CN113072639B - Purification method of high-purity recombinant hirudin - Google Patents
Purification method of high-purity recombinant hirudin Download PDFInfo
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Abstract
The invention relates to the technical field of separation of biological medicines, in particular to a purification method of high-purity recombinant hirudin, which comprises the steps of taking recombinant hirudin fermentation liquor, carrying out pretreatment, separating by adopting hydrophobic chromatography, collecting recombinant hirudin eluent I, then carrying out separation by adopting anion exchange chromatography, collecting recombinant hirudin eluent II, and drying to obtain the high-purity recombinant hirudin, wherein the hydrophobic chromatography takes phenyl bonded silica gel as a stationary phase and salt solution as a mobile phase. The method combines two liquid chromatographs of hydrophobic chromatography and anion exchange chromatography, realizes separation of recombinant hirudin and impurity components through gradient elution, effectively removes endotoxin and host protein in recombinant hirudin fermentation liquor, and prepares the recombinant hirudin with high purity and high specific activity.
Description
Technical Field
The invention relates to the technical field of separation of biological medicines, in particular to a purification method of high-purity recombinant hirudin.
Background
Leech is a traditional Chinese medicine in China, and is originally carried in Shennong Ben Cao Jing, and has the effects of breaking blood, removing blood stasis and treating dysmenorrhea. Hirudin extracted from leech salivary gland is an anticoagulant protein, has strong inhibition effect on thrombin, and can be used for inhibiting accumulation of fibrinogen and blood platelet in injured blood vessel, preventing thrombosis, and treating diseases such as disseminated intravascular coagulation. Compared with the traditional anticoagulants such as heparin, aspirin and the like, the hirudin has the advantages of small dosage, high curative effect, less adverse reaction, high safety and the like, and has good clinical application value.
Natural hirudin is a single-chain cyclic peptide compound consisting of 65-66 amino acid residues, with a molecular weight of about 7000Da, in which the N-terminal end has 3 pairs of disulfide bonds, which can be folded into a dense cyclic peptide structure, the hydrophobic domain of which is complementary to the nonpolar binding site near the catalytic center of thrombin, stabilizing the protein, and the C-terminal end has 6 acidic amino acids, which can form many ionic bonds with positively charged thrombin recognition sites. Because the content of hirudin in the leech is limited, a large amount of hirudin is difficult to extract from the leech and cannot meet the clinical use requirement, the recombinant hirudin becomes the research focus of the domestic and foreign medical world through genetic engineering. Recombinant hirudin is deso at amino acid Tyr residue 63 3- The rest structure and properties are basically the same as those of natural hirudin.
The earliest recombinant hirudin cDNA has been successfully cloned in the 80 th century of 20, and recombinant hirudin has been successfully expressed in E.coli, bacillus subtilis, yeast, eukaryotic cells and the like. The hirudin gene synthesized in the patent CN1420176A is expressed at a high level in yeast engineering bacteria, and the purity of the protein in the fermentation broth is higher. The preparation method of recombinant hirudin provided in the patent application with publication number of CN110684101A can obtain a large amount of secretory thalli and improve the expression of an active product.
However, the current research focus is mainly on the preparation and expression of recombinant hirudin, while further isolation and purification studies are relatively few. The traditional treatment mode of putting the fermentation liquor into a fermentation tank after heating is not suitable for industrial production, and the operability of removing impurities by adopting low-temperature centrifugation is poor, and the protein purity is low and the pigment content is high. The patent application with publication number CN106834395A utilizes the treatment mode of pH regulation and tank heating of fermentation liquor supernatant to raise protein purity of recombinant hirudin, and adopts low-temperature static filtration to remove impurity protein and lipid substances, but the separation process of said method is complex, and its static separation efficiency is too low, and its total activity recovery rate is low. The patent application with publication number of CN104761635A adopts membrane filtration and chromatographic separation technology to separate and purify natural hirudin, but adopts ceramic filter membrane, organic filter membrane and nanofiltration membrane to filter, so that the activity recovery rate of hirudin is low, and the nanofiltration membrane has a concentration effect, but the purity is not improved, the hydrophobicity of hirudin is reduced, and the hirudin is eluted by reverse phase chromatography after nanofiltration, so that the adsorption force of hirudin on the reverse phase chromatographic column is greatly reduced, and the separation effect is seriously affected. In addition, at present, most of reversed phase chromatographic separations adopt organic solvents such as acetonitrile, methanol and the like, so that not only can the molecular structure of protein be destroyed, and the activity is lost, but also the solvent residues can exist, and the damage to human bodies and the environment is caused.
