CN113072639A - 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 biomedicine, in particular to a method for purifying high-purity recombinant hirudin. The method combines two liquid chromatographs of hydrophobic chromatography and anion exchange chromatography, realizes the separation of recombinant hirudin and impurity components by 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 biomedicine, in particular to a method for purifying high-purity recombinant hirudin.
Background
Leeches are traditional Chinese medicines in China, originally recorded in Shen nong Ben Cao Jing, and have the effects of breaking blood, removing blood stasis and treating dysmenorrhea. Hirudin extracted from salivary gland of Hirudo is an anticoagulant protein, has strong inhibitory effect on thrombin, and can be used for inhibiting accumulation of fibrinogen and platelet in damaged blood vessel, preventing thrombosis, and treating disseminated intravascular coagulation. Compared with traditional anticoagulant drugs such as heparin, aspirin and the like, the hirudin has the advantages of small dosage, high curative effect, little 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 and having a molecular weight of about 7000Da, wherein the N-terminus has 3 pairs of disulfide bonds and 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 and is protein-stabilizing, and the C-terminus has 6 acidic amino acids capable of forming a plurality of ionic bonds with the positively charged thrombin recognition site. Because the content of hirudin in leeches is limited, a large amount of hirudin is difficult to extract from leeches and cannot meet the clinical use requirements, so that the recombination of hirudin through genetic engineering becomes the key research point in the medical field at home and abroad. The SO of the recombinant hirudin is removed from the amino acid Tyr residue at the 63 th position3-The rest structure and property are basically the same as natural hirudin.
The first recombinant hirudin cDNA was successfully cloned in 80 years of the 20 th century, and recombinant hirudin has been successfully expressed in Escherichia coli, Bacillus subtilis, yeast and eukaryotic cells. The hirudin gene synthesized in patent CN1420176A is expressed in yeast engineering bacteria at high level, and the protein purity in the fermentation liquid is higher. The patent application with publication No. CN110684101A provides a method for preparing recombinant hirudin, which can obtain a large amount of secreted thallus and improve the expression of active products.
However, the focus of the current research is mainly on the production and expression of recombinant hirudins, whereas further isolation and purification studies are relatively rare. The traditional treatment method of heating the fermentation liquor and then putting the fermentation liquor into a fermentation tank is not suitable for industrial production, and the low-temperature centrifugation is adopted to remove impurities, so that the operability is poor, the protein purity is low, and the pigment content is high. The patent application with publication No. CN106834395A utilizes the treatment of supernatant of fermentation liquid by pH adjustment and tank-loading heating to improve the protein purity of recombinant hirudin, and adopts low-temperature standing filtration to remove impurity protein and lipid substances, but the method has complicated separation process, too low efficiency of standing separation, and low total activity recovery rate. The patent application with the publication number of CN104761635A adopts a membrane filtration and chromatographic separation technology to separate and purify natural hirudin, but adopts a ceramic filter membrane, an organic filter membrane and a nanofiltration membrane to filter respectively, which not only results in low active recovery rate of the hirudin, but also has the function of concentration by the nanofiltration membrane, but also does not improve the purity, reduces the hydrophobicity of the hirudin, and is eluted by a reversed-phase chromatography after nanofiltration, thereby greatly reducing the adsorption force of the hirudin on the reversed-phase chromatography column and seriously influencing the separation effect. In addition, at present, organic solvents such as acetonitrile, methanol and the like are mostly adopted for reverse phase chromatographic separation, so that not only can the molecular structure of protein be damaged to cause activity loss, but also solvent residues can exist to cause harm to human bodies and the environment.
Disclosure of Invention
Aiming at the technical problems, the invention provides a simple, high-efficiency, energy-saving and environment-friendly method for purifying recombinant hirudin, which is suitable for large-scale industrial production, and the recombinant hirudin with high purity, high specific activity and high recovery rate can be prepared.
The above object of the present invention is achieved by the following technical solutions: a method for purifying high-purity recombinant hirudin comprises the steps of pretreating recombinant hirudin fermentation liquor, separating by using a hydrophobic chromatography, collecting recombinant hirudin eluent I, separating by using an anion exchange chromatography, collecting recombinant hirudin eluent II, and drying to obtain the high-purity recombinant hirudin, wherein the hydrophobic chromatography uses phenyl bonded silica gel as a stationary phase and salt solution as a mobile phase; anion exchange chromatography uses Diethylaminoethyl (DEAE) bonded silica gel as stationary phase and salt solution as mobile phase.
