CN105541994B

CN105541994B - method for purifying thrombopoietin or variant or derivative thereof

Info

Publication number: CN105541994B
Application number: CN201510869984.1A
Authority: CN
Inventors: 娄竞; 张波; 苏冬梅; 靳征
Original assignee: SHENYANG SUNSHINE PHARMACEUTICAL CO Ltd
Current assignee: SHENYANG SUNSHINE PHARMACEUTICAL CO Ltd
Priority date: 2015-12-01
Filing date: 2015-12-01
Publication date: 2019-12-06
Anticipated expiration: 2035-12-01
Also published as: CN105541994A

Abstract

The invention belongs to the field of biological pharmacy, and relates to a method for purifying thrombopoietin or variants or derivatives thereof, which comprises at least one column chromatography step applying anion exchange, and specifically comprises the following steps: (1) binding partially purified or unpurified thrombopoietin or a variant or derivative thereof to a chromatography column containing anion exchange chromatography packing having quaternary amine or diethylaminoethyl groups; (2) eluting the chromatographic column by using a urea solution containing NaCl, low pH and low conductivity to elute the hybrid protein; (3) the column is then washed with a buffer solution of high pH and high conductivity, thereby separating thrombopoietin from the column. After anion exchange chromatography is eluted by a urea solution containing NaCl, low pH and low conductivity, the impurity protein is obviously reduced, the purity of an intermediate can be effectively improved, and the comprehensive yield of the subsequent steps is obviously improved. The thrombopoietin purified by the purification method of the invention has higher purity, activity and stability.

Description

Method for purifying thrombopoietin or variant or derivative thereof

Technical Field

The present invention relates to the field of biopharmaceuticals, in particular to the purification of glycosylated proteins, and in particular to a process for the preparation of glycosylated proteins, such as thrombopoietin or variants or derivatives thereof, by elution with a urea solution of low pH and low conductivity.

Background

Thrombopoietin (TPO) is a glycoprotein hormone produced primarily by the liver and kidney, which regulates platelet production by the bone marrow. It stimulates the production and differentiation of megakaryocytes, dividing bone marrow cells into a large number of platelets, thereby increasing platelet numbers.

the TPO molecule consists of 332 amino acids. There are two pairs of disulfide bonds within the TPO molecule, 6 sites for N-bond glycosylation, with N-linked glycosylation occurring at asparagine residues at positions 176, 185, 213, 234, 319, 327. Variants of TPO include TPO1-163, TPO1-232, TPO1-151, TPO with 1-6 amino acids deleted from the N-terminus, derivatives of TPO include polyglycolized TPO, and the like. See, e.g., chinese patent application No.95190305.5, publication No. CN1131438A, which is incorporated herein by reference as part of the specification.

Many cell surface and secreted proteins produced by eukaryotic cells are modified with one or more oligosaccharide groups. This modification, known as glycosylation, can strongly influence the physicochemical properties of proteins and can also play an important role in protein stability, secretion and subcellular localization. Proper glycosylation is sometimes necessary for biological activity. Meanwhile, the pharmacokinetics and the drug treatment effect are also influenced by the glycosylation of the corresponding therapeutic recombinant protein molecules. Therefore, mammalian cells, improvement of cell culture conditions, site-directed gene sequence mutation, etc., all can cause the recombinant protein applied to human body treatment to generate glycosylation with different degrees, and influence the physicochemical and biological properties and clinical utility of the recombinant protein drug.

Chinese hamster ovary cells (CHO cells) have a wide range of applications in the expression of recombinant proteins. The glycosylation mechanism of CHO cell is similar to that of human cell, and the expressed and secreted recombinant protein has correct conformation, direct physiological activity, low possibility of mismatching and polymer occurrence, stable cell expression, simple subsequent purification process and high purity and activity. Several experiments have shown that the novel recombinant human erythrocytic stimulatory protein expressed by CHO cells is a protein molecule with non-uniform molecular weight and is highly glycosylated (references 1-3).

