CN113121638B

CN113121638B - Method for purifying recombinant protein

Info

Publication number: CN113121638B
Application number: CN202010044088.2A
Authority: CN
Inventors: 朱梅梅; 柳常青; 刘忠
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2022-06-14
Anticipated expiration: 2040-01-15
Also published as: CN113121638A

Abstract

The invention belongs to the field of protein chemistry, and particularly relates to a method for purifying recombinant protein, which comprises the following steps: the fermentation liquor containing the target recombinant protein is clarified and concentrated through at least one tangential flow membrane filtration step, and then is subjected to hydrophobic chromatography, reverse phase chromatography, pyrogen removal, ultrafiltration and bottling to obtain the recombinant leukocyte inhibitory factor and hirudin chimeric protein with the purity of 99.5%.

Description

Method for purifying recombinant protein

Technical Field

The invention belongs to the field of protein chemistry, and particularly relates to a method for purifying recombinant protein.

Background

Recombinant protein drugs are usually expressed by escherichia coli, yeast, mammalian cells and the like, wherein the production of protein drugs by large-scale culture combined with genetic engineering has become the mainstream of the biopharmaceutical field at present. Many protein drugs in clinic need higher dose or long-term medication, and the market demand is large, so the large-scale production of the biological pharmacy has higher requirements on the process quality and must meet strict pharmaceutical standards. Antibody or Fc fusion Protein medicines can reach purity of more than 90% by one-step Protein A affinity chromatography, and other polluting impurities such as exfoliated Protein A, Host Cell Protein (HCP), aggregates (D/A), DNA and the like are mainly removed by ion exchange and other chromatographic methods; impurities such as HCPs, aggregates and DNA of other recombinant protein drugs often require more purification steps to remove.

At present, there are several methods for separating and purifying recombinant proteins: a) one-step method, including one-step gel screening method, one-step nickel ion chelating chromatography, one-step affinity chromatography, etc.; b) a two-step method, which comprises affinity chromatography-anion exchange chromatography, affinity chromatography-cation exchange chromatography, anion exchange chromatography-molecular sieve chromatography, anion exchange chromatography-nickel ion chelate chromatography, reverse chromatography-nickel ion chelate chromatography, hydrophobic chromatography-hydroxyapatite column chromatography, hydrophobic chromatography-cation exchange chromatography, anion exchange chromatography-cation exchange chromatography and the like; c) the multi-step method comprises hydroxyapatite column chromatography-reverse chromatography, heat treatment-ion exchange-ethanol precipitation, heat treatment-activated carbon adsorption-secondary activated carbon adsorption, hydrophobic chromatography-ion exchange chromatography-molecular sieve chromatography, anion exchange chromatography-hydrophobic chromatography-hydroxyapatite column chromatography-cation exchange chromatography, anion chromatography column-aminobenzene boric acid column chromatography or phenyl cross-linked agarose gel chromatography column-ceramic hydroxyapatite chromatography column-cation chromatography column and the like.

The invention CN105418768A discloses a preparation method and application of recombinant chimeric protein, wherein, the recombinant protein is obtained by nickel ion chelation chromatography purification, and the method relates to repeated renaturation operation of protein to influence the activity of the protein; and a large amount of eluent containing nitrogen heterocyclic compounds is used in gradient elution, and the process is complicated.

As early as 1972, Hjelm et al proposed the purification of recombinant proteins by IgG affinity chromatography: cross-linked agarose gel is used as a carrier, cyanogen bromide activation technology is adopted, and IgG is used as a functional group to prepare affinity chromatography column purified recombinant protein. The method is simple to operate, and the purity of the purified recombinant protein can reach 95% by one-step chromatography operation. However, since IgG is a biological macromolecule with a large molecular weight (Mr. 150000), only a small amount of IgG can be coupled to a unit volume of the carrier to reduce steric hindrance, resulting in a low dynamic capacity of the affinity chromatography column, which is difficult to produce on a large scale and is often used for laboratory scale purification; and a large amount of amino groups on the surface of IgG are utilized during coupling of the IgG, which belongs to a multipoint coupling process, inevitably leads to nonuniform spatial orientation of immobilized protein, and increases steric hindrance.

The invention US 5075423A uses affinity chromatography-anion exchange chromatography to purify recombinant protein, and still has the disadvantages of low affinity chromatography loading and large steric hindrance. CN101220082B discloses a method for extracting and purifying recombinant protein, which uses ion exchange-molecular sieve chromatography, and the method involves repeated denaturation and renaturation to affect the activity of recombinant protein. The invention CN103695508B discloses a fermentation and purification process of staphylococcus aureus HI recombinant protein, wherein the purification process uses affinity chromatography-cation exchange chromatography, the elution uses automatic sample loading elution, and the eluent has multiple types and is complex to operate. The invention CN109055416A discloses a preparation method of soluble recombinant protein, affinity chromatography-affinity chromatography is used for purification, the problems of inclusion body protein post-treatment renaturation and unstable activity are avoided, enzyme digestion is still involved, and a large amount of eluent containing nitrogen heterocyclic compounds is used in elution, so that the operability is not strong. The invention WO 2007/035341a1 discloses a method for purifying proteins using filtration, diafiltration and a two-step sequential chromatography procedure, wherein the first step is anion exchange chromatography, hydrophobic interaction chromatography or ceramic hydroxyapatite chromatography and the second step is cation exchange chromatography, which also has the problems of multiple steps and low efficiency. The invention CN103145813A discloses a method for separating and purifying recombinant protein A, which uses heating, two times of activated carbon adsorption and hydrophobic chromatography to purify protein, still avoids using a buffer solution containing a surfactant, and the usage amount of the surfactant directly influences the recovery rate of the protein; the possibility of protein adsorption by activated carbon exists; the activity of the protein was affected using a heat-boil treatment with an overall protein recovery of 53%.

The invention US 5314993 discloses a method for purifying recombinant proteins using three steps of heating, ion exchange chromatography and ethanol precipitation, which is simple in process but not high in product recovery. Hammond et al disclose a method for large-scale purification of recombinant protein A using heat treatment, desalting with a desalting column, DEAE weak anion exchange chromatography, and phenyl hydrophobic interaction chromatography, which is complicated to operate and has a low total recovery rate. CN 105884859A discloses a method for separating and purifying recombinant protein, which uses PEG precipitation method to purify denatured inclusion body, then carries out denaturation and renaturation process, uses hydrophobic chromatography-ion exchange chromatography-molecular sieve chromatography to purify recombinant protein, still relates to the problems of repeated denaturation and renaturation.

CN101970650B and CN106148302A disclose a method for purifying recombinant protein, which comprises the steps of microfiltration clarification, ultrafiltration concentration and liquid exchange, and then sequentially passing through a high-flow-rate quaternary ammonium sepharose anion chromatographic column, an aminophenylboronic acid column chromatography or a high-efficiency phenyl cross-linked sepharose gel chromatographic column, a ceramic hydroxyapatite I type chromatographic column and a high-efficiency sulfonic sepharose cation chromatographic column, and the problems of use of expensive fillers, small treatment capacity, difficulty in realizing large-scale industrial production and the like exist respectively.

