Method for high-load and large-scale separation and purification of recombinant protein A

Abstract

The invention discloses a method for separating and purifying recombinant protein A with high loading capacity on a large scale, and provides a method for separating and purifying the recombinant protein A by using nickel ion chelation chromatography and anion exchange chromatography, the loading capacity of a nickel ion chelation chromatography sample is 40g / L, and the loading capacity of an anion exchange chromatography sample is 80g / L. According to the method, endotoxin and escherichia coli host protein are effectively removed, the purity of a recombinant protein A solution obtained through separation and purification is larger than 98% through HPLC and SDS-PAGE electrophoretic analysis, the endotoxin is smaller than 1 EU / mg, and the recovery rate is larger than 85%. The protein loading capacity is high, the technological process is simple, the cost is low, the applicability is wide, the purity and the recovery rate are high, meanwhile, the endotoxin control effect is remarkable, and therefore the protein A can be used as a raw material of immunoaffinity chromatography filler and is suitable for industrial production of recombinant protein A.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a large-scale separation and purification method for recombinant protein A. Background Art

[0002] Staphylococcus aureus protein A (abbreviated as Protein A) is a surface protein present in the cell wall of Staphylococci. It has a molecular weight of 42kDa and has five different domains that can bind to the Fc fragment of IgG. It has a strong specific affinity and can reversibly bind to the Fc fragment of IgG molecules in human and various mammalian serum. Therefore, Protein A affinity chromatography media prepared by coupling Protein A to a solid phase carrier are widely used in the separation and purification of antibodies.

[0003] Due to the wide range of uses of Protein A, market demand is increasing. Directly extracting the natural protein from Staphylococcus aureus alone not only fails to meet market demand, but the affinity chromatography medium prepared from this natural Protein A also has poor alkali resistance and low loading capacity. Therefore, in recent years, people have begun to use genetic engineering technology to produce recombinant Protein A. By mutating and modifying the natural Protein A molecule, alkali resistance and loading capacity are enhanced. Escherichia coli engineered bacteria are commonly used to express recombinant Protein A. The advantages are low cost, simple process, and high expression level. However, E. coli host protein impurities are high and endotoxin contamination is serious, which brings many challenges to the separation and purification of recombinant Protein A. Due to the very strict requirements on endotoxins in antibody drug production and clinical treatment, recombinant Protein A, as one of the important raw materials for Protein A affinity chromatography media, requires both high purity and strict endotoxin standards. Based on high quality standards, it is very important to develop a large-scale production method with a simple process flow, high production efficiency, low cost, high purity and recovery rate, and significant endotoxin control effect.

[0004] In the prior art, the main methods for isolating and purifying recombinant protein A include: (a) a one-step method, which involves obtaining the target product through a one-step gel sieve chromatography (CN 1432578A, CN 1524957A, etc.), a one-step nickel ion chelation chromatography (CN 101050464A), and a one-step affinity chromatography using the Fc fragment of IgG as a ligand (CN 101921818A); and (b) a multi-step method, which includes a two-step IgG affinity chromatography-anion exchange chromatography method (US 5075423A) and anion exchange chromatography-hydroxyapatite chromatography (CN106977591 B).

[0005] None of the above patents mention the sample loading capacity, purity and endotoxin control of the chromatography step. For gel molecular sieves, due to the small processing capacity, slow speed, and inability to remove endotoxins, they cannot be used for the industrial production of protein A at all. One-step nickel ion chelation chromatography and one-step affinity chromatography with the Fc fragment of IgG as a ligand cannot effectively remove endotoxins, and the one-step nickel ion chelation chromatography does not involve the step of removing the histidine tag, which may affect the activity of protein A. The one-step affinity chromatography with the Fc fragment of IgG as a ligand has a loading capacity of only 10-20 mg / mL filler, and IgG is used as the main raw material, so the production cost is high. The general loading capacity of IgG affinity chromatography is only 2-5 mg / mL filler, the ligand is unstable, and problems such as denaturation and ligand shedding are prone to occur during operation. Patent CN 106977591 B mentions the control of endotoxins. However, since E. coli fermentation broth contains a large amount of lipoproteins, host proteins, and nucleic acid impurities, although some impurities can be removed through simple treatment, a large number of low-isoelectric point impurities will occupy the coordination sites of the anion exchange chromatography medium, resulting in a low loading capacity of the anion exchange chromatography binding protein A. In addition, the chromatography medium may adsorb a large amount of pigments, resulting in performance degradation. In addition, the loading capacity of hydroxyapatite filler is not as good as that of anion exchange filler, and it is limited in terms of sample processing volume and production efficiency. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for high-load, large-scale separation and purification of recombinant protein A, so as to provide an industrial production method for recombinant protein A with high protein loading capacity, simple process flow, low cost, wide applicability, high purity and recovery rate, and significant endotoxin control effect.