Because the thrombopoietin expressed by CHO cell engineering cells has non-uniform glycosylation degree, a subsequent purification step is required to separate isomers with different glycosylation degrees effectively and stably so as to produce a product with stable quality.

disclosure of Invention

The invention aims to provide a method for purifying thrombopoietin, which has the characteristics of simple and convenient operation, low cost, high recovery rate, stable process and the like.

The invention relates to a method for purifying thrombopoietin or variants or derivatives thereof, characterized in that the method comprises at least one step of column chromatography using anion exchange, wherein the anion column chromatography uses quaternary amine or diethylaminoethyl groups as the ligand packing.

According to the invention, the column chromatography step comprises a urea solution elution process with low pH and low conductivity, and comprises the following specific steps:

(1) Binding the thrombopoietin or variant or derivative thereof after the initial purification or without purification to a chromatographic column for anion exchange chromatography;

(2) eluting the column with a low pH and low conductivity urea solution to elute the contaminating proteins;

(3) and washing the column with a buffer solution of high pH and high conductivity, thereby separating thrombopoietin from the column.

The method for purifying thrombopoietin or variants or derivatives thereof of the present invention comprises the following optional combination of 3-5 steps: cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, hydrophobic chromatography, gel filtration chromatography and complex ion exchange chromatography. According to the present invention, each of the above-mentioned chromatography steps may be carried out once, twice or more, and for example, anion exchange chromatography includes anion exchange chromatography a, or a combination of anion exchange chromatography a and anion exchange chromatography b. The sequence of the above-described chromatography steps can be combined arbitrarily according to experience and need by the person skilled in the art, according to the invention.

According to the invention, anion exchange chromatography a uses a filler with a quaternary amine group or diethylaminoethyl group as a functional group, the loading is carried out at pH7.0 + -0.5 (preferably pH7.0 + -0.2), the hetero-protein is eluted by using a urea solution with pH4.5 + -0.5 and conductivity 4.0 + -1.0 mS/cm, and the target protein is eluted at pH7.0 + -0.5 (preferably pH7.0 + -0.2) and conductivity 16.0 + -2.0 mS/cm. The anion exchange chromatography a can effectively clean and remove the foreign protein by using a urea solution with low pH and low conductivity, thereby achieving the purpose of improving the purity.

According to the invention, cation exchange chromatography adopts sulfopropyl or carboxymethyl group as filler of ligand, hydrophobic chromatography adopts phenyl or butyl group as filler of ligand, reverse phase chromatography adopts butyl group as filler of ligand, anion exchange chromatography a and b respectively and independently adopt quaternary amino or diethylaminoethyl group as filler of ligand, gel filtration chromatography adopts agarose or dextran as filler of matrix, and composite ion exchange chromatography adopts filler containing phenyl and carboxyl as ligand.

according to the present invention, the variants of TPO include TPO1-163, TPO1-232, TPO1-151, TPO in which 1-6 amino acids are deleted from the N-terminus, and the derivatives of TPO include polyglycolized TPO, etc.

In a preferred embodiment, the present invention elutes the hetero protein using a urea solution containing NaCl (e.g., 0.04mol/L NaCl), low pH, and low conductivity, and elutes the target protein using a buffer solution containing NaCl (e.g., 0.15mol/L NaCl), high pH, and high conductivity. However, it is easily understood by those skilled in the art that the present invention is not limited to NaCl, and LiCl or KCl may be used as long as suitable conductivity is provided.

According to the invention, the low pH is 4.5 + -0.5 and the low conductivity is 4.0 + -1.0 mS/cm; high pH of 7.0 + -0.5, preferably 7.0 + -0.2, high conductivity of 16.0 + -2.0 mS/cm; and the high-pH and high-conductivity buffer solution is a Tris & HCl buffer solution containing 0.15mol/L NaCl, wherein the pH is 7.0 +/-0.2, and the conductivity is 16.0 +/-2.0 mS/cm. However, it will be readily understood by those skilled in the art that the present invention is not limited to Tris-HCl buffer containing NaCl, and that other common buffers, for example, phosphate buffer, may be used as long as the appropriate pH and conductivity are provided.

