A method for purifying a recombinant human bone morphogenetic protein-2 dimer

Abstract

The application discloses a purification method of recombinant human bone morphogenetic protein-2 dimers. The purification method comprises the following steps: S1, carrying out affinity capture on the denatured recombinant human bone morphogenetic protein-2 inclusion body, and then adopting a renaturation liquid to perform on-column linear gradient renaturation; S2, adopting an arginine-containing buffer to elute the affinity chromatography column after S1 treatment, dropping the eluted protein liquid into an incubation buffer to perform incubation, and obtaining a protein mixture; S3, performing cation chromatography on the protein mixture, and adopting buffers containing different concentrations of arginine to flush the cation chromatography column, so that the recombinant human bone morphogenetic protein-2 dimers are obtained. The recombinant human bone morphogenetic protein-2 dimers are purified by the method, the process connection is reasonable, the production cycle is short, the obtained protein has high yield and low cost, the process is stable, and the method is very suitable for large-scale commercial production; meanwhile, the purification process also provides a reference for the research and production of similar recombinant protein drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for purifying a recombinant human bone morphogenetic protein-2 dimer, and belongs to the field of bioengineering. BACKGROUND

[0002] Bone morphogenetic protein (BMP) is also known as bone formation protein, which is a group of highly conserved functional proteins with similar structures, belonging to the TGF-β family. BMP can stimulate DNA synthesis and cell replication, thereby promoting mesenchymal cells to differentiate into osteoblasts. It is also the main factor for inducing bone and cartilage formation in vivo, and is expressed during limb growth, endochondral ossification, early fracture, and cartilage repair, and plays an important role in embryonic development and regeneration of the skeleton.

[0003] The BMP family includes BMP-1, 2, 4, 6, 7, 9, 12, 14, etc. Among them, BMP-2 has obvious effect of inducing undifferentiated fibroblasts to differentiate into osteoblasts, and can regulate the differentiation of osteoblasts and chondroblasts, induce ectopic bone formation and promote fracture healing. BMP-2 is a starting factor for bone formation, which can accelerate bone reconstruction, is an ideal target gene for bone repair gene therapy, and is the only cytokine that can induce ectopic bone formation, so it has become the most important growth factor in the study of bone tissue engineering. BMP-2 is synthesized in vivo in the form of a precursor, and the signal peptide and propeptide are removed by protease to obtain a mature peptide composed of 114 amino acid residues. The mature peptide is correctly folded by 7 pairs of disulfide bonds. The homodimer or heterodimer of the mature peptide has biological activity. At present, BMP-2 has been successfully used for the repair of nonunion and bone defect, and the industrial production of BMP-2 is helpful to the society.

[0004] BMP-2 can be prepared by natural bone extraction and purification and gene recombinant induction and expression followed by separation and purification. The natural extraction method has high cost, low yield and easy inactivation, and is not suitable for industrial production, so it is gradually replaced by the gene recombinant method. The Escherichia coli expression system has become a hot technology for producing BMP-2 by gene recombinant method due to its mature technology, high expression amount and low cost. However, the recombinant human bone morphogenetic protein-2 (rhBMP-2) expressed by Escherichia coli exists in the form of inactive inclusion body in the cell, and the extraction of rhBMP-2 dimer with biological activity from the inclusion body is the key in the research and development of recombinant protein drugs.

[0005] The common rhBMP-2 purification method has many steps and a complex process, and is only suitable for small-scale production in a laboratory and cannot realize industrialization. The renaturation process research, such as the dilution renaturation of foreign scientist Vallejo and the dialysis method renaturation of Chen Suming, needs large-scale renaturation liquid, has complex operation, long renaturation period and cannot realize large-scale production. The purification process research, such as the ion exchange and hydrophobic chromatography purification process of Xu Fang and the two-step ion exchange process of Liu Guoan, has problems of low recovery rate, high cost and poor purity.