Disclosure of Invention
Aiming at the technical problems, the invention provides a simple, efficient, energy-saving and environment-friendly purification method of recombinant hirudin, which is suitable for large-scale industrial production, and can prepare the recombinant hirudin with high purity, high specific activity and high recovery rate.
The above object of the present invention is achieved by the following technical solutions: a purification method of high-purity recombinant hirudin comprises pretreating recombinant hirudin fermentation broth, separating by hydrophobic chromatography, collecting recombinant hirudin eluent I, separating by anion exchange chromatography, collecting recombinant hirudin eluent II, and drying to obtain high-purity recombinant hirudin, wherein the hydrophobic chromatography uses phenyl bonded silica gel as stationary phase and salt solution as mobile phase; anion exchange chromatography uses Diethylaminoethyl (DEAE) bonded silica gel as the stationary phase and salt solution as the mobile phase.
The invention firstly uses mild hydrophobic chromatography to remove partial impurities while keeping the molecular structure of hirudin protein complete and the activity stable, then uses anion exchange chromatography to remove residual host hetero protein, and uses salt solution as mobile phase to carry out linear gradient elution to obtain high-purity recombinant hirudin.
The protein is easy to be denatured under the influence of physical and chemical factors such as temperature, stirring, pH value, organic solvent and the like in the separation and purification process, so that the recovery rate is low and even inactivated. The hydrophobic chromatography is a relatively mild purification method, the recombinant hirudin can keep the structure and activity of protein molecules stable through hydrophobic chromatography separation, and the activity recovery rate can reach more than 80%. The invention uses the silica gel matrix bonded with phenyl groups as a hydrophobic chromatography stationary phase, and the separation is realized by different acting forces between each component in the recombinant hirudin fermentation liquor and the hydrophobic groups of the stationary phase. The length of alkyl chain on the surface of the carbon octadecyl silica gel filler which is commonly used at present has great influence on adsorption retention and activity recovery of protein and polypeptide substances, the longer the alkyl chain is, the stronger the hydrophobicity of a stationary phase is, the stronger the binding force between the protein and the polypeptide and the stationary phase is, the elution concentration of a mobile phase organic solvent is required to be increased for smoothly eluting hirudin protein, and the organic solvent can cause aggregation of the polypeptide chain, so that protein molecules are irreversibly adsorbed and the biological activity is lost. The phenyl bonding silica gel used in the invention has weaker hydrophobicity than C18 silica gel column, and the recombinant hirudin and impurity components have larger selection difference during hydrophobic separation, better resolution, higher purification specific activity and recovery rate.
Preferably, the pretreatment of the present invention comprises: the pH value of the recombinant hirudin fermentation liquor is adjusted to 2.5-3.5 for acid precipitation, the supernatant is heated to 76-83 ℃ for constant temperature preservation for 5-10 min, and then the ice water bath is used for cooling, so that the crude recombinant hirudin is obtained.
Further preferably, the supernatant after acid precipitation is heated to 80 ℃ and stored at constant temperature for 8-10 min.
