kinetic correlation coefficient R of D254 resin at low salt concentration²Quasi-second order kinetic correlation coefficient R²Closer to 1, and fitted q_eThe value is close to the value measured by the experiment, which shows that the quasi-first-level dynamics better conforms to the adsorption process of resin to heparin, and the quasi-second-level dynamics better conforms to the adsorption form of resin when the salt concentration is high. The straight line fitted by the internal diffusion kinetic model is high in fitting degree under low concentration, indicates that the particles are internally diffused and plays a certain role in adsorption.

The adsorption capacity of the D301 resin is gradually increased along with the time, the saturated adsorption capacity is reached within about 8h, and the adsorption capacity of the resin to heparin is gradually reduced along with the increase of the salt concentrationThe low is very obvious, and when the salt concentration is more than 2mol/L, the heparin is hardly adsorbed. Determining the adsorption kinetics of heparin under different salt concentrations within 8h, performing adsorption research through quasi-first-order kinetics, quasi-second-order kinetics and internal diffusion kinetics, and performing linear fitting; quasi-second order kinetic correlation coefficient R of D301 resin²Quasi first order kinetic correlation coefficient R²More closely 1, and fit out q_eAnd the measured value is closer to the value measured by the experiment. Therefore, the quasi-secondary dynamics better conforms to the adsorption process of resin to heparin, the straight line fitted by the internal diffusion dynamics model is high in fitting degree, and the fact that the particle internal diffusion plays an important role in adsorption under the condition that salt exists is shown.

(4) Static adsorption and desorption experiments: 50mmol/L Tris-HCl buffer solution is prepared, and all the adsorption and desorption experiments are completed in 50mmol/L Tris-HCl buffer system with pH of 8.5. The pretreated resin was equilibrated with buffer and then drained from the funnel. 2.0g of the 4 resins are weighed respectively and put into a 50mL conical flask, and then 10mL of 50mg/mL heparin solution is added. Placing into a constant temperature oscillator, fully adsorbing at a constant temperature of 25 ℃ for 8h at 150rpm, absorbing upper layer liquid, measuring heparin concentration by a concentrated sulfuric acid oxidation method, and calculating adsorption capacity and adsorption rate according to the difference of the heparin concentration before and after the test:

wherein C is₀The initial concentration of heparin before adsorption is mg/mL; c₁Is the equilibrium concentration mg/mL of adsorbed heparin; v₁Is the volume of heparin solution, mL; m is the mass of the resin, g.

Washing the 4 kinds of statically adsorbed resins with distilled water to remove impurities, filtering to remove dry water, and collecting. Transferring to 4 clean conical flasks, adding 10mL of 2mol/L NaCl, fully desorbing at 25 ℃ for 8h at a constant temperature of 150rpm, sucking the supernatant, measuring the heparin concentration in the solution, and calculating the desorption amount and the desorption rate according to the difference between the heparin concentration before and after the test and the mass of the resin:

wherein C is₂The concentration of the desorbed heparin solution is mg/mL;

(5) respectively diluting the heparin solution into different gradient concentrations (5, 10, 15, 20, 25, 30, 40 and 50mg/mL), respectively taking 10mL of the diluted solution, respectively adding 2.0g of the pretreated resin, placing the diluted solution into a 150rpm shaking table, carrying out isothermal adsorption at 25 ℃ for 8h, and detecting the concentration of the heparin solution. Then, the adsorption isotherms of the D204 resin at different temperatures were measured again at 35 ℃ and 45 ℃. And drawing an adsorption isotherm by taking the concentration of heparin in the solution in equilibrium as an abscissa and the adsorption amount of the resin in adsorption equilibrium as an ordinate.

The experimental results show that:

the adsorption capacity of the 4 resins D204, D208, D254 and D301 is 89.66 + -0.99 mg/g, 83.98 + -1.61 mg/g, 66.96 + -1.08 mg/g and 55.31 + -0.57 mg/g respectively. Wherein the adsorption performance of the D204 resin to heparin is the best, the adsorption rate reaches 95.52 percent, and the adsorption rate of the D301 resin is the lowest and is 56.78 percent.

The desorption capacities D204, D208, D254 and D301 are 84.43 +/-1.52 mg/g, 71.99 +/-1.67 mg/g, 58.46 +/-1.76 mg/g and 43.56 +/-1.04 mg/g respectively, the desorption rates of the four resins to heparin all reach more than 79 percent, wherein the desorption effect of the D204 resin is the best, the desorption rate reaches 92.68 percent, the desorption rate of the D301 resin is the lowest, and only 79.29 percent can be desorbed;