CN1517365A discloses a bifunctional chimeric protein with leukocyte inhibiting function and thrombin activity, wherein Q-Sepharose FF (Pharmacia) and hydroxyapatite column chromatography are used for secondary column chromatography during purification, the service life of hydroxyapatite medium is short, large-scale column packing is difficult, the requirement on buffer solution condition is strict, and the bifunctional chimeric protein is not suitable for large-scale production.

In conclusion, the purification method of the recombinant protein of the domestic patent application rarely involves endotoxin removal, and the gel screening method cannot be used for industrial production of the protein due to small treatment amount, low speed and incapability of removing endotoxin. In the purification methods such as affinity chromatography and metal ion chelate chromatography, it is necessary to wash the chromatography medium with a large amount of a buffer solution containing a surfactant (the volume of which is ten to several tens of times the volume of the chromatography medium) in order to remove endotoxin, which is disadvantageous in terms of environmental protection and protein recovery. In addition, chromatographic media such as nickel ion chelating chromatographic media, affinity chromatographic media and the like are generally expensive and have low stability; protein A affinity chromatography can fall off to a certain extent in the repeated use process, Protein A remained in the product can cause adverse immune response reaction, and the complex of the Protein A and the antibody can activate the immune response of Fc-carrying white blood cells and a complement system in vivo to generate oxidation and anaphylactic reaction.

In recombinant protein production, aggregates are often produced during fermentation, purification, and formulation processes due to physical and chemical factors, such as temperature and time of culture, low pH, shear forces, and surface adsorption. The aggregate can not only cause the patient to generate immune response, but also cause the human to generate immune tolerance to the drug, thereby greatly reducing the drug effect of the drug. In addition, aggregates can affect the biological activity, stability, and shelf life of protein drugs. The removal of aggregates is mainly achieved by a chromatographic process, such as ion exchange chromatography, and if the aggregate content is higher than 10%, the removal capacity of single-step IEC or HIC is very limited, and multiple chromatographic steps are often needed to effectively reduce the aggregates, so that the protein yield is reduced and the cost is increased. Gagnon et al propose to remove aggregates by hydroxyapatite chromatography (US Patent 2010/0145029A 1; US Patent 2005/0107594A 1), which is highly capable of removing aggregates to the extent of 60% to less than 0.1%, but the hydroxyapatite medium has a short service life, is difficult to pack on a large scale, and has strict requirements for buffer conditions, and thus is not suitable for use in large-scale production.

In addition, the cell lines used in the gene recombination technique are living organisms, and thus need to be cultured using a complex growth medium, which is generally a mixture containing salts, sugars, amino acids, vitamins, trace elements, peptone, and the like. The isolation of the desired protein from a mixture of compounds that cultures the cells and from the byproducts of the cells themselves to a purity sufficient for use as a human therapeutic remains a difficult challenge. The recombinant protein still has considerable technical difficulty in expression and purification processes, and the degree of meeting the requirements of people is not achieved, so that the development of a novel expression and purification technology and the improvement of the purification efficiency of the inclusion body have important significance.

The invention CN 103379949A relates to the technology of processing a sample by a capture chromatography resin, inactivating viruses in the sample, and processing by at least one deep filter and an ion exchange membrane, and the purification method has the limitations, the embodiment mode is antibody and Fc fusion protein, but the purification method can not be applied to certain chimeric proteins.

Disclosure of Invention

In view of the defects of narrow application range, long process route, complex operation, higher production cost, incapability of realizing large-scale industrial production of recombinant protein and the like of the recombinant protein purification method in the prior art, the application provides the method which has wide application range and short process route and is expected to realize industrial production of the recombinant protein with higher purity.

The technical scheme of the invention is as follows: constructing an escherichia coli expression strain through gene cloning to obtain a recombinant protein engineering strain, fermenting to obtain a recombinant expressed target protein, clarifying or concentrating a fermentation broth through one or more tangential flow membrane filtration steps, performing a chromatography capture step, and further refining to obtain the recombinant protein with high purity.

A method of purifying a recombinant protein comprising the steps of:

after primary purification treatment, a fermentation liquid containing the target recombinant protein is subjected to at least one tangential flow membrane filter in sequence to process a sample to provide a filtered sample containing the target protein, an eluate after filtration is treated by hydrophobic chromatography resin to provide a first eluate containing the target protein, the first eluate is treated by reversed-phase resin to provide a second eluate containing the target protein, and then pyrogen removal and ultrafiltration are carried out to obtain the recombinant target protein with high purity.

A method for purifying recombinant protein, which comprises the following steps:

A. and (3) ultrafiltration: filtering the primary purified sample by a filter provided with a tangential flow membrane to obtain filtrate, namely a filtered sample containing the target protein;

B. hydrophobic chromatography: loading the hydrophobic chromatography packing material into a column for balancing, loading the filtration sample containing the target protein obtained in the step A, washing the chromatography column by using a balance buffer solution until the absorbance of the column reaches a baseline value, eluting the eluent, and collecting the effluent containing the target recombinant protein;

C. reversed phase chromatography: diluting the effluent containing the target recombinant protein obtained in the step B, filtering, loading the effluent onto a balanced reversed-phase chromatographic column, flushing the chromatographic column with a balancing solution to a baseline absorbance, performing gradient elution, and collecting the effluent containing the target recombinant protein;

D. removing pyrogen: c, loading the effluent containing the target recombinant protein obtained in the step C after the weak ion exchange chromatography is carried out on the column, balancing the effluent to a baseline absorbance by using a balancing solution, eluting, and collecting the effluent containing the target recombinant protein;

E. ultrafiltration and bottling: d, the effluent liquid containing the target recombinant protein obtained in the step D is subjected to ultrafiltration concentration by a filter provided with a tangential flow membrane, and the filtrate is collected, so that the target recombinant protein is obtained.

Preferably, the tangential flow filter membrane in the step A is a cellulose membrane or a polyether sulfone membrane, and the molecular weight cutoff is 10-300 KD; the molecular weight cut-off is further preferably 100-200 KD.

Preferably, the filler for hydrophobic chromatography in step B is Phenyl Sepharose or Source.

Further preferably, the filler for hydrophobic chromatography in step B is Phenyl Sepharose Fast Flow, Phenyl Sepharose High Performance, Source 15PHE, Source 15ISO or Source 15 ETH.

Preferably, the eluent in the step B is 0.05-0.5M ammonium sulfate, sodium sulfate, ammonium acetate, sodium chloride or potassium chloride solution.

Further preferably, the eluent in the step B is 0.05-0.5M ammonium sulfate, sodium sulfate or ammonium acetate solution.

More preferably, the buffer solution in step B is 0.1-0.3M ammonium sulfate, sodium sulfate or ammonium acetate solution.

Preferably, the loading limit of the chromatographic column in the step B is 4-14 g of sample per liter of chromatographic resin.

Further preferably, the loading limit of the chromatographic column in the step B is 6-10 g of sample per liter of chromatographic resin.

Preferably, the packing material for reverse phase chromatography in step C is Source RPC.

Further preferably, the filler for reverse phase chromatography in step C is Source 15RPC or Source 30 RPC.