The isoelectric point of the recombinant hirudin is 3.9, and the pH value is adjusted to 2.5-3.5, so that the polypeptide can release protons to be negatively charged, at the moment, part of impurities can be removed, and then the heat-labile protein is removed through heat treatment. The activity of the recombinant hirudin protein molecule at 80 ℃ can be maintained for about 10min, so that the heat treatment time is not longer than 10min, and the temperature is required to be reduced by an ice water bath immediately after the heat treatment.
Preferably, the hydrophobic chromatography uses mobile phase A-mobile phase B as mobile phase, wherein mobile phase A is sodium chloride-phosphate buffer solution with pH value of 4.5-5.5, and mobile phase B is phosphate buffer solution with pH value of 4.5-5.5.
Further preferably, the elution procedure for hydrophobic chromatography is: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B.
According to the invention, salt solutions with different concentrations are used as a mixed mobile phase for gradient elution, wherein the mobile phase A contains sodium chloride, so that the salt concentration is higher when the elution is started, and the sodium chloride concentration in the mobile phase is gradually reduced in the elution process. By increasing the salt concentration, the strong interaction force generated between the recombinant hirudin and the surface of the stationary phase medium can be enhanced to be adsorbed, and the salt concentration is reduced along with the gradient elution procedure, so that the desorption of the recombinant hirudin is realized. The impurities with low hydrophobicity such as fermentation medium components, pigments, host proteins and the like flow out preferentially under the elution of high salt concentration, then the recombinant hirudin protein is eluted under the action of the mobile phase with low salt concentration, and part of the impurities with higher hydrophobicity still remain to be eluted by the regeneration liquid.
Further preferably, the concentration of sodium chloride in mobile phase A of the hydrophobic chromatography is 0.08 to 1.5mol/L.
Further preferably, the concentration of phosphate in both mobile phase A and mobile phase B of the hydrophobic chromatography is 30 to 60mmol/L.
Preferably, the sampling flow rate of the crude recombinant hirudin is 70-100 mL/min when the crude recombinant hirudin is subjected to hydrophobic chromatography.
Further preferably, the column is equilibrated with mobile phase a prior to sample introduction and then eluted according to the elution procedure.
Preferably, the elution rate of the mobile phase is 70 to 100mL/min during the hydrophobic chromatography.
Preferably, after the completion of the elution procedure, the column regeneration by hydrophobic chromatography is completed by eluting with a regeneration liquid I.
More preferably, the regeneration liquid I is 70% -90% ethanol.
After the collection of the recombinant hirudin eluent I is completed, the regeneration liquid is used for column regeneration, so that the components with strong adsorption force combined on the surface of the separation medium can be washed off, the impurity residues are avoided to pollute the chromatographic column, and the service life of the chromatographic column is prolonged. And the ethanol for column regeneration is easy to recycle, has little pollution, is energy-saving and environment-friendly, and can further reduce the production cost.
Preferably, the anion exchange chromatography uses mobile phase A-mobile phase B as mobile phase, wherein mobile phase A is Tris-HCl buffer solution with pH value of 6.5-7.5, and mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 6.5-7.5.
Further preferably, the elution procedure for anion exchange chromatography is: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B.
Anion exchange chromatography uses ion exchangers as stationary phases, and charged ions of the same type can freely exchange with each other and compete for binding, such as between proteins or between proteins and other molecules, and ions of the same charge type can competitively bind to stationary phase media with opposite charges. In the present invention, recombinant hirudin with negative charge below isoelectric point pH value has large volume of negative ions capable of being exchanged with negative chloride ion to combine with the stuffing surface of chromatographic column and the chloride ion in the mobile phase to reverse the process. Due to the different charge characteristics of different components, along with the increase of chloride ion concentration in the mobile phase in the gradient elution process of the ion exchange chromatography, the recombinant hirudin protein with weak binding force flows out preferentially, and the impurity protein with strong binding force is eluted later, so that the purpose of separation is achieved. After hydrophobic chromatography separation, the residual host hetero protein and endotoxin components can be removed by combining anion exchange chromatography, so that the specific activity of the recombinant hirudin is further improved, and the recombinant hirudin with high purity is obtained.