as the initial concentration of heparin increases, the adsorption capacity of the four resins to the heparin is rapidly increased, because the resins and the heparin can be rapidly adsorbed due to the superior specific surface area of the pore structure. When heparin is used initiallyWhen the initial concentration reaches a certain concentration, the adsorption capacity is hardly increased any more, and the adsorption rate gradually slows down as the active sites are occupied by heparin, and finally the adsorption equilibrium is approached. Maximum simulated monolayer adsorption amounts of D204, D208, D254 and D301 on heparin at 25 ℃ are respectively calculated to be 110.21mg/g, 88.06mg/g, 69.90mg/g and 58.90mg/g by Langmuir adsorption isothermal model fitting. Langmuir adsorption model correlation coefficients (R) for D204, D208, D254, D301²0.9901, 0.9466, 0.9045 and 0.9623 are all higher than the correlation coefficient (R) of Freundlich model²0.9335, 0.7633, 0.7723, 0.8351). The resin is more consistent with Langmuir adsorption on heparin and belongs to monomolecular adsorption. When the initial concentration of heparin reaches a certain concentration, the active adsorption sites on the surface of the resin determine the adsorption capacity of the resin, and the number of the active adsorption sites on the surface of the resin is constant, so that the adsorption capacity of the resin does not correspondingly increase with the increase of the concentration of the heparin. R of four resins_LThe values are all between 0 and 1, which indicates that the reaction is favorable for the adsorption of resin to heparin.

With the temperature rising from 25 ℃ to 45 ℃, the adsorption quantity of the D204 resin to the heparin is gradually increased from 110.21mg/g to 192.55mg/g, k_LThe temperature is gradually reduced along with the increase of the temperature, and the increase of the temperature is favorable for the adsorption of the resin to the heparin.

From the above results, it was concluded that the adsorption performances of the four kinds of adsorption resins were compared, and D204 resin was screened as a solid adsorption medium for expanded bed countercurrent chromatography.

Example 2 study of heparin adsorption Performance by expanded bed countercurrent chromatography

Adding the D204 resin into a countercurrent chromatographic separation column to construct expanded bed countercurrent chromatography, researching the dispersion state of an adsorption medium in the expanded bed countercurrent chromatography, and inspecting related operating parameters.

(1) Construction of expanded bed countercurrent chromatography

The structure schematic diagram of the expanded bed countercurrent chromatography is shown in figure 1, and the experimental device is shown in figure 2. Adding pretreated resin into a column according to the sequence of a solution, a constant flow pump, an expanded bed countercurrent chromatography and a part of collectors, enabling the resin to be settled in the tube, winding a hose on the column anticlockwise, connecting all the parts well, sealing, discharging bubbles in the tube, fixing the bubbles by a binding belt, matching the other side with the same balance weight, controlling the flow rate by the constant flow pump, setting the flow rate direction to be from top to bottom, and controlling the rotating speed by a motor. The liquid enters the expanded bed countercurrent chromatography under the action of the constant flow pump, heparin is adsorbed in the separation column by setting different flow rates and rotating speeds, then the heparin flows out from the sample outlet, and the solution is collected by a partial collector.

(2) Determination of flow velocity direction of expanded bed countercurrent chromatography

Adding 2.0g of pretreated D204 resin into a hose with an effective volume of 100mL (phi is 5mm), allowing the resin to settle in the hose, discharging bubbles in the hose, fixing by a ribbon, connecting and sealing a joint, setting the flow rate to be 1mL/min, setting the rotation rate to be clockwise, operating for 1h according to the experimental conditions in Table 2, and observing the state of the stationary phase in the column.

TABLE 2 Experimental conditions

The results show that: the resin state was observed after 1h by setting different rotation speeds and flow directions under the flow rate condition of 1 mL/min. Comparative experiment group

a. b, c, when the flow direction is downward, upward and downward, the resin gradually moves upwards in the column, and the resin moves farther upwards along with the increase of the rotating speed, which shows that the centrifugal force provided by the rotating speed and the thrust provided by the flow speed both move the resin upwards (outlet end), and at the moment, the chromatographic column medium cannot be kept in a dispersed state in the column; the flow velocity direction of the experiment groups d, e and f is changed from bottom to top, the chromatographic column medium moves upwards (inlet end) along with the increase of the rotating speed, and the flow velocity and the rotating speed can be adjusted at the moment to ensure that the chromatographic column medium is in a stable dispersion state in the chromatographic column; however, at 400rpm, the centrifugal force provided by the rotation speed is still slightly larger than the thrust provided by the flow speed, so that the resin moves slowly in the tube, and at the flow speed of 1mL/min, the rotation speed needs to be reduced to keep the chromatographic column medium in balance in the chromatographic column. The flow velocity direction is determined from top to bottom in the following experiment.

(3) Determination of expanded bed counter-current chromatography rotational speed range

After the flow velocity direction is determined to be from top to bottom, in order to further determine the flow velocity and the rotating speed range which can be kept in the resin, different flow velocities and rotating speeds are set for experimental observation, wherein the resin is 2.0g D204 resin, the effective column volume is 30mL, the rotating speed is gradually changed between 200rpm and 500rpm, the flow velocity is gradually changed between 1mL/min and 10mL/min, after 2 hours of operation, the state of the resin is observed, and the flow velocity and the rotating speed range are determined.