The invention firstly uses mild hydrophobic chromatography to remove partial impurities while keeping the molecular structure and activity of the hirudin protein complete, then removes residual host hybrid protein by anion exchange chromatography, and respectively uses salt solution as mobile phase to carry out linear gradient elution to obtain the high-purity recombinant hirudin.
The protein is easy to denature 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 the protein is inactivated. The hydrophobic chromatography is a relatively mild purification method, the recombinant hirudin can keep the structure and the activity of protein molecules stable through hydrophobic chromatography separation, and the activity recovery rate can reach more than 80%. The invention takes silica gel matrix bonded with phenyl group as the hydrophobic chromatographic stationary phase, and the components in the recombinant hirudin fermentation liquid are separated from the hydrophobic group of the stationary phase by different acting forces. The alkyl chain length of the surface of the currently commonly used carbon octadecyl silica gel filler has great influence on the adsorption retention and activity recovery of protein and polypeptide substances, the longer the alkyl chain is, the stronger the hydrophobicity of the stationary phase is, and the stronger the binding force between the protein and the polypeptide and the stationary phase is, so that the elution concentration of a mobile phase organic solvent is required to be increased for successfully eluting the hirudin protein, and the organic solvent can cause polypeptide chain aggregation, so that the irreversible adsorption of protein molecules and the loss of biological activity are caused. The hydrophobicity of the phenyl bonded silica gel used in the invention is weaker than that of the C18 silica gel column, the selection difference of the recombinant hirudin and the impurity components is larger during hydrophobic separation, the resolution is better, and the specific purification activity and the recovery rate are higher.
Preferably, the pretreatment of the present invention comprises: firstly, adjusting the pH value of recombinant hirudin fermentation liquor to 2.5-3.5, carrying out acid precipitation, then taking supernatant, heating to 76-83 ℃, preserving at constant temperature for 5-10 min, and then cooling by using ice water bath to obtain crude recombinant hirudin.
Preferably, the supernatant after acid precipitation is heated to 80 ℃ and preserved for 8-10 min at constant temperature.
The isoelectric point of the recombinant hirudin is 3.9, the polypeptide can release protons to be negatively charged by adjusting the pH value to 2.5-3.5, partial impurities can be removed at the moment, and heat-labile proteins can be removed by heat treatment. The activity of the recombinant hirudin protein molecule can be maintained for about 10min at 80 ℃, so that the heat treatment time does not exceed 10min, and the temperature is immediately reduced by using an ice water bath after the heat treatment.
Preferably, the hydrophobic chromatography takes a mobile phase A-a mobile phase B as a mobile phase, wherein the mobile phase A is a sodium chloride-phosphate buffer solution with the pH value of 4.5-5.5, and the mobile phase B is a phosphate buffer solution with the pH value of 4.5-5.5.
Further preferably, the elution procedure of the hydrophobic chromatography is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B.
The invention uses salt solutions with different concentrations as mixed mobile phases to carry out 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, stronger interaction force generated between the recombinant hirudin and the surface of a stationary phase medium can be enhanced to be adsorbed, and the salt concentration is reduced along with a gradient elution procedure, so that desorption of the recombinant hirudin is realized. Impurities with low hydrophobicity such as fermentation medium components, pigments, host proteins and the like preferentially flow out under the condition of high-salt concentration elution, then recombinant hirudin protein is eluted under the action of a low-salt concentration mobile phase, and part of impurities with higher hydrophobicity still remain and need to be eluted through a regeneration liquid.
More preferably, the concentration of sodium chloride in the mobile phase A of the hydrophobic chromatography is 0.08-1.5 mol/L.
More preferably, the concentration of phosphate in the mobile phase A and the mobile phase B of the hydrophobic chromatography is 30-60 mmol/L.
Preferably, the sampling flow rate of the crude recombinant hirudin for hydrophobic chromatographic separation is 70-100 mL/min.
Further preferably, after sample injection, the column equilibrium is carried out by using the mobile phase A, and then elution is carried out according to an elution program.
Preferably, the elution rate of the mobile phase in the hydrophobic chromatography is 70 to 100 mL/min.
Preferably, after the elution procedure is completed, elution is performed with the regeneration liquid I to complete the column regeneration of the hydrophobic chromatography.
More preferably, the regeneration liquid I is 70% to 90% ethanol.
