CN106977591B - Method for separating and purifying recombinant staphylococcal protein A - Google Patents

Method for separating and purifying recombinant staphylococcal protein A Download PDF

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CN106977591B
CN106977591B CN201710308472.7A CN201710308472A CN106977591B CN 106977591 B CN106977591 B CN 106977591B CN 201710308472 A CN201710308472 A CN 201710308472A CN 106977591 B CN106977591 B CN 106977591B
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张海珍
余波光
李乐军
谢江明
陈校园
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Guangzhou Kangsheng Biotechnology Co Ltd
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Abstract

The invention discloses a method for separating and purifying recombinant staphylococcal protein A, which adopts two fillers with mixed mechanisms, effectively removes a series of impurities such as endotoxin, escherichia coli residual protein (HCP), escherichia coli residual DNA, protein aggregates and the like in a two-step chromatography and ultrafiltration mode, adopts the fewest steps to obtain the purest target sample, saves the process control cost, and has the characteristics of good stability, clear process control index, suitability for large-scale industrial production and the like. The protein purity of the purified protein solution is more than 97 percent by using high performance liquid phase and SDS-PAGE electrophoresis; the protein recovery rate is more than 75 percent; the endotoxin is reduced to below 1 EU/mg; the residual DNA of HCP and colibacillus is less than 5ppm, and can be used as raw material for synthesizing clinical staphylococcal protein A immunoadsorbent.

Description

Method for separating and purifying recombinant staphylococcal protein A
The technical field is as follows:
the invention relates to the technical field of biomedicine, in particular to a method for separating and purifying recombinant staphylococcal protein A.
Background art:
staphylococcal protein A (protein A or SPA for short) is a protein with polypeptide single-chain structure, contains 5 highly similar immunoglobulin Fc segment binding regions, and can be specifically combined with human immunoglobulin (IgG). SPA is coupled to agarose carrier, prepared into carrier-SPA compound and loaded into column to make staphylococcal protein A immunoadsorption column, when human plasma passes through the column, IgG type pathogenic antibody in plasma will be specifically adsorbed, and some diseases and symptoms caused by the change of IgG type antibody quality and quantity can be obviously treated and relieved. The staphylococcus protein A immunoadsorption column is widely applied to the treatment of autoimmune diseases, organ transplantation, malignant tumors and other diseases, and has wide market prospect.
Due to the wide use of staphylococcal protein A, the market demand is increasing. The natural protein is directly extracted from staphylococcus aureus, so that the market demand cannot be met. Therefore, in recent years, the method of genetic engineering has been turned to produce recombinant staphylococcal protein A. Currently, staphylococcal protein A is mainly expressed by genetic engineering bacteria-escherichia coli, and is purified by a chromatography technology to finally obtain a product meeting the requirements. The culture solution of the escherichia coli contains various protein impurities and non-protein impurities such as nucleic acid, lipid and the like, so that the downstream purification is seriously examined. The endotoxin is the most influential factor, which causes adverse effects such as fever and shiver of human body, and the pharmaceutical industry has high requirements for the content of endotoxin in pharmaceutical products, for example, the content of endotoxin in an injection preparation must be ensured to be less than 5EU per hour per kg of body weight. However, the removal of endotoxin from protein systems is still a problem in the industry, and the main reasons are that the protein systems are relatively complex, and strong forces exist between a plurality of proteins and endotoxin, and the proteins and the endotoxin are combined together and are difficult to separate, so that at present, no universal rule for removing endotoxin from the protein systems exists, and related researches are only applicable to a certain protein system. The influence of impurity host protein and host DNA on the organism is large, 100ppm of escherichia coli residual protein (HCP) can cause the immune response of a patient, and the host DNA is always the focus of attention of domestic and foreign drug administration because of the special potential safety risk. When the staphylococcus protein A immunoadsorption column is used for clinical treatment, circulation needs to be established in vitro, and the staphylococcus protein A immunoadsorption column belongs to three types of medical instruments and has high risk. Staphylococcal protein A is one of important raw materials, and the quality of staphylococcal protein A directly influences the safety of the adsorption column. Therefore, the development of a method for purifying staphylococcal protein A, which has simple and convenient process, can be amplified and has higher purity, is particularly important.