[0006] In summary, the current rhBMP-2 purification process has problems of low protein yield, complex process route, long production period, large material consumption and high cost, and is not suitable for large-scale production. Large-scale production of rhBMP-2 for its wide clinical use is a current research difficulty. SUMMARY

[0007] The purpose of the present application is to provide a purification method of recombinant human bone morphogenetic protein-2 dimer (rhBMP-2), which has high protein yield, low cost, stable process and is very suitable for large-scale commercial production.

[0008] The purification method of recombinant human bone morphogenetic protein-2 dimer provided by the present application comprises the following steps:

[0009] S1, affinity capture is performed on the denatured recombinant human bone morphogenetic protein-2 inclusion body, and then linear gradient renaturation is performed on the column using a renaturation liquid;

[0010] S2, the affinity chromatography column after step S1 is eluted using a buffer containing arginine, and the eluted protein liquid is dropped into an incubation buffer for incubation to obtain a protein mixture;

[0011] S3, cation chromatography is performed on the protein mixture, different concentrations of arginine-containing buffer are used to flush the cation chromatography column respectively, and the recombinant human bone morphogenetic protein-2 dimer is obtained.

[0012] In the above purification method, in step S1, the recombinant human bone morphogenetic protein-2 inclusion body is denatured using a denaturation dissolving liquid;

[0013] The denaturation dissolving liquid is composed of Tris-HCl with a final concentration of 50 mmol / L, urea with a final concentration of 8 mol / L, DTT with a final concentration of 100 mmol / L, EDTA with a final concentration of 5 mmol / L and water, and has a pH range of 7.2-9.0;

[0014] The denaturation condition is that the temperature is 18-26℃, the stirring speed is 100 rpm and the stirring time is 10-12 hours.

[0015] In the purification method described above, the condition for the linear gradient reversibility in step S1 is as follows:

[0016] The chromatographic equilibration buffer consists of Tris-HCl with a final concentration of 40–60 mmol / L, urea with a final concentration of 4–8 mol / L, and water, with a pH range of 7.0–8.7.

[0017] The chromatographic refolding buffer consisted of Tris-HCl at a final concentration of 40–60 mmol / L, urea at a final concentration of 50 mmol / L, 2-cyclohexylaminoethanesulfonic acid at a final concentration of 100 mmol / L, arginine at a final concentration of 200 mmol / L, oxidized glutathione at a final concentration of 0.5 mmol / L, reduced glutathione at a final concentration of 1 mmol / L, and water, with a pH range of 7.2–9.0.

[0018] The refolding time is 10-24 hours, and the proportion of the chromatographic refolding solution is linearly gradiented from 0% to 100%, while the proportion of the chromatographic equilibration solution is reduced from 100% to 0%.

[0019] The flow rate of the chromatographic refolding solution is 1-2 mL / min;

[0020] The affinity chromatography column used can have a bed volume of 60 mL, an inner diameter of 27 mm, and be packed with heparin agarose.

[0021] In the purification method described above, in step S2, the buffer solution is composed of Tris-HCl with a final concentration of 50 mmol / L, urea with a final concentration of 2 mol / L, arginine with a final concentration of 800 mmol / L, and water, with a pH range of 7.0 to 8.7.

[0022] The incubation buffer consists of sodium acetate / acetic acid or sodium citrate / citric acid at a final concentration of 50 mmol / L, urea at a final concentration of 2 mol / L, arginine at a final concentration of 50 mmol / L, and water, with a pH range of 3.4 to 6.6.

[0023] In the purification method described above, in step S2, the collection range of the protein solution begins when the intensity of the ultraviolet absorption peak UV280 increases to 50 mAu and ends when it decreases to 50 mAu.

[0024] The elution time is 10–30 minutes;

[0025] The flow rate of the buffer solution is 10 ± 5 mL / min;

[0026] The incubation conditions are: 50-60 rpm for 120-720 min;

[0027] The elution buffer is added in an amount of 8-10 times the elution volume.

