CN113072638B - Method for removing endotoxin in recombinant hirudin protein solution - Google Patents
Method for removing endotoxin in recombinant hirudin protein solution Download PDFInfo
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Abstract
The invention relates to the technical field of separation and purification of biological medicines, in particular to a method for removing endotoxin in a recombinant hirudin protein solution, which adopts anion exchange chromatography for separation and comprises the following steps: sample introduction: sampling the recombinant hirudin protein solution, wherein the sampling amount is 25-40% of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to balance the column; and (3) elution: isocratic elution is carried out by the eluent, and the outflow part is collected to obtain recombinant hirudin eluent; column regeneration: isocratic elution is carried out by using the regeneration liquid, and an outflow part is collected to obtain endotoxin eluent. According to the method, isocratic elution is carried out by increasing the salt concentration in a mobile phase according to the strength of the adsorption action of the recombinant hirudin and the endotoxin with an anion exchanger under a neutral condition, a good removal effect is realized by regulating and controlling the sample injection amount, the higher protein recovery rate is ensured, and the regeneration of a chromatographic column is realized.
Description
Technical Field
The invention relates to the technical field of separation and purification of biological medicines, in particular to a method for removing endotoxin in a recombinant hirudin protein solution.
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 injured blood vessel, preventing thrombosis, and treating disseminated intravascular coagulation. Compared with traditional anticoagulant drugs such as heparin, aspirin and the like, hirudin has the advantages of small dosage, high curative effect, few adverse reactions, 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 stable to proteins, and the C-terminus has 6 acidic amino acidsAcids, which are capable of forming a plurality of ionic bonds with positively charged thrombin recognition sites. 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 position 3- 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, for example, has high-level expression in engineered yeast, and the protein purity in the fermentation broth is high. The patent application with publication No. CN110684101A provides a method for preparing recombinant hirudin, which can obtain a large amount of secreted bacteria and improve the expression of active products.
However, the focus of the present research is mainly on the production and expression of recombinant hirudins, and relatively few further studies on isolation and purification. Recombinant hirudin prepared by expression and fermentation of microorganisms such as bacteria or fungi has impurity components such as host protein, pigment, culture medium and the like, and endotoxin generated by thallus lysis, and the accumulation of the substances on a human body can cause a series of toxic reactions, such as fever, inflammation, even shock and the like. Therefore, the recombinant hirudin prepared by fermentation culture must remove endotoxin in protein solution by a reasonable purification process, so as to avoid harm to human health.
Generally, drugs and biological products have strict limitations on endotoxin, and few methods for removing endotoxin by recombinant hirudin have been studied. The patent application with publication No. CN106834395A utilizes the treatment of supernatant of fermentation liquor by pH regulation and heating in upper tank to raise the protein purity of recombinant hirudin, and adopts low-temperature standing and filtering to remove impurity protein and lipid substances, and the endotoxin belongs to lipopolysaccharide component, and is difficult to effectively remove by using the standing separation method, and said method is complex in separation process, and the recovery rate of the obtained recombinant hirudin protein is low, so that it can not be applicable to industrial production. Therefore, on the basis of the hirudin purification process, the exploration of a method for effectively removing endotoxin still has great practical significance.
Disclosure of Invention
Aiming at the technical problems, the invention provides a simple and efficient method for removing endotoxin in a recombinant hirudin protein solution, which is suitable for large-scale industrial production, and the recombinant hirudin with high protein recovery rate and good safety is prepared.
The above object of the present invention is achieved by the following technical solutions:
a method for removing endotoxin in recombinant hirudin protein solution adopts anion exchange high performance liquid chromatography for separation, and comprises the following steps:
sample introduction: sampling the recombinant hirudin protein solution, wherein the sampling amount is 25-40% of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to balance the column;
and (3) elution: isocratic elution is carried out by the eluent, and the outflow part is collected to obtain recombinant hirudin eluent;
column regeneration: isocratic elution is carried out by using the regeneration liquid, and an outflow part is collected to obtain endotoxin eluent.
Anion exchange chromatography uses an ion exchanger as a separation stationary phase, 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 adsorption force of the recombinant hirudin and the surface of the ion exchanger is weaker, the recombinant hirudin preferentially flows out of the chromatographic column during elution, and the endotoxin component with strong adsorption force with the stationary phase needs to be washed off by the regeneration liquid with stronger elution capacity.