After the recombinant hirudin eluent I is collected, the regeneration liquid is used for column regeneration, so that components with strong adsorption force combined on the surface of the separation medium can be washed away, impurity residues are prevented from polluting the chromatographic column, and the service life of the chromatographic column is prolonged. And the ethanol used 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 takes a mobile phase A-a mobile phase B as a mobile phase, wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 6.5-7.5, and the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 6.5-7.5.
Further preferably, the elution procedure for anion exchange chromatography is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B.
Anion exchange chromatography uses an ion exchanger as a stationary phase, and charged ions of the same type can freely exchange with each other and compete for binding, for example, between proteins or between a protein and other molecules, ions of the same charge type can competitively bind to a stationary phase medium with opposite charge. In the invention, the recombinant hirudin with the pH value lower than the isoelectric point has negative charges, the anions with large volume can be exchanged with the chloride ions with the negative charges to be combined with the surface of the chromatographic column packing, and then the process is reversed by the chloride ions in the mobile phase. Because the charge characteristics of different components are different, in the gradient elution process of the exchange ion chromatography, along with the increase of the concentration of chloride ions in a mobile phase, recombinant hirudin protein with weak binding force preferentially flows out, and impurity protein with strong binding force is eluted later, so that the separation purpose is achieved. After hydrophobic chromatographic separation, anion exchange chromatography is combined, so that residual host impurity protein and endotoxin components can be removed, the specific activity of the recombinant hirudin is further improved, and the high-purity recombinant hirudin is obtained.
More preferably, the concentration of Tris-HCl in the mobile phase A and the mobile phase B of the anion exchange chromatography is 8-20 mmol/L.
More preferably, the concentration of NaCl in the mobile phase B of the anion exchange chromatography is 150 to 350 mmol/L.
Preferably, the flow rate of the sample injection when the recombinant hirudin eluent I is subjected to anion exchange chromatography is 70-100 mL/min.
Further preferably, after sample injection, the column equilibrium is carried out by using the mobile phase A, and then elution is carried out according to an elution program.
Preferably, the elution rate of the mobile phase in anion exchange chromatography is 70 to 100 mL/min.
Preferably, after completion of the elution procedure, elution is performed with the regeneration liquid II to complete column regeneration for anion exchange chromatography.
Preferably, the regeneration liquid II is NaCl-Tris-HCl buffer solution with the pH value of 6.5-7.5.
Still more preferably, the concentration of NaCl in the regeneration 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 both adopt salt solution as mobile phase, have small influence on the activity of protein, have lower cost compared with the common organic solvent mobile phase, have small pollution to the environment, avoid the harm of solvent residue to human body and improve the stability and safety of the product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method combines two liquid chromatographs of hydrophobic chromatography and anion exchange chromatography, realizes the separation of recombinant hirudin and impurity components by 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;
(2) the invention adopts salt solution as mobile phase to carry out gradient elution during hydrophobic chromatographic separation, the higher salt concentration can enhance the adsorption effect of the recombinant hirudin and the surface of a separation medium at the beginning, and components with different hydrophobicity flow out successively along with the reduction of the salt concentration, thereby realizing the preliminary separation of the recombinant hirudin and impurities;
(3) the crude hirudin has negative charges on the surface when the surface is lower than the isoelectric point, can exchange with chloride ions in a mobile phase and is fixed on the surface of a filler, and the concentration of the chloride ions is increased in the gradient elution process, and is competitively combined with an ion exchanger, so that recombinant hirudin protein with weak binding force is preferentially eluted, the purity and the specific activity of the recombinant hirudin protein are further improved, and residual host foreign protein and endotoxin are removed;
(4) the method selects reasonable stationary phase and mobile phase to carry out elution separation in sequence, has good selectivity, high resolution and high separation speed, reduces the use of organic solvent, avoids the health hidden trouble caused by environmental pollution and solvent residue, ensures that a chromatographic column used for separation does not need to be regenerated by strong acid and strong alkali, has long service life, is green, safe, environment-friendly and low in cost, and is suitable for large-scale industrial production.
Drawings
FIG. 1 is a HPLC analysis chart of recombinant hirudin eluate II of example 1.
Detailed Description
The technical solution of the present invention is further described and illustrated by the following specific examples. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified. It should be understood that the specific embodiments described herein are merely to aid in the understanding of the invention and are not intended to limit the invention specifically.