The results show that: at the rotating speed of 200rpm and the flow rate of 1mL/min, the resin can be dispersedly retained in the column after 2h, the thrust of the flow rate is gradually increased along with the increase of the flow rate to 2 and 3mL/min, the resin gradually moves from the inlet end to the outlet end, the resin can be dispersedly retained in the column, and at the time of 4 and 5mL/min, the resin is completely accumulated at the outlet end due to the fact that the thrust of the flow rate is larger than the centrifugal force provided by the rotating speed, and the resin cannot be in an expansion state. At the rotating speed of 300rpm and the flow rate of 1mL/min, the resin is accumulated at the inlet end after 2h, most of the resin can be maintained in the column in a dispersed mode, the resin is uniformly dispersed in the column along with the increase of the flow rate from 2mL/min to 4mL/min, and at the flow rate of 5mL/min, the resin is completely accumulated at the outlet end due to the thrust of the flow rate, and the resin cannot be in a dispersed state. At the rotation speed of 400rpm and the flow rate of 2mL/min, the resin is accumulated at the inlet end after 2h, the flow rate is increased to 3-8mL/min, and the resin can be uniformly dispersed in the column. At the rotating speed of 500rpm and the flow rate of 8mL/min, the resin is accumulated at the inlet end after 2h, and when the flow rate is increased to 10mL/min, the resin can be uniformly dispersed in the column;

the flow rate ranges at different speeds are summarized in table 3. Finally, the flow rate is determined to be 1-3mL/min at 200rpm, so that the resin can be stably in a diffusion state in the column; at 300rpm, the flow rate is 1-4mL/min, so that the resin can be stably diffused in the column; at 400rpm, the flow rate is 3-8mL/min, so that the resin can be stably diffused in the column; at 500rpm, the flow rate is 10mL/min, so that the resin can be stably diffused in the column; at 500rpm, the flow rate can still continue to increase, but subsequent experiments do not continue to increase the flow rate in view of the pressure-bearing capacity of the high flow rate to the hose and the interface.

TABLE 3 flow Rate Range at different rotational speeds

(4) Expanded bed countercurrent chromatography breakthrough curve study

Influence of the rotating speed on the counter-current chromatographic penetration curve of the expanded bed: filling 5.0g D204 resin into a 30mL hose, exhausting air bubbles in the hose, connecting interfaces, balancing with a Tris-HCl buffer solution (pH 8.5) for 2 hours, preparing a 50mg/mL heparin aqueous solution sample, setting the flow rate to be 1.5mL/min, the rotation speed to be 300, 400 and 500rpm, collecting the effluent at an outlet end by using a fractional collector, detecting the concentration of heparin by using a concentrated sulfuric acid oxidation method, and drawing a penetration curve according to the ratio of the concentration of the heparin at the outlet end to the concentration of the sample;

the results show that: setting the flow rate to be 1.5mL/min and the rotating speed to be 300, 400 and 500rpm to obtain a penetration curve, and summarizing data in a table 4; under the same flow rate condition, the penetration point is advanced along with the increase of the rotating speed, because in the running process, the resin in a diffusion state in the column gradually moves towards the inlet end along with the increase of the rotating speed, the contact time of the resin and the solution is reduced, the adsorption rate of the resin is reduced, and the penetration point is reached earlier. The dynamic adsorption capacity therefore decreases from 275mg/g to 125mg/g as the speed of rotation increases from 300rpm to 500 rpm. However, since the resin is still incompletely dispersed in the column at this time, the expanded bed state cannot be effectively realized by considering only the rotation speed. To achieve a stable expanded bed separation effect, research needs to be carried out on the premise that the flow rate and the rotation speed are matched and the resin is kept in a dispersed motion in the column.

TABLE 4 dynamic adsorption Capacity at different rotational speeds

Influence of flow velocity on the penetration curve of the expanded bed countercurrent chromatography: filling 5.0g D204 resin into a 30mL hose, exhausting bubbles in the hose, connecting each interface, preparing a 50mg/mL heparin aqueous solution sample according to the set flow rate of 1, 2 and 5mL/min, collecting the effluent at the outlet end by using a fraction collector, detecting the heparin concentration by using a concentrated sulfuric acid oxidation method, and drawing a penetration curve according to the ratio of the heparin concentration at the outlet end to the sample concentration; in order to make the resin in the column in a diffusion state, the rotating speed, the flow rate and the rotating speed are set according to the parameters in the table 3;

the results show that: the dynamic adsorption capacities are summarized in Table 5. As can be seen from Table 5, the time to reach the breakthrough point decreased significantly, from 176min to 9.5min, as the flow rate increased from 0.5mL/min to a high flow rate of 10 mL/min. In the process, when the medium-low flow rate is 0.5-5mL/min, the dynamic adsorption capacity is almost unchanged at about 180 mg/g; the rotation speed was gradually increased from 300rpm to 500rpm, and the dynamic adsorption capacity was expected to be increased from 180mg/g to 250 mg/g. This is probably because the contact and exchange opportunities of the resin with heparin in the column are significantly increased with the increase of the flow rate and the rotation speed, but the actual adsorption amount still needs to be analyzed by the dynamic elution experiment.