Preferably, the gradient eluent in the step C is composed of a phase A and a phase B respectively, wherein the phase A is a mixed solution of 5-40 mM trisodium citrate, 70-130 mM sodium acetate, sodium chloride or ammonium acetate and 5-10% acetonitrile or isopropanol, and the phase B is a mixed solution of 5-40 mM trisodium citrate, 35-65 mM ammonium sulfate, sodium chloride or ammonium acetate and 50-70% acetonitrile or isopropanol.

Further preferably, the gradient eluent in the step C is composed of a phase A and a phase B respectively, wherein the phase A is a mixed solution of 10-30 mM trisodium citrate, 80-120 mM sodium acetate, sodium chloride or ammonium acetate and 5-10% acetonitrile or isopropanol, and the phase B is a mixed solution of 5-40 mM trisodium citrate, 40-60 mM sodium acetate, sodium chloride or ammonium acetate and 50-70% isopropanol or acetonitrile.

Preferably, the gradient eluent in the step C elutes 10-15 CV from 0-30% B phase to 50-80% B phase.

Preferably, the loading limit of the chromatographic column in the step C is 10-20 g of sample per liter of chromatographic resin.

Further preferably, the loading limit of the chromatographic column in the step C is 8-12 g of sample per liter of chromatographic resin.

Preferably, the filler of the weak ion exchange chromatography described in step D is DEAE.

Further preferably, the filler for weak ion exchange chromatography in step D is DEAE Sephadex A-25, DEAE Sephadex A-50 or CM Sephadex C-25.

Preferably, the eluent in the step D is a mixed solution of 0.05-0.7M sodium chloride and any one of 10-35 mM trisodium citrate, ammonium acetate and sodium dihydrogen phosphate solution.

Further preferably, the eluent in the step D is a mixed solution of 0.1-0.5M sodium chloride and any one of 20-30mM trisodium citrate, ammonium acetate and sodium dihydrogen phosphate solution.

Preferably, the loading limit of the chromatographic column in the step D is 8-22 g of sample per liter of chromatographic resin.

Further preferably, the loading limit of the chromatographic column in the step D is 12-17 g of sample per liter of chromatographic resin.

Preferably, the model of the tangential flow filter membrane in the step E is PLC6C-C or PLCGC-C, and the molecular weight cut-off is 5-50 KD.

Further preferably, the model of the tangential flow filtration membrane in the step E is PLC6C-C or PLCGC-C, and the molecular weight cut-off is 10-30 KD.

In a preferred technical solution, the method comprises the following steps:

A. and (3) ultrafiltration: clarifying and filtering the primary purified sample by a cellulose membrane PLCHK-C100 KD, PLCHK-C200 KD or polyether sulfone membrane BioMax 100KD tangential flow membrane filter, concentrating by 20-30 times, replacing the concentrated protein solution and buffer solution by an ultrafiltration membrane according to the volume ratio of 1: 3-5, combining filtrates to obtain a filtered sample of the recombinant protein, and collecting and storing the filtered sample in an environment at 2-8 ℃;

B. hydrophobic chromatography: loading a filtration sample containing the recombinant protein obtained in the step A on a Phenyl Sepharose High Performance or Phenyl Sepharose Fast Flow packing column at room temperature, balancing the filtration sample with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with the pH of 7.0-8.0, loading the filtration sample containing the recombinant protein obtained in the step A, wherein the loading limit of the chromatography column is 6-10 g of sample per liter of chromatography resin, washing the chromatography column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with the pH of 7.0-8.0 after loading the filtration sample until the effluent detection value is a baseline absorbance value (A280), eluting with 0.1-0.3M ammonium sulfate, sodium sulfate or ammonium acetate buffer solution with the pH of 7.0-8.0, and collecting the effluent containing the recombinant protein;

C. reversed phase chromatography: loading the column with Source 15RPC or Source 30RPC, pre-washing the resin column with purified water, equilibrating with phase A (10-30 mM trisodium citrate, any one of 80-120 mM sodium acetate, sodium chloride or ammonium acetate, and 5-10% acetonitrile or isopropanol solution at pH 6.0-8.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium acetate, sodium chloride or ammonium acetate, and 100% acetonitrile or isopropanol, adjusting pH to 6.0-8.0, loading the filtered effluent onto a reverse phase chromatography column at a target concentration of 10-30 mM trisodium citrate, 80-120 mM sodium acetate, sodium chloride or ammonium acetate, and 5-10% acetonitrile or isopropanol, loading the column at a loading limit of 8-12 g per liter of resin, and loading the resin column with phase A (10 mM trisodium citrate, 100mM sodium acetate, pH 6.0-8.0, 100mM sodium acetate, etc.) to obtain a solution, Washing the chromatographic column with any one of sodium chloride or ammonium acetate and a solution of 10% acetonitrile or isopropanol) until the effluent detection value is a baseline absorbance value (A280), then carrying out gradient elution with a phase A (10-30 mM trisodium citrate with the pH of 6.0-8.0, any one of 80-120 mM sodium acetate, sodium chloride or ammonium acetate and a solution of 10% acetonitrile or isopropanol) and a phase B (10-30 mM trisodium citrate with the pH of 6.0-8.0, any one of 40-60 mM sodium acetate, sodium chloride or ammonium acetate and a solution of 50-70% acetonitrile or isopropanol) and a solution of 0-30% phase B to 50-80% phase B for 10-15 CV), and collecting the effluent containing the recombinant protein;

D. removing pyrogen: loading a column with DEAE Sephadex A-25 or DEAE Sephadex A-50, the column diameter being 10cm and height being 10cm (bed volume 0.78L), pre-washing the column with purified water, equilibrating with 25mM trisodium citrate buffer solution having pH of 6.0-8.0, diluting the effluent obtained in step C with 1M trisodium citrate buffer solution, adjusting pH to 6.0-8.0 to contain a target concentration of 25mM trisodium citrate, filtering and loading onto an ion exchange chromatography column, the loading limit of the ion exchange chromatography column being 12-17 g sample per liter of resin, then washing the column with 25mM trisodium citrate buffer solution having pH of 6.0-8.0, until the effluent detection value is a baseline absorbance value (A280), eluting with a mixed solution of 0.1-0.5M sodium chloride and any one of 20-30mM trisodium citrate, ammonium acetate and sodium dihydrogen phosphate buffer solution having pH of 6.0-8.0, collecting the effluent containing the recombinant protein;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the recombinant protein obtained in the step D by using a PLC 6C-C10 KD, PLCGC-C-20KD or PLCGC-C-30KD tangential flow membrane filter, concentrating by 3-5 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the target recombinant protein.

The recombinant protein is a recombinant protein, a fusion protein, a chimeric protein or an amino acid fragment and the like which are well known in the art.

In a preferred embodiment, the recombinant protein is a chimeric protein of a leukocyte inhibitory factor and hirudin.