According to the invention, thrombopoietin is glycosylated protein, and the molecular weight is 70-120 kD.

Compared with the purification process without urea or only low-pH urea, the purification method provided by the invention has the advantages that the impurity protein is eluted by the combined action of NaCl and urea under the condition of low conductivity, the purity of the intermediate can be improved to the greatest extent, the purification pressure of the subsequent steps is reduced, and the production cost is obviously reduced.

In the step a of the anion exchange chromatography of the embodiments 1 to 5, the low pH urea solution added with NaCl is used for eluting the hybrid protein, and the hybrid protein is fully eluted under the combined action of the NaCl and the low pH urea, so that a good effect is obtained. The difference between example 6 and example 1 is that only low pH urea is used to elute the heteroprotein in step a of anion exchange chromatography without adding NaCl, and the results show that the final sample purity and the total activity yield are not as good as those in example 1, especially the purity of the target protein in one step is very different.

Drawings

In order to more clearly describe the technical solution of the present invention, the following brief description is provided with reference to the accompanying drawings. It should be apparent that these drawings depict only some specific embodiments of the invention herein. The present invention includes, but is not limited to, these figures.

Figure 1 shows the cation exchange chromatography chromatogram of example 1.

FIG. 2 shows the anion exchange chromatography a chromatogram of example 1.

FIG. 3 shows a reverse phase chromatographic chromatogram of example 1.

FIG. 4 shows the anion exchange chromatography b chromatogram of example 1.

FIG. 5 shows a gel filtration chromatography chromatogram of example 1.

figure 6 shows the hydrophobic chromatography chromatogram of example 2.

Figure 7 shows the composite ion exchange chromatography chromatogram of example 2.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the present invention will be described below with reference to examples. The description is intended to be illustrative of the features and advantages of the invention, and should not be taken to limit the scope of the invention.

Example 1

the novel recombinant human Thrombopoietin (TPO) engineering cell strain is cultured in suspension to obtain culture solution. The "culture solution" in the present invention refers to a culture solution containing a target protein separated from cells and cell debris by centrifugation, filtration, ultrafiltration or the like according to a method generally used in the art.

The specific process steps are as follows:

(1) And a cation exchange chromatography column (SP Sepharose FF, 5X 10cm, 200ml), wherein 10CV (i.e., 10 column volumes) is equilibrated with 0.05mol/L of a citric acid buffer (CB, pH 6.0. + -. 0.2), the harvested culture solution is loaded, 10CV is equilibrated with 0.05mol/L of CB (pH 6.0. + -. 0.2) after loading, the hetero-protein is eluted with 0.05mol/L of CB to 0.1mol/L of NaCl (pH 6.0. + -. 0.2), and the target protein is eluted with 0.05mol/L of CB to 0.25mol/L of NaCl (pH 6.0. + -. 0.2) (see FIG. 1 for an elution peak image). The purity of the target protein is 39.1 percent, and the activity recovery rate is 61.2 percent.

(2) Anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), equilibration of 10CV with 0.02mol/L Tris HCl (pH7.0 + -0.2), loading of diluted cation exchange chromatography active peaks, equilibration of 10CV with 0.02mol/L Tris HCl (pH7.0 + -0.2) after loading, elution of the hetero-proteins with 6mol/L urea solution-0.04 mol/L NaCl (pH 4.5 + -0.5, conductivity 4.0 + -1.0 mS/cm), elution of the urea with 0.02mol/L Tris HCl-0.04mol/L NaCl (pH7.0 + -0.2, conductivity 6.0 + -1.0 mS/cm), and elution of the proteins with 0.02mol/L Tris HCl-0.15mol/L (pH7.0 + -0.2, conductivity 16.0 + -2.0 mS/cm) (see FIG. 2 for elution chart of target protein). The purity of the target protein is 81.9 percent, and the activity recovery rate is 68.9 percent.