The sample amount in the anion exchange chromatography separation also has a remarkable influence on the separation effect. Because the maximum sample loading amount of the chromatographic column is influenced by factors such as the size of filler particles, the bonding density of the surface of the filler, the size of the chromatographic column and the like, the sample loading amount is often determined according to the saturated adsorption amounts of different chromatographic columns. However, the inventors have found in practice that the total efficiency of the purification recovery and the removal of impurities of recombinant hirudin protein is not high even if the sample is introduced at the maximum loading. The sample amount of the present invention is far less than the maximum sample loading amount of the chromatographic column, because the sample amount is continuously increased, the endotoxin removing effect is greatly reduced although the recovery rate of the recombinant hirudin protein is not reduced. If the sample amount is less than 25% of the maximum sample loading amount, the endotoxin removing effect is obviously improved, but the recovery rate of the recombinant hirudin protein is obviously reduced. Therefore, the invention realizes the effect of removing the endotoxin in the recombinant hirudin protein solution and the optimal hirudin recovery rate by reasonably regulating and controlling the sample amount.
More preferably, the anion exchange chromatography of the present invention is carried out in an amount of 30% of the maximum loading of the column.
Preferably, anion exchange chromatography in the present invention uses Diethylaminoethyl (DEAE) bonded silica as the stationary phase.
Preferably, the equilibration solution, the eluent and the regeneration solution are all buffer solutions with pH values of 6.5-7.5.
Preferably, the eluent and the regeneration liquid of the invention are both Tris-HCl buffer solution containing NaCl, and the concentration of chloride ions in the eluent is less than that in the regeneration liquid.
Endotoxin is a negatively charged lipopolysaccharide substance, and has strong binding force with anion exchanger at pH > 2. Under the separation condition of the invention which is close to neutrality, the adsorption strength of the endotoxin on the ion exchange chromatographic packing is far larger than that of the recombinant hirudin. Therefore, the recombinant hirudin preferentially flows out of the chromatographic column under the action of the eluent with lower salt concentration, and then is eluted by the regeneration liquid with high salt concentration for desorption, so that the endotoxin component with strong adsorption in the chromatographic column flows out along with the regeneration liquid, and the effective separation of the recombinant hirudin and the endotoxin impurities is realized.
More preferably, in the eluent of the invention, the concentration of NaCl is 100-150 mmol/L, and the concentration of Tris-HCl is 8-20 mmol/L.
More preferably, in the regeneration liquid of the present invention, the concentration of NaCl is 1.0 to 2.0mol/L, and the concentration of Tris-HCl is 8 to 20mmol/L.
Preferably, the recombinant hirudin protein solution used in the invention is prepared by purifying recombinant hirudin fermentation liquor, and the purity is more than or equal to 95%.
Preferably, the sample injection flow rate of the invention is 70-100 ml/min.
Preferably, the equilibrium solution of the present invention is Tris-HCl buffer solution having a concentration of 8 to 20mmol/L and a pH of 6.5 to 7.5.
Preferably, the sample can be loaded and separated again after the regeneration solution is used for elution and the balance solution is used for cleaning.
The invention uses anion exchanger to do chromatographic separation, increases the salt concentration of mobile phase step by step to do isocratic elution, the recombination hirudin with weak binding force flows out preferentially, and then dissociates endotoxin with high salt concentration, realizes effective separation and completes the regeneration of chromatographic column. The recovery rate of the recombinant hirudin protein prepared by the method can reach more than 95 percent, and the endotoxin content in 100 mu g of recombinant hirudin is less than or equal to 0.25EU/ml.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the anion exchange chromatography is adopted to remove endotoxin in the recombinant hirudin protein solution, a good removal effect is realized by regulating and controlling the sample volume, and meanwhile, a higher protein recovery rate is ensured;
(2) According to the strength of the adsorption action of recombinant hirudin and endotoxin with an anion exchanger under a neutral condition, isocratic elution is carried out by increasing the salt concentration in a mobile phase, the recombinant hirudin with weak adsorption force preferentially flows out, and an endotoxin component with strong acting force is dissociated under the action of a regeneration liquid with high salt concentration;
(3) The method is simple, quick and efficient, ensures the recovery rate of the recombinant hirudin protein while effectively removing endotoxin, realizes the regeneration of the chromatographic column, prolongs the service life of the chromatographic column, is favorable for reducing the cost and is suitable for industrial production.
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 all those commonly used in the art, and the methods used in the examples are all those conventional in the art, unless otherwise specified. It should be understood that the specific embodiments described herein are only for the purpose of facilitating understanding of the present invention, and are not intended to limit the present invention specifically.