TABLE 5 dynamic adsorption capacity of heparin on expanded bed countercurrent chromatography at different flow rates

(5) Experimental determination of dynamic adsorption capacity of expanded bed countercurrent chromatography as in example 1;

(6) expanded bed countercurrent chromatography elution Curve Studies

Expanding a different flow rate elution curve of a bed countercurrent chromatography: after the resin is subjected to the penetrating adsorption by flow rates of 1, 2 and 5mL/min, Tris-HCl buffer (pH 8.5) is used for washing 2 column volumes to remove unadsorbed heparin, and elution is carried out by using 2mol/L NaCl after effluent liquid is detected to be colorless by a concentrated sulfuric acid oxidation method, wherein the flow rates during elution are respectively 1, 2 and 5 mL/min. Collecting the eluted solution by using a partial collector, detecting the concentration by using a concentrated sulfuric acid oxidation method, and drawing an elution curve;

the results show that: in the expanded bed countercurrent chromatography, when the flow rate is 1mL/min, the time taken for reaching the penetration point is the longest, and after the penetration point is reached, the rise of the penetration curve is more gentle, which indicates that the longer the adsorption time of heparin in the column is, the larger the exchange capacity in the column is; the breakthrough point is more advanced and the breakthrough curve is steeper as the flow rate increases. 1. The dynamic adsorption capacities of the three flow rates of 2 and 5mL/min were 100mg/g, 150mg/g and 200 mg/g, respectively. On the premise of sufficient exchange between the resin and the heparin, the dynamic adsorption capacity of the countercurrent chromatography of the expanded bed is increased along with the increase of the flow rate;

elution curves were experimentally determined for three flow rates of 1, 2, 5mL/min and the elution amounts are summarized in Table 6. It can be seen that the dynamic adsorption capacity of the expanded bed countercurrent chromatography under the three flow rate conditions of 1, 2 and 5mL/min is significantly higher than that of the fixed bed column chromatography, under the conditions of 300rpm and 1mL/min, the elution amount reaches 91.66mg/g, and nearly reaches the static adsorption capacity of 110.21mg/g, because the adsorption time of the expanded countercurrent chromatography is longer at 1mL/min, and the resin can be fully contacted with the heparin; and the planetary motion of the instrument causes the resin to be fully contacted with the heparin in the counter-current chromatographic operation process of the expanded bed, and the temperature of the chromatographic column is increased in the operation process of the instrument, so that the adsorption of the heparin in the column is facilitated. The elution of heparin gradually decreases with increasing flow rate, because the resin and heparin are not sufficiently adsorbed by exchange with each other as the flow rate increases and the time taken for penetration decreases, but only the effect of the countercurrent chromatography captures the heparin in the solenoid chromatographic column, and after the elution of the non-adsorbed heparin, the amount of heparin adsorbed on the resin decreases, resulting in a decrease in the amount of heparin adsorbed. From the comparison of adsorption rates, the adsorption rate of the expanded bed countercurrent chromatography reaches 91.66% at 1mL/min, the adsorption rate of the expanded bed countercurrent chromatography is 45.16% compared with that of the fixed bed column chromatography, the expanded bed countercurrent chromatography can have higher adsorption rate at the same flow rate, and the flow rate of the general column chromatography is 0.1-0.5 mL/min.

TABLE 6 heparin dynamic adsorption Capacity comparison

The retention state of heparin in the expanded bed countercurrent chromatography at different flow rates shows that the dynamic adsorption capacity of heparin gradually increases with the increase of the flow rate, and is influenced by the following three reasons: 1. the heparin is fully contacted with the resin in the separation column and is adsorbed on the resin through ion exchange, 2. the planetary motion enables the separation column to continuously move, the effect similar to the liquid-liquid separation of the countercurrent chromatography is generated, the heparin is captured in the liquid phase of a solenoid by the resin, and 3. the temperature is increased in the running process of the instrument, and the adsorption capacity of the heparin on the adsorption medium is improved. After that, after the elution of heparin which was not adsorbed on the resin with the buffer solution, the elution amounts of heparin obtained by elution with 2mol/L NaCl at flow rates of 1, 2 and 5mL/min were 91.66, 66.42 and 52.81mg/g, and the heparin remained in the solution increased with the increase of the flow rate.