[0007] Technical solution: To solve the above technical problems, the present invention provides the following technical solutions:

[0008] (1) Cell disruption and pretreatment: Take cells containing recombinant protein A and add them to a first phosphate buffer at a mass: volume ratio of 0.5-2 kg:10 L, preferably 1 kg:10 L; stir evenly with a shearing machine, then use a high-pressure homogenizer to disrupt the cells at a flow rate of 500 L / H and a homogenization pressure of 900±50 bar. After cell disruption, centrifuge and collect the supernatant to obtain a protein loading solution. The first phosphate buffer contains 20-50 mM Na2HPO4-NaH2PO4, 300-500 mM NaCl, pH 7.5-8.5, preferably 50 mM Na2HPO4-NaH2PO4, 500 mM NaCl, pH 7.5-8.5.

[0009] (2) The first step of chromatography: using an equilibrium solution to equilibrate a nickel ion chelate chromatography column, wherein the nickel ion chelate chromatography column is filled with Saifen Technology Agarosix MC90-NTA Ni chromatography filler, whose matrix is ​​high-flow agarose with an average particle size of 90 μm, and the protein loading solution obtained in step (1) is loaded onto the nickel ion chelate chromatography column, with a loading capacity of 40-50 g / L filler, preferably a loading capacity of 40 g / L filler, and then continued to equilibrate with the first equilibrium solution, rinsed with the first washing solution, and finally eluted with the first elution solution to obtain the first step chromatography collection solution; the above-mentioned first equilibrium solution contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, pH 7.5-8.5; the first washing solution contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, 10-20mM imidazole, pH7.5-8.5; the first eluent contains 20-50mM Na2HPO4-NaH2PO4, 100-300mM NaCl, 300-500mM imidazole, pH7.5-8.5; preferably: the first equilibrium solution contains 50mM Na2HPO4-NaH2PO4, 200mM NaCl, pH7.5-8.5; the first washing solution contains 50mM Na2HPO4-NaH2PO4, 200mM NaCl, 15mM imidazole, pH7.5-8.5; the first eluent contains 50mM Na2HPO4-NaH2PO4, 200mM NaCl, 300mM imidazole, pH7.5-8.5.

[0010] (3) Ultrafiltration and enzyme digestion: The first chromatography collection liquid obtained in step (2) is replaced into a second phosphate buffer using an ultrafiltration membrane with a molecular weight cutoff of 5KDa, and then TEV enzyme is added for enzyme digestion, the protein mass to TEV enzyme mass ratio is (100-200):1, preferably the protein mass to TEV enzyme mass ratio is 200:1, the enzyme digestion temperature is 30 degrees Celsius, the enzyme digestion time is 4 hours, and the protease digestion liquid is obtained; the second phosphate buffer contains 20-50mM Na2HPO4-NaH2PO4, 100-300mM NaCl, pH7.5-8.5, preferably 50mM Na2HPO4-NaH2PO4, 150mM NaCl, pH7.5-8.5;

[0011] The TEV enzyme carries a 6X His-tag and has a molecular weight of 28 kDa. It is a His-tagged cysteine ​​protease of tobacco etch virus (TEV) recombinantly expressed in Escherichia coli. It can specifically recognize the heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly / Ser and perform enzymatic cleavage between the Gln and Gly / Ser amino acid residues.

[0012] (4) Second step chromatography method: use an equilibrium solution to balance a nickel ion chelate chromatography column (consistent with the chromatography column of step (2)), then load the protease digest described in step (3) onto the nickel ion chelate chromatography column, with a sample loading of 80-90 g / L filler, use a second equilibrium solution for post-equilibrium, collect the flow-through peak during the loading and post-equilibrium process, and obtain the second step chromatography collection solution; the second equilibrium solution contains 20-50 mM Na2HPO4-NaH2PO4, pH7.5-8.5, preferably 20 mM Na2HPO4-NaH2PO4, pH7.5-8.5.