There are many reports of the current patents on recombinant staphylococcal protein a purification, such as purification by Ni column affinity chromatography (CN201010614100.5, CN201210096913.9, CN200810028707.8, CN200810028706.3, CN200810028704.4, CN201010110754.4, CN 201010110754.4); purifying by Sephacryl S200 molecular sieve method (CN03149982.1, CN200710085148.X, CN 201010225393.8); the Fc fragment of IgG is used as filler for affinity chromatography of ligand, or combined with ion exchange purification (CN201010227290.5, CN201210118134.4), GSTrapFF column affinity chromatography and Superdex 7510/300GL molecular sieve method (CN 201080013514.0). The first chromatographic column used in CN200680034577.8 is an anion exchange column, a HIC column and a ceramic hydroxyapatite column, and the second chromatographic column is a cation exchange column, or 1, 2 columns use sequential phase exchange. None of these patents mentioned above mentions the content of impurities in the protein solution. However, for the separation and purification of protein A, it is required not only to achieve high purity but also to strictly control the contents of endotoxin, protein impurities, and non-protein impurities such as nucleic acid and lipid. Patent CN103145813B mentions how to remove endotoxin, mainly using twice activated carbon adsorption-hydrophobic chromatography, because activated carbon is powder, the practical operation is not very convenient.
The invention content is as follows:
the invention aims to provide a method for separating and purifying recombinant staphylococcal protein A, which is simple and convenient to operate, effectively removes impurities such as endotoxin, protein aggregates, escherichia coli residual protein (HCP), escherichia coli residual DNA and the like, ensures that the amounts of the escherichia coli residual DNA and the HCP in a purified protein solution are both lower than 5ppm, the endotoxin is lower than 1EU/mg, the protein purity is higher than 97 percent, is suitable for industrial production, and can be used as a raw material for synthesizing a clinical staphylococcal protein A immunoadsorbent.
The term protein A or recombinant staphylococcal protein A in the present invention refers to a recombinant protein obtained by cloning, transforming and expressing in E.coli cells staphylococcal protein A gene which is full-length or contains only one or some of its domains.
The term purification refers to increasing the purity of a polypeptide or protein of interest or a target protein from a composition or sample containing the protein of interest and one or more impurities.
The term immunoglobulin (Ig) refers to a tetrapeptide chain structure made up of two identical light chains and two identical heavy chains linked by interchain disulfide bonds.
The terms "chromatographic packing", "medium", "packing" and "chromatographic medium" are synonymous and refer to materials packed into a chromatographic column for separating a multi-component mixture by virtue of the differences in the physicochemical properties of the components to complete the chromatographic process.
The invention is realized by the following technical scheme:
a method for isolating and purifying recombinant staphylococcal protein a comprising the steps of:
1) pretreating thalli: adding thallus containing staphylococcal protein A into Bis-tris buffer solution, crushing with a homogenizer, centrifuging to collect supernatant, adjusting pH to 2.0-4.0, centrifuging to collect supernatant, and adjusting pH to 6.5-7.2 to obtain protein solution;
2) first chromatographic column treatment: loading the protein solution obtained in the step 1) into a chromatographic column which is balanced by a balancing solution and is filled with a salt-resistant anion exchange filler, continuously balancing by the balancing solution, washing by a washing solution, finally eluting staphylococcal protein A by an eluent, and collecting an elution peak to obtain a first collecting solution; the equilibrium solution contains 10-30mM Bis-tris, 0.5-2 wt% TritonX-100 and 0.1mol/L NaCl, and the pH value is 6.5-7.2; the washing liquid contains 10-30mM Bis-tris, 0.1mol/LNaCl and has pH of 6.5-7.2; the eluent contains 10-30mM Bis-tris, 1mol/L NaCl and has pH of 6.5-7.2;
3) ultrafiltration displacement buffer: replacing the first collected liquid obtained in step 2) with 5KD cut-off molecular weight ultrafiltration membrane to pH 6.8-7.5 containing 5-20mM Na2HPO4-NaH2PO4And 0.1-0.3M proline, thereby obtaining a protein solution;
4) and (3) second chromatographic column treatment: loading the protein solution obtained in the step 3) into a column which is balanced by a balancing solution and is filled with hydroxyapatite chromatographic packing, continuing to balance by the balancing solution, then eluting by an eluent, and collecting an elution peak to obtain a second collecting solution; the equilibrium solution contains 5-20mM Na2HPO4-NaH2PO4And 0.1-0.3M proline, pH 6.8-7.5; the eluent contains 100mM Na2HPO4-NaH2PO4，pH 6.8-7.5；
5) Replacing ultrafiltration with a preservation solution: replacing the second collecting liquid obtained in the step 4) with a normal saline solution by ultrafiltration with an ultrafiltration membrane with the cut-off molecular weight of 5KD, thereby obtaining the separated and purified staphylococcal protein A.