Compared with the prior art, the invention has the following outstanding advantages:

1. the method for purifying the recombinant protein disclosed by the invention has a wide application range, and can obtain the chimeric protein of the leukocyte inhibitory factor and the hirudin with the purity of 99.5 percent, wherein the yield is more than 60.6 percent;

2. the tangential flow membrane filter used in the invention can effectively collect target protein, remove host cell debris and partial foreign protein, improve purification efficiency, simplify operation and reduce protein adsorption capacity;

3. the hydrophobic chromatography can effectively remove most of foreign proteins while enriching proteins, the reverse phase chromatography can further remove impurities such as dimers, isomers and the like, and the weak ion exchange chromatography can effectively remove pyrogens such as bacterial endotoxin and the like;

4. the purification method of the present invention is applicable to recombinant proteins, fusion proteins, chimeric proteins, or amino acid fragments, etc. known in the art;

5. the adopted chromatography method is easy to be used under the condition of large-scale production in a linear scale enlargement way, and meets the requirement of industrial production.

Detailed Description

The following examples are further illustrated: the protein may be a recombinant protein, a chimeric protein or an amino acid fragment known in the art, and the technical scheme to be protected in the present invention is not limited to the following examples.

In the embodiment, the detection methods of monomer purity, host cell protein, antithrombin activity, pyrogen, related substances and isomers can be detected according to a method known in the field, wherein the antithrombin activity is determined according to appendix 1-1 of Chinese pharmacopoeia, the purity is determined according to appendix 1-7 of Chinese pharmacopoeia, the content of high molecular protein is determined according to appendix 1-3 of Chinese pharmacopoeia, the content of bacterial endotoxin is determined according to general rule 1143 of the four parts of Chinese pharmacopoeia, and the isomers are determined according to appendix 1-4 of Chinese pharmacopoeia.

Expression of chimeric proteins of leukocyte inhibitory factor and hirudin: according to the Novagen company' pET system operating manual (10 th edition), a leukocyte inhibitory factor and hirudin chimeric protein plasmid is constructed according to the conventional molecular cloning technology, an escherichia coli expression strain is transferred to obtain a leukocyte inhibitory factor and hirudin chimeric protein engineering bacterium, the leukocyte inhibitory factor and hirudin chimeric protein which are recombined and expressed are obtained by fermentation, the OD600 reaches 80 under the fermentation volume of 100L, and the target protein expression rate can reach 35%;

primary purification: the wet cells obtained by fermentation were suspended in 1M trisodium citrate buffer pH7.0, homogenized at 90MPa for three times, centrifuged to collect inclusion bodies, the obtained inclusion bodies were washed and dissolved in the order of buffer A (4M urea, 50mM Tris, 1% Triton, 1M NaCl, pH7.0), buffer B (50M Tris, pH7.0) and buffer C (8M urea, 1% mercaptoethanol), and the obtained inclusion bodies were diluted with buffer D (20mM Tris, 1mM EDTA, pH7.0) to recover the primary purified sample.

Example 1

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (103.4L, 0.3g/L) by a cellulose membrane PLCHK-C100 KD tangential flow membrane filter, concentrating by 25 times, replacing a concentrated protein solution and a buffer solution by using an ultrafiltration membrane according to a volume ratio of 1:4, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, and obtaining the yield of 93.6%;

B. hydrophobic chromatography: loading a filter sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A on a Phenyl Sepharose High Performance column with the diameter of 10cm and the height of 20cm (the volume of a column bed is 1.57L) at room temperature, then balancing the column by using 5mM Tris-HCl with the pH of 7.5 and 300mM ammonium sulfate buffer solution, loading the filter sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A, washing the column by using 5mM Tris-HCl with the pH of 7.5 and 300mM ammonium sulfate buffer solution after loading until the effluent detection value is a baseline absorbance value (A280), then eluting the column by using 0.2M ammonium sulfate buffer solution with the pH of 7.5, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin at 280nm and from 200mAU to 300mAU, wherein the yield is 95.3%;

C. reversed phase chromatography: packing the column with Source 15RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the resin column with purified water and equilibrating with phase A (10 mM trisodium citrate, 100mM sodium acetate and 10% acetonitrile at pH7.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium acetate and 100% acetonitrile or isopropanol, adjusting the pH to 6.0-8.0, loading the effluent onto a reverse phase chromatography column after filtration to a target concentration comprising 10mM trisodium citrate, 100mM sodium acetate and 10% acetonitrile, and washing the column with phase A (10 mM trisodium citrate, 100mM sodium acetate and 10% acetonitrile at pH7.0) to a value of absorbance (A280), followed by phase A (10 mM trisodium citrate, 100mM and 10% acetonitrile at pH7.0) and phase B (10 mM trisodium citrate, 10mM sodium acetate and 10% acetonitrile) at pH7.0, 50mM sodium acetate and 65% acetonitrile), performing gradient elution (10% -60% of phase B elution 15CV), and collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin at 280nm from 100mAU to 200mAU, wherein the yield is 87.8%;

D. removing pyrogen: loading the column with DEAE Sephadex A-25, the column diameter being 10cm and height being 10cm (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer, pH7.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH7.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, then washing the column with 25mM trisodium citrate buffer, pH7.0, until the detected value is the baseline absorbance value (A280), eluting with 0.3M sodium chloride and 25mM trisodium citrate buffer, pH7.0, collecting the effluent containing the chimeric leukocyte inhibitor and the chimeric protein, starting at 280nm from 50mAU to 100mAU, with a yield of 90.1%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLC 6C-C10 KD tangential flow membrane filter, concentrating by 4 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the product, namely the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 97.2%, and the purity detection result of the purified final product is shown in Table 1.

Example 2

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (107.7L, 0.3g/L) by a polyethersulfone membrane BioMax 100KD tangential flow membrane filter, concentrating by 20 times, replacing a concentrated protein solution and a buffer solution by using an ultrafiltration membrane according to a volume ratio of 1:5, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, and obtaining the yield of 92.6%;

B. hydrophobic chromatography: loading a filtered sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A on a Phenyl Sepharose Fast Flow packed column with the diameter of 10cm and the height of 20cm (the volume of a column bed is 1.57L) at room temperature, then balancing the column by using 5mM Tris-HCl with the pH of 7.0 and 300mM ammonium sulfate buffer solution, washing the column by using the 5mM Tris-HCl with the pH of 7.0 and 300mM ammonium sulfate buffer solution after loading until the detected value of an effluent is a baseline absorbance value (A280), then eluting the column by using 0.1M sodium sulfate buffer solution with the pH of 7.0, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin from 200mAU to 300mAU at 280nm to obtain the yield of 94.3%;

C. reversed phase chromatography: using Source 30Q packed column having a diameter of 10cm and a height of 15cm (bed volume of 1.18L), pre-washing the resin column with purified water, and equilibrating with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 5% isopropanol at pH 6.0), before the refining step, diluting the effluent obtained in step B with a solution of 1M trisodium citrate, 3M sodium chloride and 100% isopropanol, adjusting the pH to 6.0-8.0, loading the effluent on a reverse phase chromatography column at a target concentration comprising 30mM trisodium citrate, 80mM sodium chloride and 5% isopropanol, filtering, and washing the column with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 5% isopropanol at pH 6.0) until the effluent has a baseline absorbance value (A280), and then washing the column with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 5% isopropanol at pH 6.0) and pH 6.0 with citric acid, 40mM sodium chloride and 50% isopropanol), performing gradient elution (10% -70% B phase elution 15CV), and collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin at 280nm from 100mAU to 200mAU, wherein the yield is 84.5%;

D. removing pyrogen: loading the column with DEAE Sephadex A-50, the column diameter being 10cm and height being 10cm (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer, pH8.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH8.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, then washing the column with 25mM trisodium citrate buffer, pH8.0, until the effluent is at baseline absorbance (A280), eluting with 0.1M sodium chloride and 20mM ammonium acetate buffer, pH8.0, collecting the effluent containing the chimeric protein leukocytostatic factor and hirudin starting at 50mAU and ending at 280nm and ending at 100mAU, with a yield of 89.8%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLCGC-C-20KD tangential flow membrane filter, concentrating by 3 times, bottling at the temperature of 2-8 ℃, and freezing at the temperature of-80 ℃, wherein the obtained product is the chimeric protein of the leukocyte inhibitory factor and the hirudin, the yield is 96.4%, and the purity detection result of the purified final product is shown in Table 1.