Further preferably, the concentration of Tris-HCl in both mobile phase A and mobile phase B of the anion exchange chromatography is 8 to 20mmol/L.
Further preferably, the concentration of NaCl in the mobile phase B of the anion exchange chromatography is 150 to 350mmol/L.
Preferably, the sample injection flow rate of the recombinant hirudin eluent I during anion exchange chromatographic separation is 70-100 mL/min.
Further preferably, the column is equilibrated with mobile phase a prior to sample introduction and then eluted according to the elution procedure.
Preferably, the elution rate of the mobile phase is 70 to 100mL/min during anion exchange chromatography.
Preferably, after the completion of the elution procedure, the column regeneration by anion exchange chromatography is completed by eluting with regeneration liquid II.
More preferably, the regeneration solution II is NaCl-Tris-HCl buffer solution with pH value of 6.5-7.5.
Still more preferably, the concentration of NaCl in the regenerated liquid II is 0.6-1.2 mol/L, and the concentration of Tris-HCl is 8-20 mmol/L.
The hydrophobic chromatography and the anion exchange chromatography of the invention both adopt salt solution as mobile phase, have small influence on protein activity, lower cost compared with common organic solvent mobile phase, have small environmental pollution, avoid the harm of solvent residue to human body and improve the stability and safety of products.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, two liquid chromatographs of hydrophobic chromatography and anion exchange chromatography are combined, separation of recombinant hirudin and impurity components is realized through gradient elution, endotoxin and host protein in recombinant hirudin fermentation liquor are effectively removed, and high-purity high-specific activity recombinant hirudin is prepared;
(2) According to the invention, during hydrophobic chromatographic separation, salt solution is adopted as a mobile phase for gradient elution, the adsorption effect of recombinant hirudin on the surface of a separation medium can be enhanced by adopting higher salt concentration at the beginning, and as the salt concentration is reduced, components with different hydrophobicity flow out successively, so that the primary separation of recombinant hirudin and impurities is realized;
(3) The crude hirudin provided by the invention has negative charges on the surface when the surface is lower than the isoelectric point, can be exchanged with chloride ions in the mobile phase to be fixed on the surface of the filler, and has the advantages that the concentration of the chloride ions is increased in the gradient elution process, the crude hirudin is competitively combined with an ion exchanger, the recombinant hirudin protein with weak binding force is eluted preferentially, the purity and specific activity of the recombinant hirudin protein are further improved, and the residual host impurity proteins and endotoxin are removed;
(4) The method selects reasonable stationary phase and flow dependence for elution separation, has good selectivity, high resolution and high separation speed, reduces the use of organic solvents, avoids the hidden health hazards caused by environmental pollution and solvent residues, and has the advantages that the chromatographic column used for separation does not need to be regenerated through strong acid and strong alkali, the service life is long, and the method is green, safe, environment-friendly and low in cost, and is suitable for large-scale industrial production.
Drawings
FIG. 1 shows an HPLC analysis of recombinant hirudin eluate II in example 1.
Detailed Description
The technical scheme of the invention is further described and illustrated by the following specific examples. Unless otherwise indicated, all materials used in the examples of the present invention are those commonly used in the art, and all methods used in the examples are those commonly used in the art. It should be understood that the specific embodiments described herein are merely to aid in the understanding of the present invention and are not intended to be limiting in any way.