(3) And a reversed phase chromatographic column (filler C4, pore diameter 15 μm, 125ml), balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), loading the active peak of anion exchange chromatography, balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), eluting the hetero-protein with 0.05mol/L Tris HCl-40% isopropanol (pH 6.5 + -0.2) first, and eluting the target protein with 0.05mol/L Tris HCl-50% isopropanol (pH 6.5 + -0.2) (see FIG. 3). The purity of the target protein is 93.6%, and the activity recovery rate is 85.4%.

(4) And anion exchange chromatography b column (Q Sepharose FF, 1.6X 10cm, 20ml), equilibrating 5CV with 0.05mol/L Tris-HCl (pH7.0 + -0.2), loading the reverse phase chromatographic activity peak, equilibrating 5CV with 0.05mol/L Tris-HCl (pH7.0 + -0.2) after loading, and then eluting the target protein with 0.05mol/L Tris-HCl-0.25 mol/L NaCl (pH7.0 + -0.2) (see FIG. 4 for the peak elution diagram). The purity of the target protein is 96.2 percent, and the activity recovery rate is 97.5 percent.

(5) And a gel filtration chromatography column (Superase 12, 2.6X 100cm, 530ml), which is equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm) for 10CV, the anion exchange chromatography b active peak is loaded, after which the anion exchange chromatography b active peak is equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm), and thereafter the eluted target protein is collected (the peak elution is shown in FIG. 5). The purity of the target protein is 99.3 percent, and the activity recovery rate is 83.0 percent.

The culture broth was purified according to example 1, and the final purified sample had a purity of 99.3% and a total activity recovery of 29.1%.

Example 2

The specific process steps are as follows:

(1) And a cation exchange chromatography column (SP Sepharose FF, 5X 10cm, 200ml), wherein 10CV is equilibrated with 0.05mol/L CB (pH 6.0. + -. 0.2), the harvested culture solution is loaded, 10CV is equilibrated with 0.05mol/L CB (pH 6.0. + -. 0.2) after loading, the hetero-protein is eluted with 0.05mol/L CB-0.1mol/L NaCl (pH 6.0. + -. 0.2), and the target protein is eluted with 0.05mol/L CB-0.25mol/L NaCl (pH 6.0. + -. 0.2).

(2) an anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), diluting the cation exchange chromatography active peak, loading, equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), eluting the hetero-protein with 6mol/L urea solution-0.04 mol/L NaCl (pH 4.5 + -0.5, conductivity 4.0 + -1.0 mS/cm), eluting the urea with 0.02mol/L Tris-HCl-0.04 mol/L NaCl (pH7.0 + -0.2, conductivity 6.0 + -1.0 mS/cm), and finally eluting the target protein with 0.02mol/L Tris-HCl-0.15 mol/L NaCl (pH7.0 + -0.2, 16.0 + -2.0 mS/cm).

(3) The column (Phenyl Sepharose FF, 5X 10cm, 200ml) was equilibrated with 1.5mol/L (NH4)2SO4(pH 7.0. + -. 0.5) for 10CV, the anion exchange chromatography a peak activity was adjusted for conductivity and then applied to the column, after the application, the anion exchange chromatography a peak activity was equilibrated with 1.5mol/L (NH4)2SO4(pH 7.0. + -. 0.5) for 10CV, first the impurity protein was eluted with 0.9mol/L (NH4)2SO4(pH 7.0. + -. 0.5), and then the objective protein was eluted with 0.6mol/L (NH4)2SO4(pH 7.0. + -. 0.5) (see FIG. 6 for peak elution).

(4) And a composite ion exchange chromatography column (Capto MMC, 2.6X 10cm, 53ml), wherein 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2), the hydrophobic chromatography activity peak is diluted and then subjected to loading, and after the loading, 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2), and then the target protein is eluted with 0.05mol/L Phosphate Buffer Solution (PBS) -0.25mol/L NaCl (pH 7.5. + -. 0.2) (see FIG. 7 for an elution peak).

The culture broth was purified according to example 2, and the final purified sample had a purity of 98.7% and a total activity recovery of 16.2%.