The pretreatment of the cells in step 1) is preferably: adding thallus containing staphylococcal protein A into Bis-tris buffer solution with pH of 7.2 and concentration of 10-30mM according to the mass ratio of 1kg to 10L, crushing with a high-pressure homogenizer under 800bar pressure, and centrifuging to collect supernatant; adjusting pH to 2.0-4.0 with hydrochloric acid, centrifuging, collecting supernatant, and adjusting pH to 6.5-7.2 with NaOH to obtain protein solution. Step 1) crushing thallus by using a high-pressure homogenizer at 800bar pressure instead of a heating and lysozyme method, so that the energy consumption is low, a complex heating and heat-insulating device is not needed, and continuous operation and industrial amplification can be realized.
The salt-resistant anion exchange filler in the step 2) is TOYOPEARL NH2-750F salt-tolerant anion exchange packing of particle size 30-60 μm, endotoxin at pH>3.1 solutions with partial negative charges, bound to TOYOPEARL NH by charge2And (3) loading endotoxin-contaminated protein on anion exchange packing of-750F, removing partial endotoxin by using a balance solution and a washing solution, eluting the target protein by using a gradient elution method, and removing the endotoxin remained on the column by using a high-salt buffer solution and NaOH.
Step 2), preferably, the equilibrium solution contains 20mM Bis-tris and 1% TritonX-100, 0.1mol/LNaCl, pH 7.0; the washing solution contained 20mM Bis-tris, 0.1mol/LNaCl, pH 7.0; the eluate contained 20mM Bis-tris and 1mol/L NaCl, pH 7.0.
Step 4) the hydroxyapatite chromatography packing particle size is 40 μm, CHT type I is preferred. Hydroxyapatite is the most widely used inorganic chromatographic filler at present, has high alkali resistance and good biological safety, and can be separated with high resolution due to the unique separation mechanism.
Step 4), preferably, the equilibration solution contains 5mM Na2HPO4-NaH2PO4And 0.2M preserved fruitAmino acid, pH 7.0; the eluent is 100mM Na2HPO4-NaH2PO4，pH 7.0。
The material of the ultrafiltration membrane in step 3) and step 5) is preferably polyether sulfone (PES).
The invention has the following beneficial effects:
1. according to the invention, by utilizing the fact that the protein A is extremely stable under an acidic condition and can tolerate an extreme condition of lowest pH of 2.0, after the pH of the crushed supernatant is reduced to 2.0-4.0 in the step 1), the residual DNA of escherichia coli, part of foreign proteins and cell debris contained in the crushed supernatant can be coprecipitated, the turbidity of the solution is greatly reduced, so that the protein A is more tightly combined with an anion exchange column, and the protein yield is improved; meanwhile, the residual quantity of residual DNA of the escherichia coli is greatly reduced, and the protein purity is improved; and a method for filtering and removing impurities by using a deep filter is also avoided, and the production cost is greatly reduced.
2. The first step of the purification of the present invention is carried out using TOYOPEARL NH2the-750F salt-resistant anion exchange filler has both hydrophobic effect and ion exchange effect, and is highly salt-resistant, so that the protein solution obtained in the step 1) can be directly loaded without dilution, the purification process is simplified, and the protein yield is increased; meanwhile, the particle size of the filler is 30-60 mu m, and is smaller than the average particle size of 90 mu m of the traditional sepharose, the separation effect is better, more impure proteins can be removed, and the purification pressure of the next step is reduced. In addition, the ion exchange filler can effectively remove endotoxin in the broken supernatant of escherichia coli, and the content of the endotoxin is reduced to be below 100EU/mg from 5000-10000 EU/mg.