The invention provides a purification method of high-purity recombinant hirudin, which comprises the following steps:
(1) Pretreatment: firstly, regulating the pH value of the recombinant hirudin fermentation liquor to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 76-83 ℃ and preserving for 5-10 min at constant temperature to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) Hydrophobic chromatography separation: taking phenyl bonded silica gel as a stationary phase, taking crude recombinant hirudin to perform sample injection at a flow rate of 70-100 mL/min, performing column balancing by using a mobile phase A with a volume which is 3-6 times of that of the column, and performing linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 70-100 mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 4.5-5.5, the concentration of sodium chloride is 0.08-1.5 mol/L, the concentration of phosphate is 30-60 mmol/L, and the mobile phase B is phosphate buffer solution with the pH value of 4.5-5.5 and the concentration of 30-60 mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent I; eluting the chromatographic column by using a regeneration liquid I with the volume of 3-6 times of the column volume after the elution is finished, and removing the impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 70-90% ethanol; then the mobile phase B with the volume of 2 to 3 times of the column volume is used for cleaning, redundant organic solvent ethanol is removed, and finally the mobile phase A with the volume of 3 to 6 times of the column volume is used for cleaning, thus finishing the regeneration of the phenyl bonded silica gel chromatographic column;
(3) Anion exchange chromatography separation: taking DEAE bonded silica gel as a stationary phase, taking recombinant hirudin eluent I to sample at a flow rate of 70-100 mL/min, carrying out column balancing by using a mobile phase A with a volume which is 3-6 times of that of the column, and then carrying out linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 70-100 mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 6.5-7.5 and concentration of 8-20 mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 6.5-7.5, naCl concentration of 150-350 mmol/L and Tris-HCl concentration of 8-20 mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent II, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column by using a regeneration liquid II with the volume of 3-6 times of the column volume after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with the volume of 3-6 times of the column volume to finish the regeneration of the chromatographic column; wherein the regenerating solution II is NaCl-Tris-HCl buffer solution with pH value of 6.5-7.5, the concentration of NaCl is 0.6-1.2 mol/L, and the concentration of Tris-HCl is 8-20 mmol/L.
The recombinant hirudin fermentation broth used in the invention is fermentation broth expressed by escherichia coli or saccharomycetes fermentation secretion.
The high-purity recombinant hirudin stock solution prepared by the invention has the protein content of more than or equal to 2mg/mL, the biological titer of more than or equal to 36000ATU/mL, the specific activity of more than or equal to 18000ATU/mg, the total activity recovery rate of 70 percent, and the purity of more than or equal to 98 percent detected by SDS-PAGE electrophoresis and HPLC-C18.
Example 1
(1) Pretreatment: firstly, regulating the pH value of recombinant hirudin fermentation liquor secreted and expressed by saccharomycetes to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 80 ℃ and preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) Hydrophobic chromatography separation: taking phenyl bonded silica gel as a chromatographic column (particle diameter of 5 mu m, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm) filler, taking crude recombinant hirudin to sample at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 5.0, the concentration of sodium chloride is 1mol/L, the concentration of phosphate is 50mmol/L, and the mobile phase B is phosphate buffer solution with the pH value of 5.0 and the concentration of 50mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent I; eluting the chromatographic column with 5 times of regeneration liquid I with the volume of 80% ethanol after the elution is finished, and removing the impurity components strongly adsorbed on the surface of the separation medium; then the mobile phase B with the volume of 2 times of the column is used for cleaning, redundant organic solvent ethanol is removed, and finally the mobile phase A with the volume of 5 times of the column is used for cleaning, so that the regeneration of the phenyl bonded silica gel chromatographic column is completed;
(3) Anion exchange chromatography separation: taking DEAE bonded silica gel as a chromatographic column (particle diameter 5 μm, aperture 300 angstrom, diameter 40mm×length 500 mm) filler, taking recombinant hirudin eluent I to sample at a flow rate of 80mL/min, carrying out column balancing by using mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 7.0 and concentration of 10mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent II, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column with 5 times of column volume of regeneration liquid II after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning with 5 times of column volume of mobile phase A to complete the regeneration of the chromatographic column; wherein the regeneration liquid II is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 1mol/L, and the concentration of Tris-HCl is 10mmol/L.