The invention provides a method for removing endotoxin in recombinant hirudin protein solution, which adopts anion exchange high performance liquid chromatography for separation and comprises the following steps:
sample introduction: sampling recombinant hirudin protein solution with the purity of more than or equal to 95% at the flow rate of 70-100 ml/min, wherein the sampling amount is 25-40% of the maximum sample loading amount of the chromatographic column, washing with Tris-HCl buffer solution balance liquid with the concentration of 8-20 mmol/L and the pH value of 6.5-7.5 to perform column balance, and the washing volume is 3-6 times of the column volume;
and (3) elution: eluting with Tris-HCl buffer solution containing NaCl and with the pH value of 6.5-7.5 as a mobile phase, wherein the concentration of NaCl is 100-150 mmol/L and the concentration of Tris-HCl is 8-20 mmol/L, and collecting the effluent part to obtain recombinant hirudin eluate;
regeneration: eluting with 3-6 times column volume of regeneration liquid, wherein the regeneration liquid is Tris-HCl buffer solution with pH value of 6.5-7.5 and containing NaCl, the concentration of NaCl is 1.0-2.0 mol/L, and the concentration of Tris-HCl is 8-20 mmol/L, and removing strong endotoxin adsorbed in the chromatographic column; finally, the column is washed by 3 to 6 times of the volume of the equilibrium solution, and the column can be used for sample loading and separation again.
The recombinant hirudin protein solution is obtained by purifying recombinant hirudin fermentation liquor which is obtained by fermenting, secreting and expressing escherichia coli or saccharomycetes, and the purification process is a conventional chromatographic separation method and is not described again.
After the endotoxin is removed by the method, the protein recovery rate of the recombinant hirudin is more than or equal to 95 percent, and the content of the endotoxin in 100 mu g of the recombinant hirudin is less than or equal to 0.25EU/ml by the detection of a limulus reagent gel method.
Example 1
This example provides a method for removing endotoxin from recombinant hirudin protein solution by anion exchange high performance liquid chromatography, which uses Diethylaminoethyl (DEAE) bonded silica gel matrix as the packing of chromatographic column (particle size 5 μm, pore diameter 300 angstrom, diameter 40mm × length 500 mm), and comprises the following steps:
sample introduction: sampling recombinant hirudin protein solution with the purity of 95% at the flow rate of 80ml/min, wherein the sampling amount is 30% of the maximum sample loading amount of the chromatographic column, and washing with Tris-HCl buffer solution equilibrium liquid with the concentration of 10mmol/L and the pH value of 7.0 to perform column balance, wherein the washing volume is 5 times of the column volume;
and (3) elution: eluting with Tris-HCl buffer solution containing NaCl and having pH of 7.0 as mobile phase, wherein the concentration of NaCl is 120mmol/L and the concentration of Tris-HCl is 10mmol/L, and collecting the eluate to obtain recombinant hirudin eluate;
regeneration: eluting with 5 times column volume of regeneration solution, wherein the regeneration solution is Tris-HCl buffer solution with pH of 7.0 and containing NaCl at concentration of 1.5mol/L and Tris-HCl at concentration of 10mmol/L, and removing endotoxin strongly adsorbed in the chromatographic column; finally, 5 times of column volume of the equilibrium solution is used for washing, and the column can be used for sample loading and separation again.
The medium filler and the recombinant hirudin protein solution of the chromatographic column used in this example are produced by Ningbo vas Vandard Biotech Limited.
Example 2
This example provides a method for removing endotoxin from recombinant hirudin protein solution by anion exchange high performance liquid chromatography, which uses DEAE bonded silica gel matrix as the packing of chromatographic column (particle size 5 μm, pore size 300 angstrom, diameter 40mm × length 500 mm), and comprises the following steps:
sample introduction: sampling recombinant hirudin protein solution with the purity of 95% at the flow rate of 80ml/min, wherein the sampling amount is 30% of the maximum sample loading amount of the chromatographic column, and washing with Tris-HCl buffer solution equilibrium solution with the concentration of 10mmol/L and the pH value of 7.0 to perform column equilibrium, wherein the washing volume is 5 times of the column volume;
and (3) elution: eluting with Tris-HCl buffer solution containing NaCl and with pH of 7.0 as mobile phase, wherein the concentration of NaCl is 200mmol/L and the concentration of Tris-HCl is 10mmol/L, and collecting the eluate to obtain recombinant hirudin eluate;
regeneration: eluting with 5 times column volume of regeneration solution, wherein the regeneration solution is Tris-HCl buffer solution with pH of 7.0 and containing NaCl at concentration of 2.0mol/L and 10mmol/L, and removing strong endotoxin in chromatographic column; finally, 5 times of column volume of the equilibrium solution is used for washing, and the column can be used for sample loading and separation again.
Example 3
The difference between the sample injection amount and the sample loading amount of the chromatographic column in the embodiment 1 is only 25%, and the rest operation methods and the process conditions are the same as those in the embodiment 1.
Example 4
The difference between the embodiment and the embodiment 1 is only that the sample injection amount is 40% of the maximum sample loading amount of the chromatographic column, and the rest operation method and the process conditions are the same as the embodiment 1.