3. When the adsorbent packing is synthesized downstream, the adsorbent packing is connected to agarose and cellulose carriers mainly through protein amino. The commonly used ion exchange column uses Tris-HCl buffer, which leaves a small amount of residue in the final protein solution, since Tris (structural formula:
Figure BDA0001286424560000061
) The formula contains amino groups, so trace amounts of tris (mM) also interfere with the synthesis of downstream adsorbents, resulting in reduced adsorption performance. The first chromatographic column adopts Bis-tris (structural formula is shown in the specification)
Figure BDA0001286424560000062
) The buffer solution replaces a commonly used Tris-HCl buffer solution, so that the binding force of the protein A and the chromatographic column can be obviously improved, and the recovery rate of the protein is increased. Meanwhile, the Bis-tris molecular structure does not contain free amino groups and does not compete with amino groups on proteins for binding sites, so that the adsorption filler has no influence on the downstream protein connected to the carrier synthetic protein A.
4. The invention adopts hydroxyapatite as the final fine purification step, the average grain diameter of the hydroxyapatite is 40 mu m, impurities such as dimer, endotoxin, trace escherichia coli residual DNA, HCP and the like can be further removed, and the product purity is improved.
5. Aggregation of proteins during protein purification due to concentration, pH, temperature, etc. exists in the form of a dimer or polymer, which is a major factor affecting protein purity. L-proline is one of eighteen amino acids of human body synthetic protein, and can inhibit protein aggregation and enhance protein stability. The addition of a small amount of proline can promote the protein to be in the native conformation, so that the protein A is better separated from the dimer-like substance, and more protein impurities are removed through fine separation.
In conclusion, the method is simple to operate, adopts 2 types of fillers with mixed mechanisms, effectively removes a series of impurities such as endotoxin, escherichia coli residual protein (HCP), escherichia coli residual DNA, protein aggregates and the like in a two-step chromatography and ultrafiltration mode, adopts the fewest steps to obtain the purest target sample, saves the process control cost, and has the characteristics of good stability, clear process control indexes, suitability for large-scale industrial production and the like. The protein purity of the purified protein solution is more than 97 percent by using high performance liquid phase and SDS-PAGE electrophoresis; the protein recovery rate is more than 75 percent; the endotoxin is reduced to below 1 EU/mg; the residual DNA of the HCP and the Escherichia coli is lower than 5ppm, and the HCP and the residual DNA of the Escherichia coli can be used as raw materials for synthesizing a clinical staphylococcal protein A immunoadsorbent, can realize annual output kilogram-level large-scale production, and simultaneously provides reference for industrial production of other protein purification.
Description of the drawings:
FIG. 1 shows the result of SDS-PAGE gel electrophoresis of recombinant staphylococcal protein A; wherein, the lane 1 is a low molecular weight protein marker, the molecular weights from top to bottom are respectively 98kDa, 66.2kDa, 45kDa, 31kDa, 20kDa and 14.4kDa, and the molecular weight of the recombinant staphylococcal protein A is about 20 kDa; lane 2 is the final protein solution obtained in example 1; lane 3 is the final protein solution obtained in example 2.
FIG. 2 is an HPLC chromatogram of recombinant staphylococcal protein A in example 1.
FIG. 3 is an HPLC chromatogram of recombinant staphylococcal protein A in example 2.
The specific implementation mode is as follows:
the following is a further description of the invention and is not intended to be limiting.
Example 1:
1) pretreating thalli: 1kg of staphylococcus protein A-containing bacterial cells (such as staphylococcus protein A-expressing genetically engineered bacterium Escherichia coli) are added with 10L of 10mM Bis-tris buffer solution with pH7.2 to suspend the bacterial cells, and then the bacterial cells are crushed by a high-pressure homogenizer at 800bar for 2 times, and the supernatant is collected by centrifugation to obtain about 10L. The pH of the resulting solution was adjusted to 2.0 by adding hydrochloric acid, centrifuged, and the supernatant was adjusted to pH6.5 with sodium hydroxide to obtain a protein solution.
2) First chromatographic column treatment: will be charged with TOYOPEARL NH2Column packed with-750F packing was equilibrated to 5 column volumes with an equilibration solution containing 10mM Bis-tris and 0.5 wt% TritonX-100, 0.1mol/LNaCl, pH6.5, and the protein solution obtained in step 1 was loaded into a column containing TOYOPEARL NH equilibrated with the equilibration solution2And (2) in a chromatographic column of-750F salt-tolerant anion exchange filler, continuously balancing 5 times of column volume by using a balance liquid, then washing 5 times of column volume by using a washing liquid containing 10mM Bis-tris, 0.1mol/L NaCl and pH6.5, finally eluting the target protein staphylococcal protein A by using an eluent containing 10mM Bis-tris, 1mol/L NaCl and pH6.5, and collecting an elution peak to obtain a first collection liquid.