The invention provides a purification method of high-purity recombinant hirudin, which comprises the following steps:
(1) pretreatment: firstly, adjusting the pH value of recombinant hirudin fermentation liquor to 2.5-3.5, carrying out acid precipitation, taking supernatant, heating to 76-83 ℃, preserving at constant temperature for 5-10 min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) hydrophobic chromatographic separation: taking phenyl bonded silica gel as a stationary phase, sampling crude recombinant hirudin at a flow rate of 70-100 mL/min, performing column balance by using a mobile phase A with the column volume of 3-6 times, and performing linear gradient elution by using a mobile phase A-a mobile phase B at a flow rate of 70-100 mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a 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 a 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 when the baseline rises for the second time, collecting the main elution peak part until the absorption peak falls back to the baseline 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 after the elution is finished, and removing impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 70-90% of ethanol; cleaning with a mobile phase B with the volume 2-3 times of that of the column, removing redundant organic solvent ethanol, and finally cleaning with a mobile phase A with the volume 3-6 times of that of the column to complete the regeneration of the phenyl bonded silica gel chromatographic column;
(3) anion exchange chromatography separation: taking DEAE bonded silica gel as a stationary phase, sampling recombinant hirudin eluent I at a flow rate of 70-100 mL/min, performing column balance by using a mobile phase A with the volume of 3-6 times of the column volume, and performing linear gradient elution by using the mobile phase A-mobile phase B at a flow rate of 70-100 mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 6.5-7.5 and the concentration of 8-20 mmol/L, the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 6.5-7.5, the NaCl concentration is 150-350 mmol/L, and the Tris-HCl concentration is 8-20 mmol/L; collecting signal with ultraviolet detector with detection wavelength of UV254nm, collecting main elution peak part when baseline rises to obtain recombinant hirudin eluate II, and freeze drying to obtain high-purity recombinant hirudin; after elution is finished, eluting the chromatographic column by using a regeneration liquid II with the column volume of 3-6 times, removing impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with the column volume of 3-6 times to complete regeneration of the chromatographic column; wherein the regeneration liquid II is NaCl-Tris-HCl buffer solution with the 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 liquid used in the invention is fermentation liquid secreted and expressed by escherichia coli or yeast.
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 potency 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, adjusting the pH value of recombinant hirudin fermentation liquor secreted and expressed by saccharomycetes to 2.5-3.5, carrying out acid precipitation, heating supernatant to 80 ℃, preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) hydrophobic chromatographic separation: taking phenyl bonded silica gel as a chromatographic column (the particle diameter is 5 mu m, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm) filler, taking crude recombinant hirudin to inject at the flow rate of 80mL/min, firstly using a mobile phase A with 5 times of the column volume to carry out column equilibrium, and then using the mobile phase A-mobile phase B to carry out linear gradient elution at the flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a 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 a phosphate buffer solution with the pH value of 5.0 and the concentration of 50 mmol/L; collecting signals by using an ultraviolet detector, wherein the detection wavelength is UV254nm, and when the baseline rises for the second time, collecting the main elution peak part until the absorption peak falls back to the baseline to obtain recombinant hirudin eluent I; eluting the chromatographic column by using a regeneration liquid I with 5 times of the column volume after the elution is finished, and removing impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 80% ethanol; then cleaning with a mobile phase B with 2 times of column volume to remove redundant organic solvent ethanol, and finally cleaning with a mobile phase A with 5 times of column volume to complete the regeneration of the phenyl bonded silica gel chromatographic column;
(3) anion exchange chromatography separation: using DEAE bonded silica gel as a chromatographic column (the particle diameter is 5 μm, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm) filler, taking recombinant hirudin eluent I to inject at the flow rate of 80mL/min, firstly using a mobile phase A with 5 times of the column volume to carry out column equilibrium, and then using the mobile phase A-mobile phase B to carry out linear gradient elution at the flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 7.0 and the concentration of 10mmol/L, the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10 mmol/L; collecting signal with ultraviolet detector with detection wavelength of UV254nm, collecting main elution peak part when baseline rises to obtain recombinant hirudin eluate II, and freeze drying to obtain high-purity recombinant hirudin; after the elution is finished, eluting the chromatographic column by using a regeneration liquid II with 5 times of the column volume, removing impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with 5 times of the column volume 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 10 mmol/L.