[0028] In the above purification method, in step S3, the cation chromatography is performed as follows:

[0029] The cation chromatography column is washed with a washing buffer to remove the recombinant human bone morphogenetic protein-2 monomer;

[0030] The cation chromatography column is eluted with an elution buffer, and the eluate is collected to obtain the recombinant human bone morphogenetic protein-2 dimer;

[0031] The volume of the chromatography column bed can be 62 mL, the inner diameter of the column can be 27 mm, and the chromatography medium can be SP Sepharose.

[0032] In the above purification method, in step S3, the washing buffer is composed of sodium acetate / acetic acid with a final concentration of 50 mmol / L or sodium citrate / citric acid with a final concentration of 50 mmol / L, urea with a final concentration of 2 mol / L, arginine with a final concentration of 300-550 mmol / L, and water, with a pH range of 3.4-6.6;

[0033] The elution buffer is composed of sodium acetate / acetic acid with a final concentration of 50 mmol / L, urea with a final concentration of 2 mol / L, arginine with a final concentration of 600-900 mmol / L, and water, with a pH range of 3.4-6.6.

[0034] In the above purification method, the washing time is 12-40 minutes;

[0035] The elution time is 12-40 minutes;

[0036] The flow rate of the elution buffer and the washing buffer is 10±5 mL / min.

[0037] In the above purification method, in step S3, the eluate is collected from the point when the intensity under the ultraviolet absorption peak UV280 rises to 50 mAu to the point when the intensity falls to 50 mAu.

[0038] Compared with the prior art, the method has the following beneficial effects:

[0039] 1. The on-column refolding process eliminates the generation of protein aggregation and precipitation, saves the filtration step, facilitates the recovery of denaturant, and has the advantages of small processing volume, short refolding time, high protein concentration, and high refolding rate.

[0040] 2. The column renaturation can realize the purposes of renaturation and preliminary purification simultaneously, and the two-step process is combined into one step, so that the equipment investment is reduced, the operation is simple, the cost is saved, and the production cycle is effectively shortened.

[0041] 3. The linear gradient renaturation on the column is adopted, compared with other column renaturation, the concentration of the renaturation liquid is slowly increased, the protein folding speed is slowly increased, the protein is beneficial to form a natural conformation, and the protein quality is improved.

[0042] 4. The arginine elution is adopted in the affinity chromatography, because the arginine has the effect of preventing protein aggregation, the arginine can form a salt bridge with negative charged residues and a hydrophobic bond to inhibit the aggregation between protein molecules, the solubility of the folding intermediate is increased, and the arginine elution greatly reduces the formation of the recombinant human bone morphogenetic protein-2 multimers.

[0043] 5. The eluent is directly dropped into a buffer system suitable for generating the recombinant human bone morphogenetic protein-2 dimers after the affinity chromatography, the action condition is mild, and appropriate incubation conditions can promote the folding to generate the thermodynamically stable dimers. The sample after the incubation does not need to be subjected to other treatments and can be subjected to the next chromatography stage, so that the sample treatment link is omitted, the working time is saved, and the production efficiency is improved.

[0044] 6. The arginine elution is adopted in the cation chromatography, compared with the sodium chloride elution, the eluted protein has high purity and stable state, after the step of the chromatography, the purity of the recombinant human bone morphogenetic protein-2 dimers can be more than 95%, and the yield can be more than 30%.

[0045] 7. The recombinant human bone morphogenetic protein-2 dimers with high purity can be obtained by using only two steps of purification, compared with other processes, the process is relatively stable, the production cycle is short, the purification efficiency is high, and the process is suitable for large-scale industrial production.

[0046] In summary, the recombinant human bone morphogenetic protein-2 dimers are purified by using the method, the process connection is reasonable, the production cycle is short, the protein yield is high, the cost is low, the process is stable, and the method is very suitable for large-scale commercial production; meanwhile, the purification process also provides a reference for the research and production of similar recombinant protein drugs. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is an affinity chromatography graph, P1 is the recombinant human bone morphogenetic protein-2 monomer protein peak after renaturation.