3) Ultrafiltration displacement buffer: subjecting the first collected solution obtained in step 2) to equal volume ultrafiltration with PES ultrafiltration membrane having cut-off molecular weight of 5KD, replacing with pH6.85mM Na2HPO4-NaH2PO4And 0.1M proline buffer to obtain protein solution.
4) And (3) second chromatographic column treatment: the column filled with hydroxyapatite chromatographic packing was treated with 5mM Na2HPO4-NaH2PO4And 0.1M proline, pH6.8, adding the protein solution obtained in step 3) to a column containing hydroxyapatite chromatographic packing and equilibrated with the equilibration solution, continuously equilibrating the column with 5 times of column volume with the equilibration solution, and then equilibrating with 100mM Na2HPO4-NaH2PO4Eluting with eluent with pH6.8, and collecting the elution peak to obtain a second collected solution.
5) Replacing ultrafiltration with a preservation solution: and (3) performing equal-volume ultrafiltration on the second collected liquid obtained in the step (4) by using an ultrafiltration membrane with the cut-off molecular weight of 5KD for 5 times of volume, and replacing with a normal saline solution to obtain a protein solution, namely the separated and purified staphylococcal protein A.
And (5) determining the protein purity and impurities in the protein solution: protein purity was determined by electrophoresis and high performance liquid chromatography, and as a result, referring to fig. 1 and 2, respectively, protein purity was 97.1% by reverse phase chromatography (fig. 2). The endotoxin content was measured by gel method using limulus reagent. The amount of residual protein in E.coli cells was measured using an E.coli host residual protein (E.coli P) ELISA kit. Residual amount of residual DNA in Escherichia coli Using Quant-iTTM
Figure BDA0001286424560000091
The dsDNA kit performs the assay. The results are shown in Table 1.
TABLE 1 determination of the respective impurities in the protein solution of step 5
Measurement items Results
Purity of protein 97.1％
Protein recovery 78％
Endotoxin ＜1EU/mg
HCP ＜2ppm
Residual DNA of Escherichia coli ＜5ppm
Example 2
1) Pretreating thalli: 1kg of staphylococcal protein A-containing bacterial cells (e.g., Staphylococcus protein A-expressing Escherichia coli), 10L of Bis-tris buffer solution (pH 7.2, 30 mM) was added to the cells to suspend the cells, and the cells were disrupted by a high-pressure homogenizer at 800bar for 2 times, and the supernatant was collected by centrifugation to give about 10L. The pH of the resulting solution was adjusted to 4.0 by adding hydrochloric acid, centrifuged, and the supernatant was adjusted to pH7.2 with sodium hydroxide to obtain a protein solution.
2) First chromatographic column treatment: will be charged with TOYOPEARL NH2Column with-750F packing was equilibrated 5 column volumes with equilibration solution (containing 20mM bis-tris and 1 wt% Triton X-100, 0.1mol/LNaCl, pH7.0), and the protein solution obtained in step 1 was loaded into a column containing TOYOPEARL NH equilibrated with equilibration solution2And (3) in a chromatographic column of-750F salt-tolerant anion exchange filler, continuously balancing 5 times of column volume by using a balance liquid, then washing 5 times of column volume by using a washing liquid (containing 20mM Bis-tris, 0.1mol/L NaCl, pH7.0), finally eluting the target protein staphylococcal protein A by using an eluent (containing 20mM Bis-tris and 1mol/L NaCl, pH7.0), and collecting an elution peak to obtain a first collection liquid.
3) Ultrafiltration displacement buffer: subjecting the first obtained in step 2)Collecting the filtrate, ultrafiltering with PES ultrafiltration membrane having cut-off molecular weight of 5KD, with 5 times volume of equal volume, replacing with pH7.0, and containing 5mM Na2HPO4-NaH2PO4And 0.2M proline buffer to obtain protein solution.