[0013] (5) The third step chromatography method: the anion exchange chromatography column is equilibrated with an equilibration liquid, wherein the anion exchange chromatography column is filled with a chromatographic filler of Monomix Mab60-Q produced by Saifen Technology with a particle size of 60 μm, and then the second step chromatography collection liquid of step (4) is diluted with pure water to a conductivity of less than 5 mS / cm, and then loaded onto the anion exchange chromatography column with a sample loading of 80-90 g / L filler, equilibrated with a third equilibration liquid, and finally eluted with a second eluent to obtain the third step chromatography collection liquid. The third equilibrium solution contains 20-50mM Na2HPO4-NaH2PO4, pH7.5-8.5, preferably 20mM Na2HPO4-NaH2PO4, pH7.5-8.5; the second eluent contains 20-50mM Na2HPO4-NaH2PO4, 100-300mM NaCl, pH7.5-8.5, preferably 20mM Na2HPO4-NaH2PO4, 200mM NaCl, pH7.5-8.5.

[0014] In step (2) and step (4), the nickel ion chelation chromatography column is filled with Agarosix MC90-NTANi chromatography filler of Saifen Technology, which has an average particle size of 90 μm, uses 6% highly cross-linked agarose as the matrix, and uses nitrilotriacetic acid (NTA) as the ligand to chelate nickel ions. It can specifically adsorb recombinant protein A with a 6X His tag, while most endotoxins and Escherichia coli HCP flow through and are removed. The loosely bound impurities are removed by using a washing solution containing a low concentration of imidazole, and the high-purity recombinant protein A is washed out using an elution solution containing a high concentration of imidazole, and then other impurities on the column are removed using 0.1 M NaOH.

[0015] In step (3), the material of the ultrafiltration membrane package is preferably polyethersulfone (PES).

[0016] In step (3), TEV enzyme cleavage can cut the 6X His tag and enzyme cleavage site sequence on the recombinant protein A. At this time, the sample is loaded onto the nickel ion chelate chromatography column in flow-through mode, and the recombinant protein A that flows through is collected. High concentration imidazole buffer and 0.1M NaOH are used to remove the undigested recombinant protein A hanging on the column, the cut 6X His tag, the TEV enzyme with the His tag, and other impurities with a small amount of His tag.

[0017] In step (5), the anion exchange chromatography column is filled with Monomix Mab60-Q chromatography filler produced by Saifen Technology, with a particle size of 60 μm. Since the isoelectric point of endotoxin is about 3.1, while the isoelectric point of recombinant protein A is about 5, in a pH 7.5-8.5 buffer, endotoxin has a negative charge and its binding ability with the anion exchange filler is greater than that of recombinant protein A. Therefore, the recombinant protein A is first eluted using a buffer with a certain salt concentration, and then the endotoxin remaining on the column is removed using a high salt concentration buffer and 0.5 M NaOH, thereby effectively removing endotoxin.

[0018] Technical effects: The beneficial effects of the present invention are as follows:

[0019] (1) The first step of chromatography in the present invention uses Agarosix MC90-NTA Ni chromatography filler from Saifen Technology, which has an average particle size of 90 μm, a 6% high-cross-linked agarose matrix, and nitrilotriacetic acid (NTA) as a ligand to chelate nickel ions. The His-tagged protein has a loading capacity greater than 50 mg / mL and can withstand 0.1 M NaOH cleaning and regeneration. It has high flow rate, low back pressure, high dynamic loading capacity, good chemical stability and mechanical properties, low nonspecific adsorption, high recovery rate, and is easy to scale up, which can shorten production time and improve production efficiency. The loading capacity of the first step of chromatography reaches 40-50 g / L, which is more than 2-20 times that of IgG affinity filler. At the same time, by optimizing the washing solution, a high-purity recombinant protein A eluate is obtained, which reduces the pressure of the next step of purification.

[0020] (2) The present invention uses TEV enzyme cleavage to efficiently and quickly cut the His tag of recombinant protein A, and the enzyme cleavage rate can reach more than 95%. The protein solution after enzyme cleavage is loaded onto the nickel ion chelate chromatography column in flow-through mode. In the flow-through mode, the recombinant protein A without the tag flows through without hanging on the column, and the filler load is not high. The sample loading capacity can reach 80-90g / L. At the same time, the uncut recombinant protein A, the cut 6X His tag, the TEV enzyme with the His tag, and other impurities with a small amount of His tag can be removed, further reducing the pressure of the next purification step. The most important point is that the activity of the recombinant protein A will not be affected after the tag is removed.