[0048] Figure 2 It is a cation chromatography sample protein purity detection graph, from left to right, P1 is the recombinant human bone morphogenetic protein-2 multimers, P2 is the recombinant human bone morphogenetic protein-2 dimers, and P3 is the target protein recombinant human bone morphogenetic protein-2 monomer.

[0049] Figure 3 Cationic chromatogram, from left to right, P1 is recombinant human bone morphogenetic protein-2 monomer, P2 is recombinant human bone morphogenetic protein-2 dimer, and P3 is the target protein recombinant human bone morphogenetic protein-2 multimer.

[0050] Figure 4 Cationic chromatogram, from left to right, P1 is recombinant human bone morphogenetic protein-2 monomer, P2 is recombinant human bone morphogenetic protein-2 dimer, and P3 is the target protein recombinant human bone morphogenetic protein-2 multimer.

[0051] Figure 5 The sample obtained from Example 1 of the present application and the control product both have significant alkaline phosphatase activity. DETAILED DESCRIPTION

[0052] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0053] The materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0054] The recombinant human bone morphogenetic protein-2 (rhBMP-2) inclusion bodies used in the following examples are obtained according to the method disclosed in Chinese Patent Application (200510132792.9): the recombinant bacteria BL21 / pET-28a-BMP2-h are cultured in LB medium at 37°C until the OD600 value reaches 0.8, and then 1 mM IPTG is added, and the induction culture is carried out under the same conditions for 4 hours. After the culture is completed, the bacterial bodies are collected by centrifugation, which are BL21 / pET-28a-BMP2-h inclusion bodies, and 15% SDS-PAGE electrophoresis detection shows that the size is 13.4 KD.

[0055] Example 1, Arginine purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) (on-column gradient concentration renaturation)

[0056] I. Denaturation of rhBMP-2 inclusion bodies

[0057] Inclusion body solubilization solution: 50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L DTT, 5 mmol / L EDTA, pH 7.8. Each concentration is the final concentration of the corresponding component in the solubilization solution, and the solvent is water.

[0058] Denaturation: 1 gram of recombinant human bone morphogenetic protein-2 inclusion bodies are solubilized with 10 mL of inclusion body solubilization solution, and after denaturation at 25°C, 100 rpm stirring for 12 hours, 0.45 nm membrane filtration is performed, and the supernatant is collected as the denatured rhBMP-2 protein solution.

[0059] II. Affinity chromatography of denatured rhBMP-2

[0060] The affinity chromatography was performed by using a purification instrument of Cytiva company.

[0061] The specifications of the affinity chromatography column were as follows: column bed volume 60 mL, column inner diameter 27 mm.

[0062] The chromatography medium was Heparin Sepharose (Cytiva company).

[0063] The equilibrium buffer M-A was 50 mmol / L Tris-HCl, 8 mol / L urea, pH 7.8.

[0064] The refolding buffer F-A was 50 mmol / L Tris-HCl, 2 mol / L urea, 100 mmol / L 2-cyclohexylaminoethanesulfonic acid, 200 mmol / L arginine, 1 mmol / L reduced glutathione, pH 7.8.

[0065] The elution buffer M-B was 50 mmol / L Tris-HCl, 2 mol / L urea, 1 mol / L arginine, pH 7.8.

[0066] The incubation buffer M-C was 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, 50 mmol / L arginine, pH 4.0.

[0067] The specific operation was as follows:

[0068] (1) Equilibrium: The equilibrium buffer was connected to the pump A, and the chromatography column was equilibrated with M-A at a flow rate of 10 mL / min until the baseline of the effluent was stable.

[0069] (2) Sample loading: The denatured rhBMP-2 obtained in step one was loaded onto the column treated in (1), and 100 mL was loaded at a flow rate of 10 mL / min by using pump A.