The medium packing of the chromatographic column used in the present example is produced by Ningbo Rui Va biological technology Limited. Biological activity of the recombinant hirudin eluate I and the recombinant hirudin eluate II is measured by TH substrate chromogenic method, the specific operation method refers to the specific anticoagulation activity measurement 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 FIG. 1.
TABLE 1 results of determination of biological Activity of recombinant hirudin samples in example 1
Example 2
(1) Pretreatment: firstly, adjusting the pH value of the recombinant hirudin fermentation liquor in the embodiment 1 to 2.5 to 3.5, carrying out acid precipitation, then taking the supernatant, heating to 80 ℃, preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) hydrophobic chromatographic separation: taking phenyl bonded silica gel as a chromatographic column (the particle diameter is 5 mu m, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm) filler, taking crude recombinant hirudin, injecting sample at the flow rate of 80mL/min, firstly using a mobile phase A-mobile phase B (50:50, v/v) with 5 times of the column volume to carry out column equilibrium, and then carrying out gradient elution at the flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120 min: 50% → 0% mobile phase a, 50% → 100% mobile phase B; wherein the mobile phase A is a 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 a phosphate buffer solution with the pH value of 5.0 and the concentration of 50 mmol/L; collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and when the baseline rises for the second time, collecting the main elution peak part until the absorption peak falls back to the baseline to obtain recombinant hirudin eluent I; eluting the chromatographic column with 5 times of regeneration liquid I to remove impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 80% ethanol, and then cleaning with 5 times of mobile phase A-mobile phase B (50:50, v/v) to complete the regeneration of the phenyl bonded silica gel chromatographic column;
(3) anion exchange chromatography separation: using DEAE bonded silica gel chromatographic column (particle diameter 5 μm, pore diameter 300 angstrom, diameter 40mm x length 500mm) as filler, taking recombinant hirudin eluent I to inject sample at the flow rate of 80mL/min, firstly using mobile phase A with 5 times of column volume to perform column equilibrium, then using mobile phase A-mobile phase B to perform linear gradient elution at the flow rate of 80mL/min, the elution procedure is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 7.0 and the concentration of 10mmol/L, the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10 mmol/L; collecting signal with ultraviolet detector with detection wavelength of UV254nm, collecting main elution peak part when baseline rises to obtain recombinant hirudin eluate II, and freeze drying to obtain high-purity recombinant hirudin; after the elution is finished, eluting the chromatographic column by using a regeneration liquid II with 5 times of the column volume, removing impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with 5 times of the column volume 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 10 mmol/L.
The medium packing of the chromatographic column used in the present example was produced by itself. And respectively carrying out biological activity determination on the recombinant hirudin eluent I and the recombinant hirudin eluent II by adopting a TH substrate chromogenic method, wherein the HPLC purity of the finally obtained high-purity recombinant hirudin is 98.3%, and the activity determination results of each step are shown in Table 2.
TABLE 2 results of determination of biological Activity of recombinant hirudin samples in example 2
Example 3
(1) Pretreatment: firstly, adjusting the pH value of the recombinant hirudin fermentation liquor in the embodiment 1 to 2.5 to 3.5, carrying out acid precipitation, then taking the supernatant, heating to 80 ℃, preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) hydrophobic chromatographic separation: taking phenyl bonded silica gel as a chromatographic column (with the particle size of 5 mu m, the pore diameter of 300 angstrom, the diameter of 40mm multiplied by the length of 500mm) filler, taking crude recombinant hirudin to inject at the flow rate of 80mL/min, firstly carrying out column balance by using a mobile phase A with the volume of 5 times of the column volume, and then carrying out isocratic elution by using a mobile phase B at the flow rate of 80 mL/min; wherein the mobile phase A is a 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 50 mmol/L; the mobile phase B is phosphate buffer solution with pH value of 5.0 and concentration of 50 mmol/L. Collecting signals by an ultraviolet detector, wherein the detection wavelength is UV254nm, and when the baseline rises for the second time, collecting the main elution peak part until the absorption peak falls back to the baseline to obtain recombinant hirudin eluent I; after the elution is finished, eluting the chromatographic column by using a regeneration liquid I with 5 times of column volume to remove impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid I is 80% ethanol, and then cleaning by using a mobile phase A with 5 times of column volume to complete the regeneration of the phenyl bonded silica gel chromatographic column;
(3) anion exchange chromatography separation: using DEAE bonded silica gel as a chromatographic column (the particle diameter is 5 μm, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm) filler, taking recombinant hirudin eluent I to inject at the flow rate of 80mL/min, firstly using a mobile phase A with 5 times of the column volume to carry out column equilibrium, and then using the mobile phase A-mobile phase B to carry out linear gradient elution at the flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 7.0 and the concentration of 10mmol/L, the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10 mmol/L; collecting signal with ultraviolet detector with detection wavelength of UV254nm, collecting main elution peak part when baseline rises to obtain recombinant hirudin eluate II, and freeze drying to obtain high-purity recombinant hirudin; after the elution is finished, eluting the chromatographic column by using a regeneration liquid II with 5 times of the column volume, removing impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with 5 times of the column volume 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 10 mmol/L.