[0021] (3) The present invention uses anion exchange chromatography as the last step of purification, and the filler is Monomix Mab60-Q of Saifen Technology with a particle size of 60 μm. After the first two steps of purification, most impurities and pigments as well as most endotoxins are removed, which reduces the processing pressure of anion exchange chromatography. The sample loading capacity can reach 80-90 g / L, and the endotoxin content can be reduced from 500 EU / mg to below 1 EU / mg.

[0022] In summary, the present invention uses nickel ion chelate chromatography and anion exchange chromatography to separate and purify recombinant protein A, wherein the nickel ion chelate chromatography sample loading capacity is 40-50 g / L, and the anion exchange chromatography sample loading capacity is 80-90 g / L. This method effectively removes endotoxins and Escherichia coli host proteins, and the recombinant protein A solution obtained by separation and purification has a purity greater than 98% by HPLC and SDS-PAGE electrophoresis analysis, endotoxins are less than 1 EU / mg, and the recovery rate is greater than 85%. The protein of the present invention has a high sample loading capacity, a simple process flow, low cost, wide applicability, high purity and recovery rate, and a significant endotoxin control effect, and can be used as a raw material for immunoaffinity chromatography fillers, and is suitable for the industrial production of recombinant protein A. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a chromatography purification chart in Example 1.

[0024] Figure 2 This is the secondary chromatography purification profile in Example 1.

[0025] Figure 3 This is the three-stage chromatography purification profile in Example 1.

[0026] Figure 4 This is the SDS-PAGE gel electrophoresis result of the recombinant protein A in Example 1 and Example 2.

[0027] Lane 1 is Protein Maker, with a molecular weight range of 10-245 kDa. The molecular weight of recombinant protein A with a His tag is approximately 42 kDa, and the molecular weight of recombinant protein A without the His tag is approximately 38 kDa. Lane 2 is the protein loading solution in step (2) of Example 1, lane 3 is the first chromatography collection solution in step (2) of Example 1, lane 4 is the protease cleavage solution in step (3) of Example 1, lane 5 is the second chromatography collection solution in step (4) of Example 1, lane 6 is the third chromatography collection solution in step (5) of Example 1, and lane 7 is the third chromatography collection solution in step (5) of Example 2.

[0028] Figure 5 This is the HPLC profile of the finished product recombinant protein A in Example 1.

[0029] Figure 6 This is the HPLC profile of the finished recombinant protein A in Example 2. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and examples.

[0031] The detection method related to recombinant protein A of the present invention specifically includes:

[0032] 1. SDS-PAGE electrophoresis, protein gel: SurePAGE from GenScript TM 15% protein precast gel, electrophoresis tank: BIO-RAD Tetra electrophoresis tank, electrophoresis apparatus: EPS200 electrophoresis apparatus from Tianneng Technology Co., Ltd.

[0033] 2. Purity was tested by size exclusion chromatography, HPLC: Agilent 1260 Infinity II, chromatographic column: Zenix SEC 300 (3 μm, 7.8mm ID*300mm), mobile phase: 150mM PB buffer, pH7.0, flow rate: 1mL / min, detection wavelength: 280nm, injection volume: 10μL

[0034] 3. BCA method for protein concentration detection, kit: BCA protein concentration determination kit of Biyuntian Company, product number: P0012

[0035] 4. Detection of endotoxin content by limulus amebocyte lysate colorimetric method. Kit: Endotoxin Detection Kit (limulus amebocyte lysate colorimetric method) from Biyuntian, Product No.: C0276S

[0036] The method for preparing recombinant Protein A bacteria is as follows: a recombinant Protein A encoding gene (its nucleic acid sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2) is synthesized, inserted into a pET-16b plasmid vector, and the plasmid is transformed into Escherichia coli host cells BL21(DE3). The E. coli host cells BL21(DE3) are fermented and amplified on a large scale in a 500L fermentor. After expression, the cells are collected by centrifugation to obtain recombinant Protein A bacteria. The preparation method is described in Patent CN114409801B.