Collecting heparin: mixing and collecting heparin solutions obtained by separating tubes under the conditions of three flow rates of 1, 2 and 5mL/min, precipitating with 2 times of ethanol, centrifuging at 2000rpm for 5min, discarding supernatant, washing with absolute ethanol twice, drying at room temperature, and grinding into powder to obtain purified heparin;

expanding experiment of the expanded bed countercurrent chromatography system: amplifying the system by 2 times under the experimental condition of 2mL/min, filling 10.0g of resin into a 60mL hose, exhausting bubbles in the hose, connecting various interfaces, preparing a 50mg/mL heparin aqueous solution sample, setting the flow rate to be 2mL/min and the rotating speed to be 300rpm, collecting effluent at an outlet end by using a branch collector, detecting the heparin concentration by using a concentrated sulfuric acid oxidation method, and drawing a penetration curve according to the ratio of the heparin concentration at the outlet end to the sample concentration.

After the breakthrough adsorption, the column volume was 2 column volumes washed with Tris-HCl buffer (pH 8.5) to remove the non-adsorbed heparin, and after no heparin was detected by concentrated sulfuric acid oxidation, elution was started at a flow rate of 2mL/min and a rotation rate of 300 rpm. Collecting the eluted solution by using a fraction collector, detecting the concentration by using a concentrated sulfuric acid oxidation method, and drawing an elution curve;

the results show that: after the 2-fold system is expanded, the time of the penetration point is obviously delayed from 42.5min to 60min, which shows that after the system is amplified, the resin is more fully contacted with heparin in the countercurrent chromatography of the expanded bed, the adsorption quantity is increased, and the dynamic adsorption capacity of the countercurrent chromatography of the expanded bed is increased after the penetration point is delayed;

the relevant parameters of the elution curve after the system is expanded are listed in table 7, the actual elution amount is 136.48mg/g, linear amplification relation can be well formed on the adsorption amount by the expanded bed countercurrent chromatography, and after the expanded bed countercurrent chromatography separation system is expanded, the resin can be more fully contacted with heparin in the column, and the adsorption rate reaches 55.59%, which is improved compared with the prior art.

TABLE 7 heparin dynamic adsorption Capacity comparison

(7) Influence of sample size on adsorption Effect

Influence of sample loading amount on adsorption effect: filling 5.0g D204 resin into a 30mL hose, exhausting air bubbles in the hose, connecting each interface, setting the flow rate to be 2mL/min and the rotation speed to be 300rpm, loading samples respectively at 50mg, 100mg and 200mg, washing 2 column volumes by using Tris-HCl buffer solution (pH 8.5) to remove unadsorbed heparin, eluting by using 2mol/L NaCl, collecting the eluted solution by using a fraction collector, detecting the concentration by using a concentrated sulfuric acid oxidation method, and drawing an elution curve;

the data are presented in table 8. When the sample amount is 50, 100 and 200mg, the obtained heparin is 13.73, 21.48 and 36.23 mg; the recovery rate of heparin gradually increases with the increase of the amount of the sample, but gradually decreases with the increase of the amount of the sample. This is because the planetary motion of the expanded bed countercurrent chromatography causes heparin to be mainly trapped in the liquid phase of the solenoid, and the recovery rate of heparin is reduced after heparin not adsorbed in the resin is removed.

Influence of cyclic sample loading quantity on adsorption effect: filling 5.0g D204 resin into a 30mL hose, exhausting air bubbles in the hose, connecting each interface, setting the flow rate to be 2mL/min, setting the rotation speed to be 300rpm, loading 200mg, taking Tris-HCl buffer solution (pH 8.5) as a mobile phase, circularly loading, detecting the concentration of effluent liquid every 30min after 1h, taking water as the mobile phase after 8h, washing the non-adsorbed heparin, eluting with 2mol/L NaCl, collecting the eluted solution by a fraction collector, detecting the concentration by a concentrated sulfuric acid oxidation method, and drawing an elution curve;

heparin recovery was calculated according to the formula:

wherein eta is the recovery rate of heparin; m is₁Is the amount of heparin eluted, mg; m is₂Is the heparin loading, mg.

The results show that 200mg of sample is loaded at the initial time of 0min, after 60min, sampling detection is carried out every 30min, detection is stopped at 510min, 4 cycles are carried out in the period, then, 2mol/L sodium chloride is used for elution, the elution amount after sample loading is cycled is 170.51mg, the recovery rate is summarized in Table 8, and the recovery rate after 4 cycles reaches 85.75%, which is about 4.7 times higher than that without cycle, and the recovery rate of heparin is obviously improved.

TABLE 8 elution with different loading amounts

(8) Conclusion

Constructing the countercurrent chromatography as an expanded bed countercurrent chromatography, and observing the operation parameters of the expanded bed countercurrent chromatography, wherein D204 resin is used as an adsorption medium to adsorb heparin; carrying out penetration research on different flow rates, and measuring dynamic adsorption capacity and elution capacity; the influence of different sample loading amounts on the adsorption effect is researched, and the efficient recovery of heparin is realized by circularly loading and then eluting, and the conclusion is as follows:

by studying the retention state of the resin in the column, the flow velocity direction of the countercurrent chromatography of the expanded bed is determined to be from top to bottom, and the rotation speed direction is determined to be clockwise, so that the resin can be in a stable expanded bed state in the column.