The medium packing of the chromatographic column used in this example is Ningbo borui, vast biological technology limited company. Biological activity determination is carried out on recombinant hirudin eluent I and recombinant hirudin eluent II by adopting a TH substrate chromogenic method respectively, the specific operation method refers to an anticoagulation specific activity determination method of hirudin in patent CN101248998B, the result is shown in table 1, the HPLC purity of the finally obtained high-purity recombinant hirudin is 98.9%, and the absorption spectrum of HPLC analysis is shown in figure 1.
TABLE 1 biological Activity assay of recombinant hirudin samples in example 1
Example 2
(1) Pretreatment: firstly, regulating the pH value of the recombinant hirudin fermentation broth in the embodiment 1 to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 80 ℃ and preserving for 10min at constant temperature to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) Hydrophobic chromatography separation: taking phenyl bonded silica gel as a chromatographic column (particle size of 5 mu m, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm) filler, taking crude recombinant hirudin to sample at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A-mobile phase B (50:50, v/v) with 5 times of column volume, and then carrying out gradient elution at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:50% → 0% mobile phase a,50% → 100% mobile phase B; wherein, the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 5.0, the concentration of sodium chloride is 1mol/L, the concentration of phosphate is 50mmol/L, and the mobile phase B is phosphate buffer solution with the pH value of 5.0 and the concentration of 50mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent I; eluting the chromatographic column with 5 times of column volume of regeneration liquid I, removing the impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 80% ethanol, and then washing with 5 times of column volume of mobile phase A-mobile phase B (50:50, v/v), thereby completing the regeneration of the phenyl bonded silica gel chromatographic column;
(3) Anion exchange chromatography separation: filling a DEAE bonded silica gel chromatographic column (particle diameter of 5 mu m, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm), taking recombinant hirudin eluent I, sampling at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 7.0 and concentration of 10mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent II, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column with 5 times of column volume of regeneration liquid II after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning with 5 times of column volume of mobile phase A to complete the regeneration of the chromatographic column; wherein the regeneration liquid II is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 1mol/L, and the concentration of Tris-HCl is 10mmol/L.
The medium packing of the chromatographic column used in this example was self-produced. Biological activity determination is carried out on recombinant hirudin eluent I and recombinant hirudin eluent II by adopting a TH substrate chromogenic method, the HPLC purity of the finally obtained high-purity recombinant hirudin is 98.3%, and the activity determination results of the steps are shown in Table 2.
TABLE 2 biological Activity assay of recombinant hirudin samples in example 2
Example 3
(1) Pretreatment: firstly, regulating the pH value of the recombinant hirudin fermentation broth in the embodiment 1 to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 80 ℃ and preserving for 10min at constant temperature to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) Hydrophobic chromatography separation: taking phenyl bonded silica gel as a chromatographic column (particle diameter of 5 mu m, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm) filler, taking crude recombinant hirudin to sample at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A with 5 times of column volume, and then carrying out isocratic elution by using a mobile phase B at a flow rate of 80 mL/min; wherein the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 5.0, the concentration of sodium chloride is 1mol/L, and the concentration of phosphate is 50mmol/L; the mobile phase B is phosphate buffer solution with pH value of 5.0 and concentration of 50mmol/L. Collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent I; eluting the chromatographic column with 5 times of column volume of regeneration liquid I after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 80% ethanol, and then, washing with 5 times of column volume of mobile phase A to complete the regeneration of the phenyl bonded silica gel chromatographic column;
(3) Anion exchange chromatography separation: taking DEAE bonded silica gel as a chromatographic column (particle diameter 5 μm, aperture 300 angstrom, diameter 40mm×length 500 mm) filler, taking recombinant hirudin eluent I to sample at a flow rate of 80mL/min, carrying out column balancing by using mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 7.0 and concentration of 10mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent II, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column with 5 times of column volume of regeneration liquid II after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning with 5 times of column volume of mobile phase A to complete the regeneration of the chromatographic column; wherein the regeneration liquid II is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 1mol/L, and the concentration of Tris-HCl is 10mmol/L.