Example 3

The specific process steps are as follows:

(1) An anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), loading the harvested culture fluid, equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), then eluting the hetero-protein with 6mol/L urea-0.04 mol/L NaCl solution (pH 4.5 + -0.5, conductivity 4.0 + -1.0 mS/cm), then eluting the urea with 0.02mol/L Tris-HCl-0.04 mol/L NaCl (pH7.0 + -0.2, conductivity 6.0 + -1.0 mS/cm), and finally eluting the target protein with 0.02mol/L Tris-0.15 mol/L NaCl (pH7.0 + -0.2, conductivity 16.0 + -2.0 mS/cm).

(2) And a reversed phase chromatographic column (filler C4, aperture diameter 15 μm, 125ml), balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), loading the active peak of anion exchange chromatography a, balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), eluting the hetero-protein with 0.05mol/L Tris HCl-40% isopropanol (pH 6.5 + -0.2) first, and eluting the target protein with 0.05mol/L Tris HCl-50% isopropanol (pH 6.5 + -0.2).

(3) And a composite ion exchange chromatography column (Capto MMC, 2.6X 10cm, 53ml), wherein 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2), the active peak of reverse phase chromatography is diluted and then applied, 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2) after application, and then the target protein is eluted with 0.05mol/L PBS-0.25mol/L NaCl (pH 7.5. + -. 0.2).

The culture broth was purified according to example 3, and the final purified sample had a purity of 98.4% and a total activity recovery of 26.9%.

Example 4

The specific process steps are as follows:

(1) An anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), loading the sample, equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2) after loading, then eluting the hybrid with 6mol/L urea-0.04 mol/L NaCl solution (pH 4.5 + -0.5, conductivity 4.0 + -1.0 mS/cm), then eluting the urea with 0.02mol/L Tris-HCl-0.04 mol/L NaCl (pH7.0 + -0.2, conductivity 6.0 + -1.0 mS/cm), and finally eluting the target protein with 0.05mol/L Tris-HCl-0.15 mol/L NaCl (pH7.0 + -0.2, conductivity 16.0 + -2.0 mS/cm).

(2) and a composite ion exchange chromatography column (Capto MMC, 2.6X 10cm, 53ml), wherein 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2), the anion exchange chromatography a active peak is diluted and then subjected to loading, 10CV is equilibrated with 0.05mol/L CB-0.15mol/L NaCl (pH 6.0. + -. 0.2) after loading, and then the target protein is eluted with 0.05mol/L PBS-0.25mol/L NaCl (pH 7.5. + -. 0.2).

(3) And anion exchange chromatography b column (Q Sepharose FF, 1.6X 10cm, 20ml), using 0.05mol/L Tris & HCl (pH7.0 + -0.2) to balance 5CV, diluting the active peak of the complex ion exchange chromatography, loading, using 0.05mol/L Tris & HCl (pH7.0 + -0.2) to balance 5CV, and using 0.05mol/L Tris & HCl-0.25mol/L NaCl (pH7.0 + -0.2) to elute the target protein.

(4) then, the gel filtration chromatography column (Superase 12, 2.6X 100cm, 530ml) was equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm) for 10CV, the anion exchange chromatography b active peak was applied, after which the anion exchange chromatography b active peak was equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm), and the eluted target protein was collected.

The culture broth was purified according to example 4, and the final purified sample had a purity of 98.5% and a total activity recovery of 18.8%.

example 5

The specific process steps are as follows:

(1) An anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2), loading the sample, equilibrating 10CV with 0.02mol/L Tris-HCl (pH7.0 + -0.2) after loading, then eluting the hybrid with 6mol/L urea-0.04 mol/L NaCl solution (pH 4.5 + -0.5, conductivity 4.0 + -1.0 mS/cm), then eluting the urea with 0.02mol/L Tris-HCl-0.04 mol/L NaCl (pH7.0 + -0.2, conductivity 6.0 + -1.0 mS/cm), and finally eluting the target protein with 0.02mol/L Tris-HCl-0.15 mol/L NaCl (pH7.0 + -0.2, conductivity 16.0 + -2.0 mS/cm).