[0070] (3) Equilibrium: The equilibrium buffer was connected to the pump A, and the chromatography column treated in (2) was equilibrated with M-A at a flow rate of 10 mL / min until the baseline of the effluent was stable.

[0071] (4) Column refolding: The equilibrium buffer was connected to the pump A, and the refolding buffer was connected to the pump B. The proportion of pump B was controlled from 0% to 100%, and the column was washed at a flow rate of 1 mL / min for 20 hours.

[0072] (5) Elution:

[0073] Elution buffer connection B pump, with M-B to 10 mL / min flow rate rinse complex after the chromatographic column, UV absorption peak UV280 to 50 mAu start collection, when the test peak back to 50 mAu end collection, elution time is 20 minutes, elution volume of about 48 mL, eluent sample by HPLC detection, this step elution sample more rhBMP-2 monomer in the form of, content of about 60%.

[0074] Figure 1 For affinity chromatography, P1 is the recombinant human bone morphogenetic protein-2 monomer protein peak after renaturation.

[0075] (6) Incubation:

[0076] When eluted, elution sample directly into 440 mL incubation solution M-C, under the temperature conditions of 18-26 ℃, 50 revolutions per minute, incubation 720 min, mild incubation conditions make rhBMP-2 monomer combination into rhBMP-2 dimer.

[0077] III. rhBMP-2 cation chromatography

[0078] Cation chromatography using Cytiva purification instrument.

[0079] Cation chromatography column packing specifications: column bed volume 62 mL, column diameter 27 mm.

[0080] Chromatographic medium: SP Sepharose (Cytiva).

[0081] Equilibrium buffer M-C: 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, 50 mmol / L arginine, pH 4.0.

[0082] Rinse buffer Y-B: 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, 400 mmol / L arginine, pH 4.0.

[0083] Elution buffer Y-C: 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, 800 mmol / L arginine, pH 4.0.

[0084] Specific operation as follows:

[0085] (1) Equilibrium: with equilibrium buffer M-C at a flow rate of 10 mL / min to balance the chromatographic column, equilibrium to the baseline of the effluent smooth.

[0086] (2) Sample: the sample obtained after incubation at a flow rate of 10 mL / min to (1) after the treatment of the chromatographic column, sample volume of 488 mL.

[0087] Figure 2 The protein purity detection map for cationic chromatography is shown from left to right as follows: P1 is recombinant human bone morphogenetic protein-2 multimer, P2 is recombinant human bone morphogenetic protein-2 dimer, and P3 is the target protein recombinant human bone morphogenetic protein-2 monomer.

[0088] (3) Equilibrium: equilibrate the chromatography column treated in (2) with equilibrium buffer M-C at a flow rate of 10 mL / min until the baseline of the effluent is stable.

[0089] (4) Removal of rhBMP-2 monomer protein

[0090] Wash the chromatography column with cationic wash buffer Y-B at a flow rate of 10 mL / min for 3 column volumes to remove the rhBMP-2 monomer protein.

[0091] (5) Elution of rhBMP-2 dimer

[0092] Elute the chromatography column with cationic elution buffer Y-C at a flow rate of 10 mL / min. Start collecting when the UV absorption peak UV280 rises to 50 mAu, and end collecting when the detection peak falls to 50 mAu. The elution time is 30 minutes, and the elution volume is 75 mL. The protein eluted in this step is rhBMP-2 dimer (P2 in Figure 3 ).

[0093] IV. Calculation of purification yield

[0094] The inclusion bodies purified by the steps I-III are detected as follows:

[0095] The purity of the dimer after cationic chromatography is analyzed by HPLC detection, and the results are shown in Figure 4 , with a purity of 95%.

[0096] The protein concentrations after affinity chromatography refolding and cationic chromatography can be measured by ultraviolet spectrophotometry, which are 2.45 mg / L and 0.77 mg / mL, respectively. The protein sample volumes of the two are 48 mL and 75 mL, respectively. The inclusion body protein concentration before refolding is measured by ultraviolet spectrophotometry to be 1.9 mg / mL, and the refolding inclusion body sample volume is 100 mL.