Example 3

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (99.5L, 0.3g/L) by a cellulose membrane PLCHK-C200 KD tangential flow membrane filter, concentrating by 30 times, replacing a concentrated protein solution and a buffer solution by using an ultrafiltration membrane according to the volume ratio of 1:3, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, and obtaining the yield of 93.4%;

B. hydrophobic chromatography: loading a filter sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A on a Phenyl Sepharose High Performance column with the diameter of 10cm and the height of 20cm (the volume of a column bed is 1.57L) at room temperature, then balancing the column by using 5mM Tris-HCl with the pH of 8.0 and 300mM ammonium sulfate buffer solution, loading the filter sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A, washing the column by using 5mM Tris-HCl with the pH of 8.0 and 300mM ammonium sulfate buffer solution after loading until the effluent detection value is a baseline absorbance value (A280), then eluting the column by using 0.3M ammonium acetate buffer solution with the pH of 8.0, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin at 280nm from 200mAU to 300mAU with the yield of 95.0%;

C. reversed phase chromatography: packing the column using Source 15RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the resin column with purified water and equilibrating with phase A (20mM trisodium citrate, 120mM ammonium acetate and 10% acetonitrile at pH8.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M ammonium acetate and 100% acetonitrile, adjusting pH8.0, to target concentrations comprising 20mM trisodium citrate, 120mM ammonium acetate and 10% acetonitrile, loading onto the reverse phase column after filtration, and then washing the column with phase A (a solution of 20mM trisodium citrate, 120mM ammonium acetate and 10% acetonitrile at pH8.0) until the effluent detects a baseline absorbance value (A280), and phase B (20mM trisodium citrate, 120mM ammonium acetate and 10% acetonitrile at pH8.0) and 20mM trisodium citrate, 120mM trisodium acetate and 10% acetonitrile at pH8.0), and then washing the column with phase A (20mM trisodium citrate, 20mM trisodium acetate and 10% acetonitrile at pH 280) and phase B (trisodium citrate at pH8.0), 60mM ammonium acetate and 70% acetonitrile), and collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin at 280nm from 100mAU to 200mAU, wherein the yield is 84.9%;

D. removing pyrogen: loading the column with DEAE Sephadex A-25, the column diameter being 10cm and height being 10cm (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer, pH 6.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH 6.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, then washing the column with 25mM trisodium citrate buffer, pH 6.0, until the effluent is at baseline absorbance (A280), eluting with 0.5M sodium chloride and 30mM sodium dihydrogen phosphate buffer, pH 6.0, collecting the effluent containing the chimeric protein leukocytostatic factor and hirudin starting at 50mAU and ending at 100mAU at 280nm, with a yield of 89.9%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLCGC-C-30KD tangential flow membrane filter, concentrating by 5 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 97.6%, the purity detection result of the purified final product is shown in table 1, and the purity detection result of the purified final product is shown in table 1.

Example 4

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (107.2L, 0.3g/L) by a cellulose membrane PLCHK-C50 KD tangential flow membrane filter, concentrating by 25 times, replacing a concentrated protein solution and a buffer solution by an ultrafiltration membrane according to the volume ratio of 1:2, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at 2-8 ℃, and obtaining the yield of 92.6 percent after the sample is filtered;

B. hydrophobic chromatography: loading a chromatographic column with a Source 15PHE (column bed volume of 1.57L) and a diameter of 10cm and a height of 20cm at room temperature, then balancing the chromatographic column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with pH7.5, loading the filtered sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A, washing the chromatographic column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with pH7.5 after loading until the effluent detection value is a baseline absorbance value (A280), eluting with 0.4M sodium chloride buffer solution with pH7.5, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin from 200mAU to 300mAU at 280nm with a yield of 93.4%;

C. reversed phase chromatography: packing the column with Source 15RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the resin column with purified water and equilibrating with phase A (a solution of 5mM trisodium citrate, 70mM sodium chloride and 10% isopropanol at pH7.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium chloride and 100% acetonitrile or isopropanol, adjusting the pH to 7.0, to a target concentration comprising 5mM trisodium citrate, 70mM sodium chloride and 10% isopropanol, filtering and loading onto a reverse phase chromatography column, then washing the column with phase A (a solution of 5mM trisodium citrate, 70mM sodium chloride and 10% isopropanol at pH7.0) to a baseline absorbance value (A280), and then with phase A (a solution of 5mM trisodium citrate, 70mM sodium chloride and 10% isopropanol at pH7.0) and B (a solution of 5mM trisodium citrate, 70mM sodium chloride and 10% isopropanol) at pH7.0), 35mM sodium chloride and 70% isopropanol) is subjected to gradient elution (30% -70% B phase elution 10CV), and effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin is collected from 100mAU to 200mAU at 280nm, wherein the yield is 83.3%;

D. removing pyrogen: loading the column with CM Sephadex C-25, column diameter 10CM and height 10CM (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer, pH7.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH7.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, washing the column with 25mM trisodium citrate buffer, pH7.0, until the effluent detection value is a baseline absorbance value (A280), eluting with 0.7M sodium chloride and 10mM trisodium citrate buffer, pH7.0, collecting the effluent containing the chimeric protein leukocytostatic factor and hirudin starting at 50mAU and ending at 280nm and ending at 100mAU, yield 88.9%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLC 6C-C5 KD tangential flow membrane filter, concentrating by 5 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the product, namely the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 95.5%, and the purity detection result of the purified final product is shown in Table 1.