[0037] Example 1:

[0038] (1) Cell disruption and pretreatment: Cells containing recombinant protein A were added to a phosphate buffer solution at a mass:volume ratio of 1 kg:10 L. The cells were stirred evenly using a shearing machine and then disrupted using a high-pressure homogenizer at a flow rate of 500 L / H and a homogenization pressure of 900 ± 50 bar. After cell disruption, the supernatant was collected by centrifugation to obtain a protein loading solution. The phosphate buffer solution contained 20 mM Na2HPO4-NaH2PO4, 500 mM NaCl, and a pH of 7.5.

[0039] (2) The first step of chromatography: the nickel ion chelate chromatography column is equilibrated with an equilibrium solution, wherein the nickel ion chelate chromatography column is filled with Saifen Technology Agarosix MC90-NTA Ni chromatography filler, whose matrix is ​​high-flow agarose with an average particle size of 90 μm, and the protein loading liquid obtained in step (1) is loaded onto the nickel ion chelate chromatography column with a loading capacity of 40 g / L filler, and then the column is further equilibrated with the equilibrium solution, rinsed with a washing solution, and finally eluted with an eluent to obtain the first step chromatography collection liquid; the above-mentioned equilibrium solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 7.5; the washing solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 15 mM imidazole, pH 7.5; the eluent contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 300 mM imidazole, pH 7.5.

[0040] (3) Ultrafiltration solution replacement and enzyme digestion: The first step chromatography collection liquid obtained in step (2) is replaced into phosphate buffer using an ultrafiltration membrane with a molecular weight cutoff of 5KDa, and then TEV enzyme is added for enzyme digestion. The protein mass to TEV enzyme mass ratio is 200:1, the enzyme digestion temperature is 30 degrees Celsius, and the enzyme digestion time is 4 hours to obtain a protease digestion liquid; the phosphate buffer contains 20mMNa2HPO4-NaH2PO4, 150mM NaCl, and pH 7.5.

[0041] (4) Second step chromatography method: Use equilibration solution to equilibrate nickel ion chelate chromatography column, then load the protease digest described in step (3) onto the nickel ion chelate chromatography column, with a sample loading of 80 g / L filler, and use equilibration solution for post-equilibration. During the loading and post-equilibration process, the flow-through peak is collected to obtain the second step chromatography collection solution; the equilibration solution contains 20 mM Na2HPO4-NaH2PO4, pH 7.5.

[0042] (5) Third step chromatography method: Use an equilibration solution to equilibrate an anion exchange chromatography column, wherein the anion exchange chromatography column is filled with a 60 μm chromatographic filler of Monomix Mab60-Q from Saifen Technology, and then dilute the collected solution from the second step chromatography in step (4) with pure water until the conductivity is less than 5 mS / cm, and then load the sample onto the anion exchange chromatography column with a sample load of 80 g / L filler, equilibrate with the equilibration solution, and finally elute with the eluent to obtain the collected solution from the third step chromatography, i.e., the finished recombinant protein A solution after separation and purification. The equilibration solution contains 20 mM Na2HPO4-NaH2PO4, pH 7.5; the eluent contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 7.5.

[0043] The purity of the finished recombinant protein A solution described in step (5) was tested by SDS-PAGE electrophoresis and high performance liquid chromatography, and the results were shown in Table 1. Figure 4 and Figure 5 , the purity was 98.3% by size exclusion chromatography (see Figure 5 The protein concentration was determined using the BCA assay, and the endotoxin content was determined using the Limulus Amebocyte Lysate (LAL) colorimetric assay. The protein purity was 98.3%, the protein recovery was 86.2%, and the endotoxin content in the protein solution was less than 1 EU / mg.

[0044] Example 2:

[0045] (1) Cell disruption and pretreatment: Cells containing recombinant protein A were added to a phosphate buffer solution at a mass:volume ratio of 1 kg to 10 L. The cells were stirred evenly using a shearing machine and then disrupted using a high-pressure homogenizer at a flow rate of 500 L / H and a homogenization pressure of 900 ± 50 bar. After cell disruption, the supernatant was collected by centrifugation to obtain a protein loading solution. The phosphate buffer solution contained 50 mM Na2HPO4-NaH2PO4, 500 mM NaCl, and a pH of 8.5.