[0097] Total refolding rate = amount of protein eluted by cationic chromatography / amount of inclusion body protein before refolding;

[0098] Amount of protein eluted by cationic chromatography = cationic elution protein concentration x cationic elution sample volume;

[0099] Amount of inclusion body protein before refolding = inclusion body protein concentration before refolding x refolding inclusion body sample volume;

[0100] The total yield of the process is 30.4% calculated by the above formula.

[0101] V. Protein activity detection

[0102] The protein activity was detected by C2C12 cell method. After rhBMP-2 and the control BMP-2 were applied to C2C12 cells for 72 hours, the alkaline phosphatase activity was detected. Both of them showed obvious color development and had significant alkaline phosphatase activity. The half-effective dilution fold of rhBMP-2 was 8.577, and the half-effective dilution fold of the control BMP-2 was 7.398, that is, the activity of rhBMP-2 was 115% of the control. Figure 5

[0103] Comparative Example 1: Purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) by sodium chloride (gradient concentration renaturation on column)

[0104] In the purification process of recombinant human bone morphogenetic protein-2, in addition to replacing arginine with sodium chloride in the steps of affinity chromatography and cation chromatography, the purification of rhBMP-2 was carried out using the same steps and conditions as in Example 1. In the process of affinity chromatography, the composition of the elution buffer was 50 mmol / L Tris-HCl, 2 mol / L urea, and 1 mol / L sodium chloride, pH 7.8. In the process of cation chromatography, the composition of the washing buffer was 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, and 200 mmol / L sodium chloride, pH 4.0. The composition of the elution buffer was 50 mmol / L sodium citrate / citric acid, 2 mol / L urea, and 400 mmol / L sodium chloride, pH 4.0.

[0105] The yield of rhBMP-2 dimer obtained in this comparative example was 13%, which was much lower than the yield of arginine elution, and the purity was 89%, which was relatively low, and the operation was complex, which was not suitable for large-scale industrial production.

[0106] Comparative Example 2: Purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) by arginine (fixed concentration renaturation on column)

[0107] I. Denaturation of rhBMP-2 inclusion bodies

[0108] The same as Example 1.

[0109] II. Affinity chromatography of rhBMP-2 after denaturation

[0110] Basically the same as Example 1, except that:

[0111] (5) Elution:

[0112] ​The elution buffer is connected to pump B, and the proportion of pump B is controlled at 100%. The renatured chromatographic column is washed with M-B at a flow rate of 10 mL / min. The collection is started when the UV absorption peak UV280 rises to 50 mAu, and the collection is ended when the detection peak falls to 50 mAu. The elution time is 20 minutes, and the elution volume is about 41 mL. The elution sample is detected by HPLC. The sample eluted in this step mainly exists in the form of rhBMP-2 monomer, and the content is about 46%.

[0113] (6) Incubation:

[0114] During elution, the elution sample is directly dropped into 369 mL of incubation solution M-C, and incubated at a temperature of 18-26°C and a rotation speed of 50 rpm for 720 min. The mild incubation condition enables the rhBMP-2 monomer to combine into rhBMP-2 dimer.

[0115] III. rhBMP-2 cation chromatography

[0116] The basic procedure is the same as that in Example 1, except that the sample amount in step (2) is 410 mL, and the elution volume in step (5) is 62 mL.

[0117] The yield of rhBMP-2 dimer obtained in this comparative example is 19%, which is lower than the yield of the method of the present application.

Claims

1. A method for purifying recombinant human bone morphogenetic protein-2 dimer, comprising the following steps: S1. The denatured recombinant human bone morphogenetic protein-2 inclusion bodies were affinity captured and then subjected to linear gradient refolding on a column using a refolding solution. The condition for the linear gradient reversibility is as follows: The chromatographic equilibration buffer consists of Tris-HCl with a final concentration of 40-60 mmol / L, urea with a final concentration of 4-8 mol / L, and water, with a pH range of 7.0-8.