Comparative example 1
Comparative example 1 differs from example 1 only in that the sample introduction amount is 10% of the maximum sample loading amount of the column, and the remaining operation method and process conditions are the same as those of example 1.
Comparative example 2
Comparative example 2 differs from example 1 only in that the sample loading is 20% of the maximum loading of the column, and the remaining operating method and process conditions are the same as in example 1.
Comparative example 3
Comparative example 3 is different from example 1 only in that the sample introduction amount is 50% of the maximum sample loading amount of the column, and the rest of the operation method and the process conditions are the same as those of example 1.
The recovery (%) of the recombinant hirudin protein was calculated according to the Lowry method of section 4 of the Chinese pharmacopoeia 2020 edition. The endotoxin content in recombinant hirudin was determined by the gel method in the bacterial endotoxin test method of general rule 1143 of section 4 of the edition of Chinese pharmacopoeia 2020. The results are shown in Table 1.
TABLE 1 recovery of recombinant hirudin protein and detection of endotoxin in examples 1 to 4 and comparative examples 1 to 3
According to the results of the above examples and comparative examples, it can be seen that when the amount of the sample is less than 25% of the maximum loading amount of the column, the protein recovery rate of the recombinant hirudin is lower as the amount of the sample is smaller, and when the amount of the sample exceeds 40% of the maximum loading amount of the column, the residual amount of endotoxin in the recombinant hirudin is further increased as the amount of the sample is increased, and therefore, when the sample is injected in the range of 25 to 40% of the maximum loading amount, the endotoxin removing effect can be achieved while ensuring a high protein recovery rate. On the other hand, in comparative example 1 and example 2, it is understood that increasing the concentration of NaCl in the eluate affects the effect of removing endotoxin, and the endotoxin content in the eluate is increased.
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 (4)
1. A method for removing endotoxin in recombinant hirudin protein solution is characterized in that anion exchange chromatography is adopted for separation, and the method comprises the following steps:
sample introduction: injecting a recombinant hirudin protein solution into a sample, wherein the sample injection amount is 25 to 40 percent of the maximum sample loading amount of the chromatographic column, and flushing with a balancing solution to carry out column balance;
and (3) elution: isocratic elution is carried out by the eluent, and the outflow part is collected to obtain recombinant hirudin eluent;
column regeneration: isocratic elution is carried out by using the regeneration liquid, and an outflow part is collected to obtain endotoxin eluent;
the balance liquid, the eluent and the regeneration liquid are all buffer solutions with pH values of 6.5 to 7.5;
the eluent and the regeneration liquid are both Tris-HCl buffer solution containing NaCl, and the concentration of chloride ions in the eluent is less than that of chloride ions in the regeneration liquid;
the anion exchange chromatography uses a diethylaminoethyl bonded silica gel matrix as a chromatographic column, and the matrix is as follows: particle size 5 μm, pore size 300 angstroms, diameter 40mm x length 500mm;
in the eluent, the concentration of NaCl is 100 to 150mmol/L, and the concentration of Tris-HCl is 8 to 20 mmol/L;
in the regeneration liquid, the concentration of NaCl is 1.0 to 2.0mol/L, and the concentration of Tris-HCl is 8 to 20mmol/L.
2. The method for removing endotoxin from a recombinant hirudin protein solution according to claim 1, wherein the equilibrium solution is Tris-HCl buffer with a concentration of 8 to 20mmol/L and a pH of 6.5 to 7.5.
3. The method for removing endotoxin from recombinant hirudin protein solution according to claim 1, wherein the recombinant hirudin protein solution is obtained by purifying recombinant hirudin fermentation broth with a purity of 90% or more.
4. The method for removing endotoxin from a recombinant hirudin protein solution according to claim 1, wherein the recombinant hirudin eluate contains 100 μ g of recombinant hirudin with an endotoxin content of 0.25EU/ml or less.
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| CN103509105B (en) * | 2012-06-29 | 2015-09-23 | 郑州英诺生物科技有限公司 | The production technique of high active hirudin is gone out based on anion-exchange column high efficiency separation |
| CN104926937A (en) * | 2015-06-19 | 2015-09-23 | 广西复鑫益生物科技有限公司平南分公司 | Method for extracting hirudin from leech saliva |
| CN106366200B (en) * | 2015-07-23 | 2020-04-10 | 武汉光谷人福生物医药有限公司 | Process for preparing recombinant glucokinase-hirudin fusion protein |
| CN107353338B (en) * | 2017-07-27 | 2020-11-17 | 宁波博睿修存生物科技有限公司 | Method for separating pigment molecules in hirudin fermentation liquor |
| CN108395475B (en) * | 2018-03-29 | 2021-09-10 | 苏州至汇生物科技有限公司 | Hirudin separation and purification method based on affinity chromatography |
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