Determining the flow speed and rotation speed range of the expanded bed state: the rotating speed is 200rpm, and the flow rate is 1-3 mL/min; when the rotating speed is 300rpm, the flow rate is 1-4 mL/min; when the rotating speed is 400rpm, the flow rate is 3-8 mL/min; the resin was stabilized in the expanded bed state in the column at a rotation speed of 500rpm and a flow rate of 10 mL/min.

At 1.5mL/min, the rotation speed was increased from 300rpm to 500rpm, and the resin in a dispersed state in the column gradually moved toward the inlet end, so that the breakthrough curve reached the breakthrough point earlier and the dynamic adsorption capacity was decreased from 275mg/g to 125 mg/g.

The flow rate has a significant effect on the breakthrough point, increasing the flow rate from 0.5mL/min to a high flow rate of 10mL/min, decreasing the time to reach the breakthrough point from 176min to 9.5min, increasing the dynamic adsorption capacity from 180mg/g to 250mg/g, increasing the dynamic adsorption capacity with increasing flow rate.

Compared with fixed bed column chromatography, the elution amount of the expanded bed countercurrent chromatography is obviously higher than that of the fixed bed column chromatography under the same flow rate. The maximum adsorption rate of the expanded bed countercurrent chromatography reaches 91.66 percent at 1mL/min, and the elution amount nearly reaches the static adsorption capacity of 110.21 mg/g. Expanded bed countercurrent chromatography retains heparin within a solenoid chromatography column such that the dynamic adsorption capacity increases with increasing flow rate, while the column chromatography dynamic adsorption capacity decreases with increasing flow rate. The result shows that the expanded bed countercurrent chromatography can effectively improve the adsorption rate of resin to heparin at higher flow rate and reduce the separation time.

The expansion experiment shows that when the flow rate is 2mL/min and the rotating speed is 300rpm, a linear amplification relation can be well formed.

The influence of the sample loading amount on the adsorption effect shows that the expanded bed countercurrent chromatography is more suitable for high-concentration sample loading separation, and when the sample loading amount is 50, 100 and 200mg, the heparin obtained by recovery is 13.73, 21.48 and 36.23 mg; the single recovery rate of 200mg of the sample is 18.11 percent, the recovery rate reaches 85.75 percent after 4 cycles, and the experimental result shows that the cycle sample loading can improve the heparin yield by about 4.7 times.

Example 3 chromatographic separation and purification of heparin anticoagulant activity and purity investigation

The anticoagulant activity of the heparin obtained in example 2 and after column chromatography separation is detected by an azure A colorimetric method, a chromogenic substrate method and a sheep plasma method respectively, the three determination methods are compared, and the anticoagulant activity and the purity of the heparin prepared by column chromatography and expanded bed countercurrent chromatography are analyzed and evaluated.

The results show that:

(1) detecting the titer of the heparin by an azure A method: as shown in Table 9, the titer of the heparin standard product is 197IU/mg, and the titer of the heparin crude product determined by the azure A colorimetric method is 118.42 + -3.06 IU/mg. After separation and purification, the anticoagulant potency of the heparin is improved, wherein the heparin potency is respectively improved from 118.42IU/mg to 156.61IU/mg and 153.68IU/mg under the flow rates of column chromatography 1 and 2 mL/min; the heparin titer is respectively improved from 118.42IU/mg to 145.13IU/mg and 156.73IU/mg under the flow rate of the expanded bed countercurrent chromatography of 1 and 2 mL/min; the obtained heparin titer is higher at the flow rate of 5mL/min, wherein the column chromatography titer reaches 182.74IU/mg, the expanded bed countercurrent chromatography reaches 192.35IU/mg, which is slightly higher than the column chromatography.

TABLE 9 determination of heparin potency by azure A colorimetry

(2) The titer is measured by a sheep plasma method: the titer of the heparin obtained by the experiment is shown in table 10, and the potency of the crude heparin product is 134.17 + -4.12 IU/mg. Compared with a azure A colorimetric method, the determination is more accurate by a sheep plasma method, the titer of the heparin separated and purified by column chromatography is 205.51 +/-7.90 IU/mg, and the titer of the heparin separated and purified by expanded bed countercurrent chromatography is 216.09 +/-11.89 IU/mg, which all reach the standard of 180IU/mg specified by pharmacopoeia. The titer of the heparin after the separation and purification of the countercurrent chromatography of the expanded bed is slightly higher than that of the heparin after the separation and purification of the column chromatography.