[0046] (2) The first step of chromatography: the nickel ion chelate chromatography column is equilibrated with an equilibration solution, wherein the nickel ion chelate chromatography column is filled with Saifen Technology Agarosix MC90-NTA Ni chromatography filler, whose matrix is ​​high-flow agarose with an average particle size of 90 μm, and the protein loading liquid obtained in step (1) is loaded onto the nickel ion chelate chromatography column with a loading capacity of 50 g / L filler, and then the equilibration solution is continued to be used for equilibration, and the washing solution is used for washing, and finally the eluent is used for elution to obtain the first step chromatography collection liquid; the above-mentioned equilibration solution contains 50 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 8.5; the washing solution contains 50 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 15 mM imidazole, pH 8.5; the eluent contains 50 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 300 mM imidazole, pH 8.5.

[0047] (3) Ultrafiltration solution replacement and enzyme digestion: The first step chromatography collection liquid obtained in step (2) is replaced into phosphate buffer using an ultrafiltration membrane pack with a molecular weight cutoff of 5KDa, and then TEV enzyme is added for enzyme digestion. The protein mass to TEV enzyme mass ratio is 200:1, the enzyme digestion temperature is 30 degrees Celsius, and the enzyme digestion time is 4 hours to obtain a protease digestion liquid; the phosphate buffer contains 50mMNa2HPO4-NaH2PO4, 150mM NaCl, pH8.5.

[0048] (4) Second step chromatography method: Use equilibration solution to equilibrate nickel ion chelate chromatography column, then load the protease digest described in step (3) onto the nickel ion chelate chromatography column, with a sample loading of 90 g / L filler, and use equilibration solution for post-equilibration. During the loading and post-equilibration process, the flow-through peak is collected to obtain the second step chromatography collection solution; the equilibration solution contains 50 mM Na2HPO4-NaH2PO4, pH 8.5.

[0049] (5) Third step chromatography method: Use an equilibration solution to equilibrate an anion exchange chromatography column, wherein the anion exchange chromatography column is filled with a 60 μm particle size of a Monomix Mab60-Q chromatography filler from Saifen Technology, and then dilute the second step chromatography collection liquid in step (4) with pure water until the conductivity is less than 5 mS / cm, and then load the sample onto the anion exchange chromatography column with a sample load of 90 g / L filler, equilibrate with the equilibration solution, and finally elute with the eluent to obtain the third step chromatography collection liquid, i.e., the finished recombinant protein A solution after separation and purification. The equilibration solution contains 50 mM Na2HPO4-NaH2PO4, pH 8.5; the eluent contains 50 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 8.5.

[0050] In step (5), the recombinant protein A solution was tested for purity using SDS-PAGE electrophoresis and high performance liquid chromatography, and the results were shown in Table 1. Figure 4 and Figure 6 , the purity was 98.5% by size exclusion chromatography (see Figure 6 ), protein concentration was determined using the BCA assay, and endotoxin was determined using the Limulus Amebocyte Lysate (LAL) colorimetric assay. Protein purity was 98.5%, protein recovery was 87.3%, and endotoxin content in the protein solution was less than 1 EU / mg.

[0051] Comparative Example 1:

[0052] This comparative example provides a method for separating and purifying recombinant protein A. The specific parameters are the same as those in Example 1, except that step (4) is removed and the protease cleavage solution obtained in step (3) is purified by anion exchange chromatography. The specific steps are as follows.

[0053] (1) Cell disruption and pretreatment: Cells containing recombinant protein A were added to a phosphate buffer solution at a mass:volume ratio of 1 kg:10 L. The cells were stirred evenly using a shearing machine and then disrupted using a high-pressure homogenizer at a flow rate of 500 L / H and a homogenization pressure of 900 ± 50 bar. After cell disruption, the supernatant was collected by centrifugation to obtain a protein loading solution. The phosphate buffer solution contained 20 mM Na2HPO4-NaH2PO4, 500 mM NaCl, and a pH of 7.5.

[0054] (2) The first step of chromatography: the nickel ion chelate chromatography column is equilibrated with an equilibrium solution, wherein the nickel ion chelate chromatography column is filled with Saifen Technology Agarosix MC90-NTA Ni chromatography filler, whose matrix is ​​high-flow agarose with an average particle size of 90 μm, and the protein loading liquid obtained in step (1) is loaded onto the nickel ion chelate chromatography column with a loading capacity of 40 g / L filler, and then the column is further equilibrated with the equilibrium solution, rinsed with a washing solution, and finally eluted with an eluent to obtain the first step chromatography collection liquid; the above-mentioned equilibrium solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 7.5; the washing solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 15 mM imidazole, pH 7.5; the eluent contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 300 mM imidazole, pH 7.5.