The medium packing of the chromatographic column used in this example was self-produced. Biological activity determination is carried out on recombinant hirudin eluent I and recombinant hirudin eluent II by adopting a TH substrate chromogenic method, the HPLC purity of the finally obtained high-purity recombinant hirudin is 96.6%, and the activity determination results of all steps are shown in Table 3.
TABLE 3 biological Activity assay of recombinant hirudin samples in example 3
Comparative example 1
The comparative example differs from example 1 only in that the chromatographic column for the reversed phase chromatography separation was a C18 column (diameter 40 mm. Times.length 500mm, particle size 5 μm, pore size 300 angstrom) and the separation medium used was a self-produced octadecyl bonded silica gel, and the other operating methods and conditions were the same as in example 1.
Biological activity determination is carried out on recombinant hirudin eluent I and recombinant hirudin eluent II by adopting a TH substrate chromogenic method, the HPLC purity of the finally obtained high-purity recombinant hirudin is 94.8%, and the sample activity determination results are shown in Table 4.
TABLE 4 biological Activity assay of recombinant hirudin samples from comparative example 1
| Detecting items | Protein content | Biological potency | Specific activity | Recovery of Activity |
| Recombinant hirudin eluent I | 1.23mg/ml | 16984IU/ml | 13808IU/mg | 53％ |
| Recombinant hirudin eluent II | 1.71mg/ml | 30562IU/ml | 17873IU/mg | 85％ |
Comparative example 2
(1) Pretreatment: firstly, regulating the pH value of the recombinant hirudin fermentation broth in the embodiment 1 to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 80 ℃ and preserving for 10min at constant temperature to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) Anion exchange chromatography separation: filling a DEAE bonded silica gel bonded chromatographic column (particle diameter of 5 μm, aperture of 300 angstrom, diameter of 40mm×length of 500 mm), taking recombinant hirudin eluent I, sampling at a flow rate of 80mL/min, carrying out column balancing by using mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 7.0 and concentration of 10mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent I, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column with 5 times of the volume of the regeneration liquid I after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning with 5 times of the volume of the mobile phase A to complete the regeneration of the chromatographic column; wherein the regenerating solution I is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 1mol/L, and the concentration of Tris-HCl is 10mmol/L;
(3) Hydrophobic chromatography separation: taking phenyl bonded silica gel as a chromatographic column (particle diameter of 5 mu m, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm) filler, taking crude recombinant hirudin to sample at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 5.0, the concentration of sodium chloride is 1mol/L, the concentration of phosphate is 50mmol/L, and the mobile phase B is phosphate buffer solution with the pH value of 5.0 and the concentration of 50mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent II; eluting the chromatographic column with 5 times of the volume of the regenerated liquid I after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, wherein the regenerated liquid II is 80% ethanol; and then the mixture is washed by a mobile phase B with the volume of 2 times of the column, the redundant organic solvent ethanol is removed, and finally the mixture is washed by a mobile phase A with the volume of 5 times of the column, so that the regeneration of the phenyl bonded silica gel chromatographic column is completed.
The medium packing of the chromatographic column used in the comparative example is self-produced. Biological activity determination is carried out on recombinant hirudin eluent I and recombinant hirudin eluent II by adopting a TH substrate chromogenic method, the HPLC purity of the finally obtained high-purity recombinant hirudin is 95.6%, and the sample activity determination results are shown in Table 5.
TABLE 5 biological Activity assay of recombinant hirudin samples from comparative example 2
| Detecting items | Protein content | Biological potency | Specific activity | Recovery of Activity |
| Recombinant hirudin eluent I | 1.45mg/ml | 13812IU/ml | 9526IU/mg | 92％ |
| Recombinant hirudin eluent II | 2.1mg/ml | 33671IU/ml | 16034IU/mg | 79％ |
Examples 1 and 2 by the method of the invention, the recombinant hirudin obtained has higher purity and specific activity; whereas example 3 did not employ gradient elution during hydrophobic chromatography, resulting in a decrease in specific activity of the product. In comparative example 1, the C18 column is adopted for hydrophobic chromatography separation, the protein is easy to inactivate in the elution process, and the activity recovery rate is low; in contrast, in comparative example 2, the anion exchange separation was performed before the hydrophobic separation, but the specific activity was reduced because the pigment molecules were eluted together with hirudin without significantly decreasing the activity recovery rate.