TABLE 10 sheep plasma method for heparin potency determination

(3) Detecting the titer of the heparin by a chromogenic substrate method: the titer of the heparin obtained by the experiment is shown in table 11, the anti-FXa titer of the heparin obtained by separation and purification at different flow rates of column chromatography is improved and reduced compared with the crude heparin product, the anti-FIIa titer is improved compared with the crude heparin product, the FXa titer of the heparin obtained by separation and purification of the expanded bed countercurrent chromatography at 1mL/min is improved and reduced compared with the crude heparin product, the anti-FIIa titer is improved compared with the crude heparin product, and the anti-FXa titer and the anti-FIIa titer of 2mL/min, 5mL/min are both improved compared with the crude heparin product. Since the heparin chain (which has at least 18 monosaccharide components and contains pentasaccharide units) must form a ternary complex with both antithrombin and thrombin in order to inhibit the activity of FIIa. Most of the sugar chains of heparin with a higher molecular weight meet this condition, while low molecular weight heparin has a portion of the heparin chains that do not. The low molecular heparin mainly plays a role in inhibiting the FXa, retains partial FVIIa activity, and has the advantages of good subcutaneous injection absorption, high bioavailability, small bleeding tendency and the like. The molecular weight of heparin adsorbed by column chromatography is larger, the molecular weight of heparin adsorbed by countercurrent chromatography is smaller, Fxa/FIIa is higher, and the anticoagulant effect is better, so that the heparin is more suitable for serving as an exogenous anticoagulant.

TABLE 11 measurement of heparin potency by chromogenic substrate method

(4) HPLC detection of heparin purity

The heparin crude product, the standard product and the heparin obtained by eluting after penetrating through different flow rates are subjected to HPLC-ELSD detection results, the heparin standard product and the heparin crude product are obtained by calculating a molecular weight standard curve, the molecular weight range of the heparin standard product is 7177-14293Da, the molecular weight range of the heparin crude product is 6793-14769Da, the heparin crude product contains higher NaCl, after penetrating through adsorption through column chromatography or expanded bed countercurrent chromatography, the heparin crude product can be purified to different degrees, and the NaCl is obviously reduced after purification.

The heparin is roughly divided into three components of Hp-1, Hp-2, Hp-3 and Cl according to the retention time of 6.72min, 7.48min, 8.15min, 10.99min and 13.29min after being separated and purified^-And Na⁺The percentages of the components are listed in table 12 according to the area of each peak, the gel filtration chromatography shows peaks according to the molecular weight, the content of heparin standard products is distributed from high molecular weight to low molecular weight and is uniform, and the content of heparin crude products is different in molecular weight and is not uniform. Compared with a chromatogram of a crude heparin product, the NaCl content of the heparin obtained by column chromatography is obviously reduced, the Hp-1 and Hp-2 with higher molecular weights are more, and the Hp-2 and Hp-3 with lower molecular weights are more in the heparin obtained by separation and purification of the expanded bed counter-current chromatography, so that the result is consistent with the Fxa/FIIa value.

TABLE 12 percentage heparin content at different flow rates

(5) Conclusion

The main quality indexes of heparin are the titer and the molecular weight, the titer of the heparin is determined by the main azure A colorimetric method, the chromogenic substrate method and the sheep plasma method, and the components of the heparin are preliminarily analyzed by HPLC-ELSD, and the specific results are as follows:

(1) the mark is 140IU/mg heparin, the titer is 118.42 plus or minus 3.06IU/mg by using an azure A colorimetric method, the titer of a heparin crude product by a sheep plasma method is 134.17 plus or minus 4.12IU/mg, the sheep plasma method is more accurate, the titer of the heparin separated and purified by column chromatography is 205.51 plus or minus 7.90IU/mg, and the titer of the heparin separated and purified by an expanded bed counter current chromatography is 216.09 plus or minus 11.89IU/mg, which all reach the standard of 180IU/mg specified by pharmacopoeia.

(2) The anti-FXa of the crude heparin product is 173.48IU/mg, the titer of the anti-FIIa is 80.32IU/mg, and the Fxa/FIIa is 2.16, after column chromatography separation and purification, the Fxa/FIIa is 1.46-1.90, and after separation and purification by expanded bed countercurrent chromatography at the flow rate of 2mL/min and 5mL/min, the heparin Fxa/FIIa is 4.03 and 3.89 respectively, which shows that the molecular weight of the heparin after column chromatography separation and purification is higher, and the molecular weight of the heparin after separation and purification by expanded bed countercurrent chromatography is lower.

(4) After separation and purification of heparin, HPLC-ELSD detection is carried out, and experimental results show that the heparin is a mixture with different molecular weights, the separated and purified heparin is roughly divided into three components Hp-1, Hp-2 and Hp-3, compared with the heparin separated and purified by column chromatography, the heparin separated and purified by the expanded bed countercurrent chromatography has smaller molecular weight and higher Fxa/FIIa, and the heparin purified by the expanded bed countercurrent chromatography has better heparin purification effect at the flow rate of 2 mL/min.