Example 5

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (105.8L, 0.3g/L) by a cellulose membrane PLCHK-C10 KD tangential flow membrane filter, concentrating by 15 times, replacing a concentrated protein solution and a buffer solution by using an ultrafiltration membrane according to the volume ratio of 1:6, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, and obtaining the yield of 92.2% after the sample is filtered;

B. hydrophobic chromatography: loading a chromatographic column with the diameter of 10cm and the height of 20cm (the volume of a column bed is 1.57L) by using a Source 15ISO (standard laboratory test) packed column, then balancing the chromatographic column by using 5mM Tris-HCl with the pH value of 7.5 and 300mM ammonium sulfate buffer solution, loading a filtered sample containing the leukocyte inhibitor and the hirudin chimeric protein obtained in the step A, washing the chromatographic column by using 5mM Tris-HCl with the pH value of 7.5 and 300mM ammonium sulfate buffer solution after loading until an effluent detection value is a baseline absorbance value (A280), eluting by using 0.5M ammonium sulfate buffer solution with the pH value of 7.5, and collecting the effluent containing the leukocyte inhibitor and the hirudin chimeric protein from 200mAU to 300mAU at 280nm with the yield of 93.1%;

C. reversed phase chromatography: packing the column using Source 15RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the resin column with purified water and equilibrating with phase A (a solution of 40mM trisodium citrate, 130mM sodium chloride and 5% isopropanol at pH7.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium chloride and 100% isopropanol, adjusting the pH to 7.0, loading onto a reverse phase chromatography column after filtration at a target concentration comprising 40mM trisodium citrate, 130mM sodium chloride and 10% isopropanol, then washing the column with phase A (a solution of 40mM trisodium citrate, 130mM sodium chloride and 5% isopropanol at pH7.0) to a baseline absorbance value (A280), and then phase A (a solution of 40mM trisodium citrate, 130mM sodium chloride and 5% isopropanol at pH7.0) and B (a solution of 40mM trisodium citrate, 130mM sodium chloride and 5% isopropanol at pH7.0), 65mM sodium chloride and 50% isopropanol) is subjected to gradient elution (0-70% B phase elution 15CV), and effluent containing the leukocyte inhibitory factor and the hirudin chimeric protein is collected from 100mAU to 200mAU at 280nm, wherein the yield is 83.7%;

D. removing pyrogen: loading the column with DEAE Sephadex A-25, the column diameter being 10cm and height being 10cm (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer, pH7.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH7.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, washing the column with 25mM trisodium citrate buffer, pH7.0, until the detected value is the baseline absorbance value (A280), eluting with 0.05M sodium chloride and 35mM trisodium citrate buffer, pH7.0, collecting the effluent containing the chimeric leukocyte inhibitor and the chimeric protein, starting from 50mAU to 100mAU at 280nm, with a yield of 87.9%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLC 6C-C40 KD tangential flow membrane filter, concentrating by 2 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the product, namely the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 95.9%, and the purity detection result of the purified final product is shown in Table 1.

Example 6

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (104.5L, 0.3g/L) by a cellulose membrane PLCHK-C300 KD tangential flow membrane filter, concentrating by 35 times, replacing a concentrated protein solution and a buffer solution by an ultrafiltration membrane according to a volume ratio of 1:3, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, wherein the yield is 92.8%;

B. hydrophobic chromatography: loading a chromatographic column with a Source 15ETH (column bed volume of 1.57L) and a chromatographic column diameter of 10cm and a height of 20cm at room temperature, then balancing the chromatographic column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with pH7.5, loading the filtered sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A, washing the chromatographic column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with pH7.5 after loading until the effluent detection value is a baseline absorbance value (A280), eluting with 0.05M ammonium sulfate buffer solution with pH3.5, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin from 200mAU to 300mAU at 280nm with the yield of 93.4%;

C. reversed phase chromatography: packing the column with Source 30RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the column with purified water and equilibrating with phase A (a solution of 10mM trisodium citrate, 100mM ammonium acetate and 10% acetonitrile at pH7.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M ammonium acetate and 100% acetonitrile, adjusting the pH to 7.0, to target concentrations comprising 10mM trisodium citrate, 100mM ammonium acetate and 10% acetonitrile, filtering and loading onto a reverse phase column, washing the column with phase A (a solution of 10mM trisodium citrate, 100mM ammonium acetate and 10% acetonitrile at pH7.0) until the effluent detects a baseline absorbance value (A280), and then washing the column with phase A (a solution of 10mM trisodium citrate, 100mM ammonium acetate and 10% acetonitrile at pH7.0) and phase B (a solution of 10mM trisodium citrate, 100mM ammonium acetate and 10% acetonitrile at pH7.0) with phase A, 50mM ammonium acetate and 50% acetonitrile), and (20% -80% of phase B elution 15CV) collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin at 280nm from 100mAU to 200mAU, wherein the yield is 83.6%;

D. removing pyrogen: loading onto a CM Sephadex C-25 column having a diameter of 10CM and a height of 10CM (bed volume 0.78L) and equilibrated with 25mM trisodium citrate buffer solution having a pH of 7.0, diluting the effluent from step C with 1M trisodium citrate buffer solution, adjusting the pH to 7.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, washing the column with 25mM trisodium citrate buffer solution having a pH of 7.0 until the effluent detection value is a baseline absorbance value (A280), eluting with 0.6M sodium chloride and 15mM trisodium citrate buffer solution having a pH of 7.0, collecting the effluent containing the chimeric protein of leukocyte inhibitory factor and hirudin starting at 50mAU and ending at 100mAU at 280nm, at a yield of 88.1%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLC 6C-C50 KD tangential flow membrane filter, concentrating by 6 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the product, namely the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 96.2%, and the purity detection result of the purified final product is shown in Table 1.

Example 7

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (110.1L, 0.3g/L) by a Hydrosart membrane 100KD tangential flow filter, concentrating by 20 times, replacing a concentrated protein solution and a buffer solution by an ultrafiltration membrane according to a volume ratio of 1:5, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at 2-8 ℃, wherein the yield is 91.4%;

B. hydrophobic chromatography: loading the filtered sample containing the chimeric protein of the leukocyte inhibitor and hirudin obtained in step A after equilibration with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution having a pH of 7.0, loading the column with the filtered sample containing the chimeric protein of the leukocyte inhibitor and hirudin obtained in step A at room temperature using Octyl Sepharose 4 Fast Flow packed column having a diameter of 10cm and a height of 20cm (a bed volume of 1.57L), washing the column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution having a pH of 7.0 after loading until the effluent detection value is a baseline absorbance value (A280), eluting with 0.1M sodium sulfate buffer solution having a pH of 7.0, collecting the effluent containing the chimeric protein of the leukocyte inhibitor and hirudin at 280nm from 200mAU to 300mAU, and obtaining a yield of 90.1%;

C. reversed phase chromatography: packing the column with Source 30RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), pre-washing the column with purified water and equilibrating with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 3% acetonitrile at pH8.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium chloride and 100% acetonitrile, adjusting pH to 8.0, to target concentrations comprising 10-30 mM trisodium citrate, 80 sodium chloride and 3% acetonitrile, filtering and loading onto a reverse phase column, washing the column with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 3% acetonitrile at pH8.0) until the effluent has a detected absorbance value (A280), and then washing the column with phase A (a solution of 30mM trisodium citrate, 80mM sodium chloride and 3% acetonitrile at pH8.0) and phase B (a solution of 30mM trisodium citrate, 80mM sodium chloride and 3% acetonitrile at pH8.0) with phase B (a solution of 30mM trisodium citrate, pH8.0), 40mM sodium chloride and 80% acetonitrile), and (0-80% B phase elution 15CV) collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin from 100mAU to 200mAU at 280nm, wherein the yield is 80.9%;

D. removing pyrogen: loading the column on an ANX Sepharose FF packed column having a column diameter of 10cm and a height of 10cm (bed volume 0.78L), pre-washing the column with purified water and equilibrating with 25mM trisodium citrate buffer at pH8.0, diluting the effluent from step C with 1M trisodium citrate buffer, adjusting pH to 8.0 to a target concentration containing 25mM trisodium citrate, filtering and loading onto an ion exchange chromatography column, washing the column with 25mM trisodium citrate buffer at pH8.0 until the effluent detection value is a baseline absorbance value (A280), eluting with 0.1M sodium chloride and 20mM ammonium acetate buffer at pH8.0, and collecting the yield of the chimeric protein containing leukocytosin and hirudin from 50U to 100U at 280nm, at 85.7%;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLCGC-C-10KD tangential flow membrane filter, concentrating by 3 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the product, namely the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 93.9%, and the purity detection result of the purified final product is shown in Table 1.