4) And (3) second chromatographic column treatment: the column packed with hydroxyapatite chromatography packing was equilibrated (pH 7.0, 5mM Na)2HPO4-NaH2PO4And 0.2M proline), loading the protein solution obtained in step 3) into a column filled with hydroxyapatite chromatographic packing and equilibrated with an equilibration solution, continuing to equilibrate the column volume by 5 times with the equilibration solution, and then eluting with an eluent (100mM Na)2HPO4-NaH2PO4pH7.0) and collecting a second collecting liquid of an elution peak.
5) Replacing ultrafiltration with a preservation solution: and (3) performing equal-volume ultrafiltration on the second collection liquid obtained in the step 4) by using an ultrafiltration membrane with the cut-off molecular weight of 5KD for 5 times of volume, and replacing with a normal saline solution to obtain a protein solution (protein A solution), namely the separated and purified staphylococcal protein A.
The protein purity and the detection method of each impurity in the protein solution of step 5 are the same as those of example 1. Referring to fig. 1 and 2, respectively, the protein purity was 97.6% by reverse phase chromatography (fig. 3). The results are shown in Table 2.
TABLE 2 determination of the respective impurities in the protein solution
Measurement items Results
Purity of protein 97.6％
Protein recovery 76％
Endotoxin ＜1EU/mg
HCP ＜1ppm
Residual DNA of Escherichia coli ＜1ppm
Example 3
Basically, the same as example 1, except that step 1) pretreatment of the cells: 1kg of the cells containing staphylococcal protein A was added to 10L of Bis-tris buffer solution (20 mM, pH 7.2) to suspend the cells, and then the cells were disrupted by a high-pressure homogenizer at 800bar for 2 times, and the supernatant was collected by centrifugation (about 10L). The resulting solution was adjusted to pH 3.0 by adding hydrochloric acid, centrifuged, and the supernatant was adjusted to pH7.2 with sodium hydroxide, thereby obtaining a protein solution.
Example 4
Basically the same as example 1 except that the first chromatographic column treatment in step 2): the equilibrium solution contains 30mM Bis-tris, 2 wt% TritonX-100, 0.1mol/LNaCl and pH7.2; the washing solution contained 30mM Bis-tris, 0.1mol/L NaCl, and the pH7.2 eluate contained 30mM Bis-tris and 1mol/L NaCl, pH 7.2.
Example 5
Essentially the same as example 2, except that the ultrafiltration buffer in step 3) was pH7.2 containing 10mM Na2HPO4-NaH2PO4And 0.2M proline in buffer. Step 4) treatment of the second column with an equilibration solution containing 10mM Na2HPO4-NaH2PO4And 0.2M proline, pH 7.2; the eluent contained 100mM Na2HPO4-NaH2PO4，pH 7.2。
Example 6
Essentially the same as example 2, except that the ultrafiltration buffer in step 3) contained 20mM Na2HPO4-NaH2PO4And 0.3M proline, pH7.5 buffer. Step 4) treatment of the second column with an equilibration solution containing 20mM Na2HPO4-NaH2PO4And 0.3M proline, pH 7.5; the eluent contained 100mM Na2HPO4-NaH2PO4，pH 7.5。
Example 7: identification of protein A Activity
The purified recombinant staphylococcal protein A is connected to an agarose carrier, a plasma adsorption experiment is carried out, and the adsorption effect of the synthesized adsorbent on IgG type antibodies in human plasma is measured, so that the activity of the protein is evaluated.
The protein A solutions obtained in examples 1 and 2 were coupled to Sepharose 6FF by sodium periodate oxidation, respectively. The specific method is described in patent 201010512304.8. Through simulating the clinical immunoadsorption step, the in vitro test on the in-plasma immunoglobulin adsorbability is carried out to evaluate the adsorbability, and further the clinical use value can be known, the specific steps are shown in patent 201010512304.8, the adsorbability of the immunoadsorbent taking the recombinant staphylococcal protein A as the ligand in the examples 1 and 2 to IgG in human plasma is 34mg/g and 37mg/g of filler respectively, compared with the protein purified by a tris-HCl system under the same condition through anion exchange chromatography, the adsorbability of the synthetic filler is 30mg/g and 33mg/g of filler respectively, and the average adsorbability is improved by 13% and 12%. The higher the adsorption performance, the higher the decline rate of the pathogenic antibody under the same treatment time or cycle number, which is more helpful for the prognosis of the disease, so that the improvement of the adsorption performance of 10-15% is also very significant.