7. The chromatographic refolding buffer consisted of Tris-HCl at a final concentration of 40–60 mmol / L, urea at a final concentration of 50 mmol / L, 2-cyclohexylaminoethanesulfonic acid at a final concentration of 100 mmol / L, arginine at a final concentration of 200 mmol / L, oxidized glutathione at a final concentration of 0.5 mmol / L, reduced glutathione at a final concentration of 1 mmol / L, and water, with a pH range of 7.2–9.

0. The refolding time is 10-24 hours, and the proportion of the chromatographic refolding solution is linearly gradiented from 0% to 100%, while the proportion of the chromatographic equilibration solution is reduced from 100% to 0%. The flow rate of the chromatographic refolding solution is 0.8-1.2 mL / min; S2. Elute the affinity chromatography column treated in step S1 with a buffer containing arginine. Add the eluted protein solution to an incubation buffer for incubation to obtain a protein mixture. The elution buffer consists of Tris-HCl at a final concentration of 50 mmol / L, urea at a final concentration of 2 mol / L, arginine at a final concentration of 800 mmol / L, and water, with a pH range of 7.0–8.

7. The incubation buffer consists of sodium acetate / acetic acid or sodium citrate / citric acid at a final concentration of 50 mmol / L, urea at a final concentration of 2 mol / L, arginine at a final concentration of 50 mmol / L, and water, with a pH range of 3.4 to 6.

6. S3. The protein mixture is subjected to cationic chromatography. The cationic chromatography column is washed and eluted with buffers containing different concentrations of arginine to obtain the recombinant human bone morphogenetic protein-2 dimer. The rinsing buffer consists of sodium acetate / acetic acid or sodium citrate / citric acid at a final concentration of 50 mmol / L, urea at a final concentration of 2 mol / L, arginine at a final concentration of 300-550 mmol / L, and water, with a pH range of 3.4-6.

6. The elution buffer consists of sodium acetate / acetic acid at a final concentration of 50 mmol / L, urea at a final concentration of 2 mol / L, arginine at a final concentration of 600-900 mmol / L, and water, with a pH range of 3.4-6.

6.

2. The purification method according to claim 1, characterized in that: In step S1, the recombinant human bone morphogenetic protein-2 inclusion bodies are denatured using a denaturing dissolving solution; The denaturing solution consisted of Tris-HCl with a final concentration of 50 mmol / L, urea with a final concentration of 8 mol / L, DTT with a final concentration of 100 mmol / L, EDTA with a final concentration of 5 mmol / L, and water, with a pH range of 7.2 to 9.

0. The denaturation conditions are: temperature 18~26℃, stirring at 100 rpm for 10~12 hours.

3. The purification method according to claim 1 or 2, characterized in that: In step S2, the collection range of the protein solution begins when the intensity of the ultraviolet absorption peak UV280 rises to 50 mAu and ends when it falls back to 50 mAu. The elution time is 10-30 minutes; The flow rate of the elution buffer is 10 ± 5 mL / min; The incubation conditions are: 40-60 rpm for 120-720 min; The amount of incubation buffer added is 8 to 10 times the elution volume.

4. The purification method according to claim 1 or 2, characterized in that: In step S3, the cation chromatography steps are as follows: The cationic chromatography column was washed with rinsing buffer to remove recombinant human bone morphogenetic protein-2 monomers; The cation chromatography column was eluted with elution buffer, and the eluent was collected to obtain the recombinant human bone morphogenetic protein-2 dimer.

5. The purification method according to claim 4, characterized in that: The rinsing time is 12-40 minutes; The elution time is 12-40 minutes; The flow rate of the elution buffer and the rinsing buffer is 10 ± 5 mL / min.

6. The purification method according to claim 5, characterized in that: In step S3, the collection range of the eluent is from when the intensity of the UV absorption peak at UV280 rises to 50 mAu and ends when it falls back to 50 mAu.

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