Example 8

A. And (3) ultrafiltration: clarifying and filtering a primary purified sample (100.8L, 0.3g/L) by a cellulose triacetate membrane 200KD tangential flow membrane filter, concentrating by 30 times, replacing a concentrated protein solution and a buffer solution by using an ultrafiltration membrane according to the volume ratio of 1:6, combining filtrates to obtain a filtered sample of the leukocyte inhibitory factor and hirudin chimeric protein, collecting and storing the filtered sample in an environment at 2-8 ℃, and cleaning the filtration membrane by using 25mM sodium hydroxide after the sample is filtered, wherein the yield is 91.6%;

B. hydrophobic chromatography: loading a filtered sample containing the chimeric protein of the leukocyte inhibitor and the hirudin obtained in the step A on a Butyl Sepharose 4 Fast Flow packed column with the diameter of 10cm and the height of 20cm (the volume of a column bed is 1.57L) at room temperature, then balancing the column by using 5mM Tris-HCl with the pH of 8.0 and 300mM ammonium sulfate buffer solution, washing the column by using 5mM Tris-HCl with the pH of 8.0 and 300mM ammonium sulfate buffer solution after loading until the detected value of the effluent is a baseline absorbance value (A280), then eluting the column by using 0.3M ammonium acetate buffer solution with the pH of 8.0, and collecting the effluent containing the chimeric protein of the leukocyte inhibitor and the hirudin from 200mAU to 300mAU at 280nm to obtain the yield of 90.9%;

C. reversed phase chromatography: packing the column with Source 30RPC, the column diameter being 10cm and the height being 15cm (bed volume 1.18L), prewashing the column with purified water and equilibrating with phase A (a solution of 20mM ammonium acetate, 120mM sodium chloride and 5% isopropanol at pH8.0), diluting the effluent from step B with a solution of 1M trisodium citrate, 3M sodium chloride and 100% isopropanol, adjusting the pH to 8.0, to target concentrations comprising 20mM trisodium citrate, 120mM sodium chloride and 5% isopropanol, filtering and loading onto a reverse phase column, washing the column with phase A (a solution of 20mM ammonium acetate, 120mM sodium chloride and 5% isopropanol at pH8.0) until the effluent detects a baseline absorbance value (A280), and then phase A (a solution of 20mM ammonium acetate, 120mM sodium chloride and 5% isopropanol at pH8.0) and phase B (a solution of 20mM ammonium acetate, 120mM sodium chloride and 5% isopropanol at pH8.0) are added, 60mM sodium chloride and 70% isopropanol), and (30% -80% B phase elution 10CV) collecting the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin at 280nm from 100mAU to 200mAU, with a yield of 80.7%;

D. removing pyrogen: loading the column with DEAE Sepharose FF having a diameter of 10cm and a height of 10cm (bed volume 0.78L), pre-washing the resin column with purified water and equilibrating with 25mM trisodium citrate buffer solution having a pH of 6.0, diluting the effluent from step C with 1M trisodium citrate buffer solution, adjusting the pH to 6.0 to a target concentration comprising 25mM trisodium citrate, loading onto an ion exchange chromatography column after filtration, washing the column with 25mM trisodium citrate buffer solution having a pH of 6.0 until the effluent detection value is a baseline absorbance value (A280), eluting with 0.5M sodium chloride and 30mM sodium dihydrogen phosphate buffer solution having a pH of 6.0, and collecting the effluent containing the chimeric protein of leukocyte inhibitory factor and hirudin starting from 50mAU to the end of 100mAU at 280 nm;

E. ultrafiltration and bottling: and D, clarifying and filtering the effluent containing the chimeric protein of the leukocyte inhibitory factor and the hirudin obtained in the step D by a cellulose membrane PLCGC-C-20KD tangential flow membrane filter, concentrating by 5 times, bottling at the temperature of 2-8 ℃, and freeze-drying at the temperature of-80 ℃ to obtain the chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the yield is 94.5%, and the purity detection result of the purified final product is shown in Table 1.

Comparative example 1

A. And (3) ultrafiltration: subjecting a sample (110.5L, 0.3g/L) containing the chimeric protein of the leukocyte inhibitory factor and the hirudin which is subjected to primary purification treatment to ultrafiltration membrane with molecular weight cutoff of 5KD, concentrating, desalting, and obtaining a yield of 91.4%;

B. ion exchange chromatography: packing the column with Q Sepharose FF, the column diameter is 10cm and the height is 15cm (the bed volume is 1.18L), pre-washing the resin column with purified water, balancing with 10mM trisodium citrate and 100mM ammonium sulfate buffer solution with pH7.0, and washing the column with 10mM trisodium citrate and 100mM ammonium sulfate buffer solution with pH7.0 until the effluent detected value is a baseline absorbance value (A280), eluting with 25mM Tris (pH8.0) and 0.35M NaCl to obtain an effluent containing the chimeric protein of the leukocyte inhibitory factor and hirudin, and the yield is 74.8%;

C. hydroxyapatite chromatography: using hydroxyapatiteLoading on a stone column, adjusting pH to 7.4, and purifying with 40mM KH₂PO₄(pH7.4)、50mM NaH₂PO₄Eluting, collecting and combining the eluates, wherein the yield is 57.6%;

D. gel filtration chromatography: sephadex G-25 is used for column packing, the obtained product is the chimeric protein of the leukocyte inhibitory factor and the hirudin, the finished product is obtained after freeze drying, the yield is 85.4 percent, and the purity detection result of the purified final product is shown in Table 1.

Comparative example 2

A. Protein a capture chromatography: use of

Treating the primary treated sample (100.6L, 0.3g/L) with Ultra Plus resin and a Quickcase chromatography column, equilibrating with 25mM Tris, 100mM sodium chloride, pH 7.2 and loading the load with clarified harvest, washing the column with equilibration buffer to baseline absorbance (A280), washing the column with 20mM trisodium citrate/citric acid, 0.5M sodium chloride, pH 6.0, eluting the product from the column with 0.1M acetic acid, pH3.5, collecting the eluate starting at 1OD and ending at 1OD at 280nm for a 1cm path length, yield 85.4%;

B. virus inactivation, depth filtration and Q-membrane chromatography: inactivating at room temperature, performing deep filtration, and performing Q membrane treatment to obtain 86.2% yield;