The protein solutions in the embodiments 3, 4, 5 and 6 are respectively detected for protein purity, HCP residual quantity, Escherichia coli residual DNA residual quantity, adsorption performance and the like according to the detection method, and the detection results all meet the requirements, namely the protein purity is more than 97%; the protein recovery rate is more than 75 percent; the adsorption performance is more than 33 mg/g; the endotoxin is reduced to below 1 EU/mg; HCP and DNA were below 5 ppm.

Claims (6)

1. A method for separating and purifying recombinant staphylococcal protein a, comprising the steps of:
1) pretreating thalli: adding thalli containing recombinant staphylococcal protein A into Bis-tris buffer solution, crushing by using a homogenizer, centrifuging to collect supernatant, adjusting pH to 2.0-4.0, centrifuging to collect supernatant, and adjusting pH to 6.5-7.2 to obtain protein solution;
2) first chromatographic column treatment: loading the protein liquid obtained in the step 1) into a chromatographic column which is balanced by balance liquid and is filled with salt-resistant anion exchange filler, wherein the salt-resistant anion exchange filler is TOYOPEARLNH2-750F salt-tolerant anion exchange packing, the particle size of which is 30-60 μm, continuously balancing with a balancing solution, washing with a washing solution, finally eluting staphylococcal protein a with an eluent, and collecting the elution peak to obtain a first collection solution; the equilibrium solution contains 10-30mM Bis-tris, 0.5-2 wt% TritonX-100 and 0.1mol/LNaCl, and the pH value is 6.5-7.2; the washing liquid contains 10-30mM Bis-tris, 0.1mol/LNaCl and has pH of 6.5-7.2; the eluent contains 10-30mM Bis-tris, 1mol/LNaCl and has pH of 6.5-7.2;
3) ultrafiltration displacement buffer: replacing the first collected liquid obtained in step 2) with 5KD cut-off molecular weight ultrafiltration membrane to pH 6.8-7.5 containing 5-20mM Na2HPO4-NaH2PO4And 0.1-0.3M proline, thereby obtaining a protein solution;
4) and (3) second chromatographic column treatment: loading the protein solution obtained in the step 3) into a column which is balanced by a balancing solution and is filled with hydroxyapatite chromatographic packing, continuing to balance by the balancing solution, then eluting by an eluent, and collecting an elution peak to obtain a second collecting solution; the equilibrium solution contains 5-20mM Na2HPO4-NaH2PO4And 0.1-0.3M proline, pH 6.8-7.5; the eluent contains 100mM Na2HPO4-NaH2PO4，pH 6.8-7.5；
5) Replacing ultrafiltration with a preservation solution: replacing the second collecting liquid obtained in the step 4) with a normal saline solution by ultrafiltration with an ultrafiltration membrane with the cut-off molecular weight of 5KD, thereby obtaining the separated and purified staphylococcal protein A.
2. The method for separating and purifying recombinant staphylococcal protein A according to claim 1, wherein the thallus pretreatment is: adding thallus containing staphylococcal protein A into Bis-tris buffer solution with pH of 7.2 and concentration of 10-30mM according to the mass ratio of 1kg to 10L, crushing with a high-pressure homogenizer under 800bar pressure, and centrifuging to collect supernatant; adjusting pH to 2.0-4.0 with hydrochloric acid, centrifuging, collecting supernatant, and adjusting pH to 6.5-7.2 with NaOH to obtain protein solution.
3. The method for separating and purifying recombinant staphylococcal protein A according to claim 1, wherein the equilibrium solution in step 2) contains 20mM Bis-tris and 1% TritonX-100, 0.1mol/LNaCl, pH 7.0; the washing solution contained 20mM Bis-tris, 0.1mol/LNaCl, pH 7.0; the eluate contained 20mM Bis-tris and 1mol/L NaCl, pH 7.0.
4. The method for separating and purifying recombinant staphylococcal protein A according to claim 1, wherein the hydroxyapatite chromatography packing of step 4) has a particle size of 40 μm.
5. The method for separating and purifying recombinant staphylococcal protein A according to claim 1, wherein the balance of step 4) contains 5mM Na2HPO4-NaH2PO4And 0.2M proline, pH 7.0; the eluent is 100mM Na2HPO4-NaH2PO4，pH7.0。
6. The method for separating and purifying recombinant staphylococcal protein A according to claim 1, wherein the ultrafiltration membrane in step 3) and step 5) is made of polyethersulfone.
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