[0055] (3) Ultrafiltration solution replacement and enzyme digestion: The first step chromatography collection liquid obtained in step (2) is replaced into phosphate buffer using an ultrafiltration membrane with a molecular weight cutoff of 5KDa, and then TEV enzyme is added for enzyme digestion. The protein mass to TEV enzyme mass ratio is 200:1, the enzyme digestion temperature is 30 degrees Celsius, and the enzyme digestion time is 4 hours to obtain a protease digestion liquid; the phosphate buffer contains 20mMNa2HPO4-NaH2PO4, 150mM NaCl, and pH 7.5.

[0056] (4) The third step of chromatography: an anion exchange chromatography column is equilibrated with an equilibration solution, wherein the anion exchange chromatography column is filled with a 60 μm particle size of a Monomix Mab60-Q chromatography filler of Saifen Technology, and then the protease digest of step (3) is diluted with pure water to a conductivity of less than 5 mS / cm, and then loaded onto an anion exchange chromatography column with a sample loading of 80 g / L filler, equilibrated with an equilibration solution, and finally eluted with an eluent to obtain a third step chromatography collection solution, i.e., a finished recombinant protein A solution that has been separated and purified. The equilibration solution contains 20 mM Na2HPO4-NaH2PO4, pH 7.5; the eluent contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 7.5.

[0057] Comparative Example 2:

[0058] This comparative example provides a method for separating and purifying recombinant protein A. The specific parameters are the same as those in Example 1, except that the anion exchange chromatography in step (5) is removed, and the protein collection liquid obtained in step (4) is regarded as the finished recombinant protein A solution. The specific steps are as follows.

[0059] (1) Cell disruption and pretreatment: Cells containing recombinant protein A were added to a phosphate buffer solution at a mass:volume ratio of 1 kg:10 L. The cells were stirred evenly using a shearing machine and then disrupted using a high-pressure homogenizer at a flow rate of 500 L / H and a homogenization pressure of 900 ± 50 bar. After cell disruption, the supernatant was collected by centrifugation to obtain a protein loading solution. The phosphate buffer solution contained 20 mM Na2HPO4-NaH2PO4, 500 mM NaCl, and a pH of 7.5.

[0060] (2) The first step of chromatography: the nickel ion chelate chromatography column is equilibrated with an equilibrium solution, wherein the nickel ion chelate chromatography column is filled with Saifen Technology Agarosix MC90-NTA Ni chromatography filler, whose matrix is ​​high-flow agarose with an average particle size of 90 μm, and the protein loading liquid obtained in step (1) is loaded onto the nickel ion chelate chromatography column with a loading capacity of 40 g / L filler, and then the column is further equilibrated with the equilibrium solution, rinsed with a washing solution, and finally eluted with an eluent to obtain the first step chromatography collection liquid; the above-mentioned equilibrium solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, pH 7.5; the washing solution contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 15 mM imidazole, pH 7.5; the eluent contains 20 mM Na2HPO4-NaH2PO4, 200 mM NaCl, 300 mM imidazole, pH 7.5.

[0061] (3) Ultrafiltration solution replacement and enzyme digestion: The first step chromatography collection liquid obtained in step (2) is replaced into phosphate buffer using an ultrafiltration membrane with a molecular weight cutoff of 5KDa, and then TEV enzyme is added for enzyme digestion. The protein mass to TEV enzyme mass ratio is 200:1, the enzyme digestion temperature is 30 degrees Celsius, and the enzyme digestion time is 4 hours to obtain a protease digestion liquid; the phosphate buffer contains 20mMNa2HPO4-NaH2PO4, 150mM NaCl, and pH 7.5.

[0062] (4) Second step chromatography method: Use equilibration solution to equilibrate nickel ion chelate chromatography column, then load the protease digest described in step (3) onto the nickel ion chelate chromatography column, with a sample loading of 80 g / L filler, and use equilibration solution for post-equilibration. During the loading and post-equilibration process, the flow-through peak is collected to obtain the finished protein A collection solution; the equilibration solution contains 20 mM Na2HPO4-NaH2PO4, pH 7.5.