(3) And a hydrophobic chromatography column (Phenyl Sepharose FF, 5 × 10cm, 200ml), wherein 10CV is equilibrated with 1.5mol/L (NH4)2SO4(pH7.0 + -0.5), the conductivity of the active peak of reverse phase chromatography is adjusted, the sample is loaded, after the 10CV is equilibrated with 1.5mol/L (NH4)2SO4(pH7.0 + -0.5), the impurity protein is eluted with 0.9mol/L (NH4)2SO4(pH7.0 + -0.5), and the target protein is eluted with 0.6mol/L (NH4)2SO4(pH7.0 + -0.5).

The culture broth was purified according to example 5, and the final purified sample had a purity of 98.0% and a total activity recovery of 19.5%.

example 6

The specific process steps are as follows:

(1) The resulting culture broth was applied to a cation exchange chromatography column (SP Sepharose FF, 5X 10cm, 200ml) and equilibrated with 0.05mol/L CB (pH 6.0. + -. 0.2) for 10CV (i.e., 10 column volumes), and then equilibrated with 0.05mol/L CB (pH 6.0. + -. 0.2) for 10CV, the hetero-protein was eluted with 0.05mol/L CB-0.1mol/L NaCl (pH 6.0. + -. 0.2), and the target protein was eluted with 0.05mol/L CB-0.25mol/L NaCl (pH 6.0. + -. 0.2). The purity of the target protein is 38.9 percent, and the activity recovery rate is 61.5 percent.

(2) An anion exchange chromatography a column (Q Sepharose FF, 5X 10cm, 200ml), which is equilibrated with 0.02mol/L Tris-HCl (pH 7.0. + -. 0.2) for 10CV, which is a sample obtained by diluting the cation exchange chromatography active peak, which is equilibrated with 0.02mol/L Tris-HCl (pH 7.0. + -. 0.2) for 10CV, followed by elution of the hetero-protein with 6mol/L urea solution (pH 4.5. + -. 0.5), followed by elution of the urea with 0.02mol/L Tris-HCl-0.04 mol/L NaCl (pH 7.0. + -. 0.2, conductivity 6.0. + -. 1.0mS/cm), and elution of the objective protein with 0.02mol/L Tris-HCl-0.15 mol/L NaCl (pH 7.0. + -. 0.2, conductivity 16.0. + -. 2.0 mS/cm). The purity of the target protein is 50.2 percent, and the activity recovery rate is 82.5 percent.

(3) And a reversed phase chromatographic column (filler C4, aperture diameter 15 μm, 125ml), balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), loading the active peak of anion exchange chromatography a, balancing 10CV with 0.05mol/L Tris HCl-5% isopropanol (pH 6.5 + -0.2), eluting the hetero-protein with 0.05mol/L Tris HCl-40% isopropanol (pH 6.5 + -0.2) first, and eluting the target protein with 0.05mol/L Tris HCl-50% isopropanol (pH 6.5 + -0.2). The purity of the target protein is 82.6%, and the activity recovery rate is 40.7%.

(4) And anion exchange chromatography b column (Q Sepharose FF, 1.6X 10cm, 20ml), using 0.05mol/L Tris & HCl (pH7.0 + -0.2) to balance 5CV, loading the reversed phase chromatography activity peak, using 0.05mol/L Tris & HCl (pH7.0 + -0.2) to balance 5CV after loading, and then using 0.05mol/L Tris & HCl-0.25mol/L NaCl (pH7.0 + -0.2) to elute the target protein. The purity of the target protein is 94.3 percent, and the activity recovery rate is 96.9 percent.

(5) Then, the gel filtration chromatography column (Superase 12, 2.6X 100cm, 530ml) was equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm) for 10CV, the anion exchange chromatography b active peak was applied, after which the anion exchange chromatography b active peak was equilibrated with 0.1mol/L PBS (pH 6.0. + -. 0.2, conductivity 20. + -.5 mS/cm), and the eluted target protein was collected. The purity of the target protein is 98.1 percent, and the activity recovery rate is 84.1 percent.