The medium packing of the chromatographic column used in the present example was produced by itself. And respectively carrying out biological activity determination on the recombinant hirudin eluent I and the recombinant hirudin eluent II by a TH substrate chromogenic method, wherein the HPLC purity of the finally obtained high-purity recombinant hirudin is 96.6%, and the activity determination results of each step are shown in Table 3.
TABLE 3 results of determination of the biological Activity of the recombinant hirudin sample in example 3
Comparative example 1
This comparative example differs from example 1 only in that the column for the reverse phase chromatographic separation was a C18 column (diameter 40 mm. times. length 500mm, particle diameter 5 μm, pore diameter 300. ANG.), the separation medium used was self-produced octadecyl-bonded silica gel, and the other procedures and conditions were the same as in example 1.
And respectively carrying out biological activity determination on the recombinant hirudin eluent I and the recombinant hirudin eluent II by a TH substrate chromogenic method, wherein 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 determination of the biological Activity of the recombinant hirudin sample of comparative example 1
| Detecting items | Protein content | Biological value | Specific activity | Recovery rate 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, adjusting the pH value of the recombinant hirudin fermentation liquor in the embodiment 1 to 2.5 to 3.5, carrying out acid precipitation, then taking the supernatant, heating to 80 ℃, preserving at constant temperature for 10min to obtain crude hirudin, and immediately cooling with ice water to obtain crude recombinant hirudin;
(2) anion exchange chromatography separation: using DEAE bonded silica gel bonded chromatographic column (particle diameter 5 μm, aperture 300 angstrom, diameter 40mm x length 500mm) as filler, taking recombinant hirudin eluent I to inject sample at the flow rate of 80mL/min, firstly using mobile phase A with 5 times of column volume to perform column equilibrium, then using mobile phase A-mobile phase B to perform linear gradient elution at the flow rate of 80mL/min, the elution procedure is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a Tris-HCl buffer solution with the pH value of 7.0 and the concentration of 10mmol/L, the mobile phase B is a NaCl-Tris-HCl buffer solution with the pH value of 7.0, the concentration of NaCl is 300mmol/L, and the concentration of Tris-HCl is 10 mmol/L; collecting signal with ultraviolet detector with detection wavelength of UV254nm, collecting main elution peak part when baseline rises to obtain recombinant hirudin eluate I, and freeze drying to obtain high-purity recombinant hirudin; after the elution is finished, eluting the chromatographic column by using a regeneration liquid I with 5 times of the column volume, removing impurity components strongly adsorbed on the surface of the separation medium, and then cleaning by using a mobile phase A with 5 times of the column volume to complete the regeneration of the chromatographic column; wherein the regeneration liquid 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 10 mmol/L;
(3) hydrophobic chromatographic separation: taking phenyl bonded silica gel as a chromatographic column (the particle diameter is 5 mu m, the pore diameter is 300 angstrom, the diameter is 40mm multiplied by the length is 500mm) filler, taking crude recombinant hirudin to inject at the flow rate of 80mL/min, firstly using a mobile phase A with 5 times of the column volume to carry out column equilibrium, and then using the mobile phase A-mobile phase B to carry out linear gradient elution at the flow rate of 80mL/min, wherein the elution procedure is as follows: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B; wherein the mobile phase A is a 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 a phosphate buffer solution with the pH value of 5.0 and the concentration of 50 mmol/L; collecting signals by using an ultraviolet detector, wherein the detection wavelength is UV254nm, and when the baseline rises for the second time, collecting the main elution peak part until the absorption peak falls back to the baseline to obtain recombinant hirudin eluent II; eluting the chromatographic column by using a regeneration liquid I with 5 times of the column volume after the elution is finished, and removing impurity components strongly adsorbed on the surface of the separation medium, wherein the regeneration liquid II is 80% ethanol; then washing with mobile phase B with 2 times of column volume to remove excessive organic solvent ethanol, and finally washing with mobile phase A with 5 times of column volume to complete the regeneration of the phenyl bonded silica gel chromatographic column.