C. hydrophobic chromatography: loading the column with Phenyl Sepharose HP, a column diameter of 10cm and a height of 20cm (bed volume 1.57L), prewashing the column with water and equilibrating with 1.0M ammonium sulfate and 18mM sodium phosphate, pH7.0, diluting the Q membrane effluent with 2.2M ammonium sulfate and 40mM sodium phosphate, pH7.0, prior to the refining step, to a target concentration containing 1.0M ammonium sulfate and 18mM sodium phosphate, washing the column to baseline absorbance (A280) with 1.1M ammonium sulfate and 2mM sodium phosphate, pH7.0 and then 0.95M ammonium sulfate and 17mM sodium phosphate, pH7.0, respectively, eluting with 0.55M ammonium sulfate and 10mM sodium phosphate, pH7.0, and collecting the eluate starting at 5OD and ending at 1cm path length at 280nm, to 1OD, yield 87.3%;

D. and (4) nanofiltration: c, performing nanofiltration on the eluate obtained in the step C, wherein the aperture of a filter membrane is 0.1 mu m, and collecting filtrate with the yield of 94.6%;

E. ultrafiltration and diafiltration: concentrating the filtrate obtained in step D to 70g/L by an ultrafiltration membrane with the molecular weight cut-off of 30KD, then continuously percolating with minimum 8 volumes of 19mM histidine at pH5.6, further concentrating the product to 195g/L after the diafiltration, washing the ultrafiltration with 19mM histidine at pH5.6 buffer solution, and combining the concentrate and the washings, wherein the yield is 94.1%;

F. filtering, bottling and freeze-drying: the filled and labeled bottles were freeze-dried at-80 ℃ at 2-8 ℃ with a 0.22 μm filter into pre-sterilized, pyrogen-free polyethylene terephthalate glycol containers in a yield of 93.3%, and the purity measurements of the purified end products are shown in Table 1.

Comparative example 3

A. And (3) ultrafiltration concentration: taking a sample (108.6L, 0.3g/L) after primary treatment, filtering the sample by an ultrafiltration membrane with the molecular weight cutoff of 3KD, concentrating the sample by 20 times to obtain filtrate, namely a filtered sample of the leukocyte inhibitory factor and the hirudin chimeric protein, collecting and storing the filtered sample in an environment at the temperature of 2-8 ℃, wherein the yield is 88.6%;

and (3) crude purification: loading a Butyl Sepharose F.F. medium into a column, adjusting the pH value of a balanced buffer solution to 25mmol Tris, 0.3M ammonium sulfate and 0.5mol/L NaCl, collecting target proteins passing through peaks, eluting heteroproteins with 25mmol/L Tris pH value of 8.0 to obtain an eluent, adjusting the pH value of the eluent to 3.0-4.0 by hydrochloric acid, precipitating, centrifuging the obtained precipitate for 15min at 4 ℃ and 12000r/min to obtain a precipitate, dissolving the precipitate with 20mmol/LPB pH value of 8.0 buffer solution until the precipitate is clear, and filtering with a 0.45 mu M membrane to obtain a filtrate with the yield of 60.3%;

and (3) fine purification: packing a column by using a Source 30Q medium, adjusting the pH value to 8.0 by using an equilibrium buffer solution A of 20mmol/L PB and 0.2mol/L NaCl, performing linear gradient elution under the elution condition of B (20mmol/L PB, 0.5mol/L NaCl and pH value of 8.0), collecting a target protein peak (about 35% B solution), adjusting the pH value of the obtained eluent to 3.0-4.0 by using phosphoric acid, performing precipitation, centrifuging the obtained precipitate for 15min at 4 ℃ and 12000r/min to obtain a precipitate, dissolving the obtained precipitate by using 10mmol/L PB and pH value 7.4 buffer solutions until the precipitate is clear, and filtering the precipitate by using a 0.45 mu m membrane to obtain a filtrate with the yield of 59.8%;

pyrogen removal and part of high molecular weight proteins: the column is filled with Superdex 75 medium, the balance buffer solution is 10mmol/L PB pH6.5, the eluent is 10mmol/L PB pH6.5, TNHH stock solution is obtained, and the yield is 84.7%.

And (3) freeze drying: the TNHH stock solution obtained above was diluted with 10mmol/L PB (pH7.5) to a TNHH protein concentration of 3.0mg/ml, followed by addition of mannitol 4.0%, followed by lyophilization at low temperature to give a final product in a yield of 91.2%.

TABLE 1 Final product purity assay of recombinant proteins

Claims

1. A method for purifying a recombinant protein, comprising: the fermentation liquor containing the target recombinant protein is processed by the following steps:

B. hydrophobic chromatography: after the hydrophobic chromatography packing is filled into a column for balancing, loading the filtration sample containing the target protein obtained in the step A, washing the chromatography column by using an equilibrium buffer solution until the absorbance of a base line is reached, eluting an eluent, and collecting and combining the effluent liquid containing the target recombinant protein;

C. reversed phase chromatography: diluting the effluent containing the target recombinant protein obtained in the step B, filtering, loading onto a reversed-phase chromatographic column, flushing the chromatographic column with a balancing solution to a baseline absorbance, performing gradient elution, collecting and combining the effluent containing the target recombinant protein;

D. removing pyrogen: c, loading the effluent containing the target recombinant protein obtained in the step C after the weak ion exchange chromatography is carried out on the column, balancing the effluent to a baseline absorbance by using a balance solution, eluting, collecting and combining the effluent containing the target recombinant protein;

E. ultrafiltration and bottling: d, the effluent liquid containing the target recombinant protein obtained in the step D is subjected to ultrafiltration concentration by a filter provided with a tangential flow membrane, and the filtrate is collected to obtain the target recombinant protein;

wherein the eluent in the step B is 0.05-0.5M of ammonium sulfate, sodium sulfate, ammonium acetate, sodium chloride or potassium chloride solution;

the gradient eluent in the step C is composed of a phase A and a phase B, wherein the phase A is a mixed solution of 5-40 mM trisodium citrate, 70-130 mM sodium acetate, sodium chloride or ammonium acetate and 5-10% acetonitrile or isopropanol, and the phase B is a mixed solution of 5-40 mM trisodium citrate, 35-65 mM ammonium sulfate, sodium chloride or ammonium acetate and 50-70% acetonitrile or isopropanol;

the eluent in the step D is a mixed solution of 0.05-0.7M of sodium chloride and any one of 10-35 mM of trisodium citrate, ammonium acetate and sodium dihydrogen phosphate solution;

the tangential flow membrane in the step A is a cellulose membrane or a polyether sulfone membrane; the filler for hydrophobic chromatography in the step B is Phenyl Sepharose or Source; c, the filler of the ion exchange chromatography in the step C is Source RPC; the filler of the weak ion exchange chromatography in the step D is DEAE; the tangential flow membrane in the step E is PLC6C-C or PLCGC-C; the recombinant protein is a chimeric protein of a leukocyte inhibitory factor and hirudin.

2. The method of claim 1, wherein the molecular weight cut-off of the tangential flow membrane in step A is 10-300 KD.

3. The method of claim 1, wherein the filler of the ion exchange chromatography in step C is Source 15RPC or Source 30RPC 1.

4. The method of claim 1, wherein the filler for weak ion exchange chromatography in step D is DEAE Sephadex A-25, DEAE Sephadex A-50 or CM Sephadex C-25.

5. The method of claim 1, wherein the molecular weight cut-off of the tangential flow membrane in step E is 5-50 KD.