The culture broth was purified according to example 6, and the final purified sample had a purity of 98.1% and a total activity recovery of 16.8%.

The samples purified by the purification methods described in the above examples 1-6 have a purity of more than 98%, a residual foreign DNA content of less than 100pg/15000IU, a residual CHO cell protein content of less than 0.05%, and a bacterial endotoxin content of less than 2EU/10000U, both determined by SDS-PAGE and HPLC.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Reference to the literature

1.Butler M.Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems.Cytotechnology.2006；50:57-76.

2.St.Amand MM,Tran K,Radhakrishnan D,Robinson AS,Ogunnaike BA.Controllability Analysis of Protein Glycosylation in Cho Cells.Andersen MR,ed.PLoS ONE.2014；9(2):e87973.

3.Yang Z,Wang S,Halim A,Schulz MA,Frodin M,Rahman SH,et al.Engineered CHO cells for production of diverse,homogeneous glycoproteins.Nat Biotechnol (2015)33(8):842-4。

Claims

1. A method of purifying thrombopoietin, comprising: the method comprises the following steps:

(1) Balancing a cation exchange chromatography column filled with agarose containing carboxymethyl with a citric acid buffer solution with pH6.0 +/-0.2, balancing the column with a citric acid buffer solution with pH6.0 +/-0.2 after the thrombopoietin to be purified is loaded, eluting the foreign protein with a citric acid buffer solution with pH6.0 +/-0.2 and containing 0.1mol/L NaCl, and eluting the target protein with a citric acid buffer solution with pH6.0 +/-0.2 and containing 0.25mol/L NaCl;

(2) Equilibrating an anion chromatography a column filled with agarose containing quaternary amine groups with a Tris-HCl buffer solution with the pH value of 7.0 +/-0.2, loading the activity peak in the step (1), then equilibrating with the Tris-HCl buffer solution with the pH value of 7.0 +/-0.2, eluting the hetero protein with a 6mol/L urea solution containing 0.04mol/L NaCl and with the pH value of 4.5 +/-0.5 and the conductivity of 4.0 +/-1.0 mS/cm, then eluting the urea with a Tris-HCl buffer solution containing 0.04mol/L NaCl and with the pH value of 7.0 +/-0.2 and the conductivity of 6.0 +/-1.0 mS/cm, and finally eluting the target protein with Tris-HCl containing 0.15mol/L NaCl and with the pH value of 7.0 +/-0.2 and the conductivity of 16.0 +/-2.0 mS/cm;

(3) Equilibrating a reversed phase chromatographic column with a C4 filler by using Tris-HCl buffer solution containing 5% isopropanol and having a pH value of 6.5 +/-0.2, loading the activity peak in the step (2), then equilibrating the activity peak by using Tris-HCl buffer solution containing 5% isopropanol and having a pH value of 6.5 +/-0.2, firstly eluting the hybrid protein by using Tris-HCl buffer solution containing 40% isopropanol and having a pH value of 6.5 +/-0.2, and then eluting the target protein by using Tris-HCl buffer solution containing 50% isopropanol and having a pH value of 6.5 +/-0.2;

(4) Balancing an agarose anion exchange chromatography b column with a quaternary amino filler by using a Tris-HCl buffer solution with the pH value of 7.0 +/-0.2, loading an activity peak in the step (3), balancing by using a Tris-HCl buffer solution with the pH value of 7.0 +/-0.2, and eluting the target protein by using a Tris-HCl buffer solution with the pH value of 7.0 +/-0.2 and containing 0.25mol/L NaCl;

(5) And (3) a gel filtration chromatographic column adopts agarose as a base frame filler, the gel filtration chromatographic column is balanced by PBS with pH6.0 +/-0.2 and conductivity of 20 +/-5 mS/cm, the active peak in the step (4) is loaded and then balanced by PBS with pH6.0 +/-0.2 and conductivity of 20 +/-5 mS/cm, and then the eluted target protein is collected.