The medium filler of the chromatographic column used in the comparative example is produced by itself. The recombinant hirudin eluate I and the recombinant hirudin eluate II are respectively subjected to biological activity determination by 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 results of recombinant hirudin samples of comparative example 2
| Detecting items | Protein content | Biological value | Specific activity | Recovery rate 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 the recombinant hirudin obtained by the method of the present invention has higher purity and specific activity; in contrast, example 3, which did not use gradient elution during hydrophobic chromatography, resulted in a decrease in specific activity of the product. Comparative example 1 adopts C18 column to carry out hydrophobic chromatographic separation, protein is easy to inactivate in the elution process, and the activity recovery rate is low; in comparative example 2, where anion exchange separation was performed before hydrophobic separation, the recovery of activity was not significantly decreased, but pigment molecules and hirudin were eluted together, thereby decreasing the specific activity.
The above embodiments are not exhaustive of the range of parameters of the claimed technical solutions of the present invention and the new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the claimed technical solutions of the present invention, and if no specific description is given for all the parameters involved in the technical solutions of the present invention, there is no unique combination of the parameters with each other that is not replaceable.
The specific embodiments described herein are merely illustrative of the spirit of the invention and do not limit the scope of the invention. The technical solutions similar or similar to the present invention can be obtained by those skilled in the art through equivalent substitution or equivalent transformation, and all fall within the protection scope of the present invention.
Claims (10)
1. A method for purifying high-purity recombinant hirudin is characterized in that recombinant hirudin fermentation liquor is taken, pretreated, separated by hydrophobic chromatography, and then a recombinant hirudin eluent I is collected and separated by anion exchange chromatography, and a recombinant hirudin eluent II is collected and dried 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 anion exchange chromatography uses diethylaminoethyl bonded silica gel as a stationary phase and a salt solution as a mobile phase.
2. The method for purifying high purity recombinant hirudin according to claim 1, characterized in that the pretreatment comprises: firstly, adjusting the pH value of recombinant hirudin fermentation liquor to 2.5-3.5, carrying out acid precipitation, then taking supernatant, heating to 76-83 ℃, preserving at constant temperature for 5-10 min to obtain crude hirudin, and then cooling with ice water to obtain crude recombinant hirudin.
3. The method for purifying high purity recombinant hirudin according to claim 1, wherein the hydrophobic chromatography uses mobile phase A-mobile phase B as mobile phase, wherein the mobile phase A is sodium chloride-phosphate buffer with pH value of 4.5-5.5, and the mobile phase B is phosphate buffer with pH value of 4.5-5.5.
4. The method for purifying high purity recombinant hirudin according to claim 3, characterized in that the elution procedure of the hydrophobic chromatography is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B.
5. The method for purifying high purity recombinant hirudin according to claim 3, wherein the concentration of sodium chloride in the mobile phase A of the hydrophobic chromatography is 0.08 to 1.5 mol/L.
6. The method for purifying high purity recombinant hirudin according to claim 1, wherein the anion exchange chromatography uses mobile phase A-mobile phase B as mobile phase, wherein the mobile phase A is Tris-HCl buffer with pH 6.5-7.5, and the mobile phase B is sodium chloride-Tris-HCl buffer with pH 6.5-7.5.
7. The method for purifying high purity recombinant hirudin according to claim 6, characterized in that the elution procedure of anion exchange chromatography is: 0-120 min: 100% → 0% mobile phase a, 0% → 100% mobile phase B.
8. The method for purifying high purity recombinant hirudin according to claim 6, wherein the concentration of sodium chloride in the mobile phase B of anion exchange chromatography is 150 to 350 mmol/L.
9. The method for purifying high purity recombinant hirudin according to claim 1, characterized in that after completion of the elution procedure, the columns of the hydrophobic chromatography and the anion exchange chromatography are eluted with regeneration liquid I and regeneration liquid II, respectively, to complete the column regeneration.
10. The method for purifying high-purity recombinant hirudin according to claim 9, wherein the regeneration liquid I is 70% -90% ethanol, and the regeneration liquid II is a sodium chloride-Tris-HCl buffer solution with pH value of 6.5-7.5.
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