[0063] The recombinant protein A-related detection method was used to detect the finished recombinant protein A solution obtained after the final purification of Example 1-2 and Comparative Example 1-2 to obtain the purity, recovery rate and endotoxin content of the recombinant protein A. The test results are shown in Table 1.

[0064] Table 1 Purity, recovery and endotoxin content of recombinant protein A

[0065]

[0066] As shown in Table 1, the three-step chromatography method employed in Examples 1-2 of the present invention yielded a purified recombinant Protein A solution with a purity exceeding 98%, a recovery exceeding 85%, and an endotoxin content of less than 1 EU / mg. In contrast, in Comparative Examples 1-2, whether the second nickel ion chelation chromatography step or the third anion exchange chromatography step was omitted, the purity, recovery, and endotoxin content of the purified recombinant Protein A solution were poor.

Claims

1. A method for high-load, large-scale separation and purification of recombinant protein A, characterized in that: The method comprises the following steps: (1) Taking the bacterial cells containing recombinant protein A, breaking them and centrifuging them to collect the supernatant to obtain a protein loading solution; (2) First step chromatography: the protein loading solution obtained in step (1) is loaded onto a nickel ion chelate chromatography column, then equilibrated with a first equilibration solution, rinsed with a first washing solution, and finally eluted with a first eluent to obtain a first step chromatography collection solution; (3) Ultrafiltration and enzyme digestion: The first chromatography collection liquid obtained in step (2) is replaced into a second phosphate buffer using an ultrafiltration membrane with a molecular weight cutoff of 5Kda, and then TEV enzyme is added for enzyme digestion to obtain a protease digestion liquid; (4) Second step chromatography: equilibrate the nickel ion chelate chromatography column with an equilibrium solution, then load the protease digestion solution of step (3) onto the nickel ion chelate chromatography column, perform post-equilibration with a second equilibrium solution, and collect the flow-through peak during the loading and post-equilibration process to obtain a second step chromatography collection solution; (5) Third step chromatography method: Use the third equilibration solution to equilibrate the anion exchange chromatography column, then dilute the second step chromatography collection liquid in step (4) with pure water until the conductivity is less than 5 mS / cm, then load it onto the anion exchange chromatography column, equilibrate it with the third equilibration solution, and finally elute it with the second elution solution to obtain the third step chromatography collection liquid, that is, obtain the purified recombinant protein A.

2. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (1), the gene sequence of the recombinant protein A is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO:

2.

3. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (1), the bacteria containing recombinant protein A are added to a first phosphate buffer, which is then broken using a high-pressure homogenizer and centrifuged to collect the supernatant; the first phosphate buffer contains 20-50 mM Na2HPO4-NaH2PO4, 300-500 mM NaCl, and pH 7.5-8.

5.

4. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (1), the ratio of recombinant protein A bacteria to phosphate buffer is: 0.5-2kg:10L.

5. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (2), the nickel ion chelate chromatography column is filled with Agarosix MC90-NTA Ni chromatography filler of Saifen Technology; the loading capacity of the protein loading solution is 40-50 g / L; The first equilibrium solution contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, pH 7.5-8.5; The first washing solution contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, 10-20 mM imidazole, pH 7.5-8.5; The first eluent contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, 300-500 mM imidazole, pH 7.5-8.

5.

6. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (3), the second phosphate buffer contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, pH 7.5-8.5; The TEV enzyme was used for enzymatic cleavage at a temperature of 30 degrees Celsius and for a time of 4 hours.

7. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (3), the mass ratio of protein to TEV enzyme is (100-200):

1.

8. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (4), the sample loading capacity of the protease cleavage solution is 80-90 g / L; The second equilibrium solution contains 20-50 mM Na2HPO4-NaH2PO4, pH 7.5-8.

5.

9. The method for high-load, large-scale separation and purification of recombinant protein A according to claim 1, characterized in that: In step (5), the anion exchange chromatography column is filled with Monomix Mab60-Q chromatography filler of Saifen Technology with a particle size of 60 μm; The third equilibrium solution contains 20-50 mM Na2HPO4-NaH2PO4, pH 7.5-8.5; The second eluent contains 20-50 mM Na2HPO4-NaH2PO4, 100-300 mM NaCl, pH 7.5-8.

5.

10. Recombinant protein A prepared by the method for high-load, large-scale separation and purification of recombinant protein A according to any one of claims 1 to 9.

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