In conclusion, it can be concluded that:

an expanded bed countercurrent chromatography separation and purification method is constructed by adding solid adsorption medium D204 resin into a separation column of countercurrent chromatography, heparin separated and purified by the method is deeply researched, heparin extraction process parameters are optimized, and compared with heparin extracted by fixed bed column chromatography, the difference of anticoagulant activity of heparin prepared by two different heparin extraction processes is comparatively researched, and the main research result is as follows:

(1) the results of adsorption kinetics research show that the adsorption kinetics of resin adsorbing heparin accords with a quasi-second-level kinetics model, and the intra-particle diffusion has certain influence on the adsorption process; the static saturated adsorption capacity and the resolving power of four resins are obtained from high to low through static adsorption and desorption experiments: d204> D208> D254> D301; d204 at 25 ℃ reaches 110.21mg/g in saturated adsorption capacity, and at 45 ℃ reaches 192.55mg/g in saturated adsorption capacity, which indicates that the temperature rise is favorable for adsorption of the ion exchange resin to heparin; the data of four resins adsorbing heparin are fitted through two models of Langmuir and Freundlich, and the process of four resins adsorbing heparin is well fitted with Langmuir adsorption isothermal curve, and belongs to monomolecular layer adsorption. The D204 resin is screened out to be used as an adsorption medium in subsequent experiments.

(2) By studying the retention state of the resin in the column, the flow velocity direction of the countercurrent chromatography of the expanded bed is determined to be from top to bottom, the rotation velocity direction is clockwise, and the appropriate sample injection flow velocity within the range of the rotation velocity of 200-500rpm is respectively determined, so that the resin is in a stable expanded dispersion state in the separation column. The penetration behavior and the elution curve of the countercurrent chromatography of the expanded bed under different heparin concentrations and flow rates are researched, and the experimental result shows that the dynamic adsorption capacity is 275mg/g under the conditions that the column volume is 30mL, the sample loading concentration is 50mg/mL, the flow rate is 1.5mL/min and the rotating speed is 300rpm and the dynamic adsorption capacity is gradually reduced along with the increase of the rotating speed under the condition of 5.0g D204 resin. The flow rate has a significant effect on the breakthrough point time, reaching the breakthrough point at 176min at a flow rate of 0.5mL/min, gradually advancing the breakthrough point time to 9.5min as the flow rate increases to 10mL/min, and increasing the dynamic adsorption capacity as the flow rate increases. The recovery rate of 200mg heparin is circularly loaded, the recovery rate of a single circulation is 18.11%, the recovery rate reaches 85.75% after 4 circulations, and the experimental result shows that the heparin yield can be obviously improved by 4.7 times by circularly loading. Compared with fixed bed column chromatography, under the same flow rate of 1, 2 and 5mL/min, the two are eluted after penetrating adsorption, and the elution amount of the expanded bed countercurrent chromatography is 1.69, 2.06 and 2.58 times of that of the fixed bed column chromatography respectively. The highest adsorption rate of the expanded bed countercurrent chromatography at 1mL/min reaches 91.66%, and the result shows that the expanded bed countercurrent chromatography effectively improves the adsorption rate of resin to heparin at higher flow rate and reduces the separation time of heparin products.

(3) The heparin crude product with the mark of 140IU/mg has the titer of 118.42 +/-3.06 IU/mg measured by a azure A colorimetric method, the titer of the heparin crude product by a sheep plasma method is 134.17 +/-4.12 IU/mg, the sheep plasma method is more accurate, the titer of the heparin separated and purified by column chromatography is 205.51 +/-7.90 IU/mg, and the titer of the heparin separated and purified by an expanded bed counter-current chromatography is 216.09 +/-11.89 IU/mg, which all reach the standard of 180IU/mg specified by the pharmacopoeia. The anti-FXa of the crude heparin product is 173.48IU/mg, the titer of the anti-FIIa is 80.32IU/mg, and the Fxa/FIIa is 2.16, after column chromatography separation and purification, the Fxa/FIIa is 1.46-1.90, and after separation and purification by expanded bed countercurrent chromatography at the flow rate of 2mL/min and 5mL/min, the heparin Fxa/FIIa is 4.03 and 3.89 respectively, which shows that the molecular weight of the heparin after column chromatography separation and purification is higher, and the molecular weight of the heparin after separation and purification by expanded bed countercurrent chromatography is lower. After separation and purification of heparin, HPLC-ELSD detection is carried out, and experimental results show that the heparin is a mixture with different molecular weights, the obtained heparin is roughly divided into three components Hp-1, Hp-2 and Hp-3, compared with the heparin separated and purified by column chromatography, the heparin separated and purified by the expanded bed countercurrent chromatography has smaller molecular weight, higher Fxa/FIIa and better anticoagulant effect, and is more suitable for being used as an exogenous anticoagulant.

The above description is only exemplary of the present invention and should not be taken as limiting, any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.