The above embodiments are not exhaustive of the point values in the parameter ranges of the technical solutions claimed in the present invention and the new technical solutions formed by equivalent substitution of single or multiple technical features in the technical solutions of the embodiments are also all within the scope of the protection claimed in the present invention, and if all the parameters involved in the solutions of the present invention are not specifically described, there is no unique combination of irreplaceable parameters.
The specific embodiments described herein are offered by way of illustration only and are not intended to limit the scope of the invention. The technical scheme similar or similar to the invention can be obtained by adopting the equivalent substitution or equivalent transformation mode by a person skilled in the art, and the technical scheme falls within the protection scope of the invention.
Claims (1)
1. A method for purifying high purity recombinant hirudin, said method comprising the steps of:
s1, pretreatment: firstly, regulating the pH value of recombinant hirudin fermentation liquor secreted and expressed by saccharomycetes to 2.5-3.5 for acid precipitation, then taking supernatant, heating to 80 ℃ and preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
s2 hydrophobic chromatography separation: taking phenyl bonded silica gel as a chromatographic column (particle diameter of 5 μm, aperture of 300 angstrom, diameter of 40mm multiplied by length of 500 mm) filler, taking crude recombinant hirudin to sample at a flow rate of 80mL/min, carrying out column balancing by using a mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using a mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is sodium chloride-phosphate buffer solution with the pH value of 5.0, the concentration of sodium chloride is 1mol/L, the concentration of phosphate is 50mmol/L, and the mobile phase B is phosphate buffer solution with the pH value of 5.0 and the concentration of 50mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and collecting main eluting peak parts when the base line rises for the second time until the absorption peak falls back to the base line, so as to obtain recombinant hirudin eluent I; eluting the chromatographic column with 5 times of regeneration liquid I with the volume of 80% ethanol after the elution is finished, and removing the impurity components strongly adsorbed on the surface of the separation medium; then the mobile phase B with the volume of 2 times of the column is used for cleaning, redundant organic solvent ethanol is removed, and finally the mobile phase A with the volume of 5 times of the column is used for cleaning, so that the regeneration of the phenyl bonded silica gel chromatographic column is completed;
s3 anion exchange chromatography separation: taking DEAE bonded silica gel as a chromatographic column (particle diameter 5 μm, aperture 300 angstrom, diameter 40mm×length 500 mm) filler, taking recombinant hirudin eluent I, sampling at a flow rate of 80mL/min, carrying out column balancing by using mobile phase A with 5 times of column volume, and then carrying out linear gradient elution by using mobile phase A-mobile phase B at a flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120min:100% → 0% mobile phase a,0% → 100% mobile phase B; wherein, the mobile phase A is Tris-HCl buffer solution with pH value of 7.0 and concentration of 10mmol/L, the mobile phase B is NaCl-Tris-HCl buffer solution with pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10mmol/L; collecting signals with an ultraviolet detector, detecting the wavelength of UV254nm, collecting main eluting peak parts when the base line rises to obtain recombinant hirudin eluent II, and finally freeze-drying to obtain high-purity recombinant hirudin; eluting the chromatographic column with 5 times of column volume of regeneration liquid II after the elution is finished, removing the impurity components strongly adsorbed on the surface of the separation medium, and then cleaning with 5 times of column volume of mobile phase A to complete the regeneration of the chromatographic column; wherein the regeneration liquid II is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 1mol/L, and the concentration of Tris-HCl is 10mmol/L.
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