Preparation method of recombinant human bone morphogenetic protein-2

Abstract

The invention relates to a preparation method of recombinant human bone morphogenetic protein-2. The preparation method comprises the following steps: culturing host cells expressing recombinant human bone morphogenetic protein-2, inducing the host cells to express the recombinant human bone morphogenetic protein-2, breaking the cells and collecting precipitates to obtain crude protein, and purifying the crude protein to recover the recombinant human bone morphogenetic protein-2. The purification comprises affinity chromatography and ion exchange chromatography in sequence, a filler for the affinity chromatography comprises heparin sepharose gel, and a filler for the ion exchange chromatography purification comprises any one or a combination of at least two of diethylamino sepharose gel, Q sepharose gel or QAE sepharose gel. The brand-new purification process is designed, the purification efficiency is improved, the endotoxin content is reduced, the purity of the target protein can reach 95% or above, and the endotoxin content is lower than 10 EU / mg.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a method for preparing recombinant human bone morphogenetic protein-2. Background Art

[0002] There are many growth factors involved in the process of bone formation. They act on the osteogenesis process by regulating cell proliferation and differentiation and changing the synthesis of cell products. Therefore, they have broad application prospects in bone tissue engineering. In tissue engineering, the members of the TGF-β superfamily, especially bone morphogenetic proteins (BMPs), have a significant osteoinductive effect on osteogenesis. Among them, human bone morphogenetic protein-2 (BMP-2) is one of the most widely studied BMPs with the strongest osteogenesis induction activity. BMP-2 has the ability to induce undifferentiated mesenchymal stem cells to differentiate and proliferate into chondrocytes and osteoblasts, promote osteoblast differentiation and maturation, participate in the growth and development of bone and cartilage and their reconstruction, and thus accelerate bone defect repair. BMP-2 can bind to receptors on the cell membrane surface, activate the Smad signaling pathway in the cell, and promote decidualization of human endometrial stromal cells. Research results also found that BMP-2 can also promote or inhibit tumor cell proliferation, invasion and metastasis, becoming a hot topic in the current exploration of tumor mechanisms and prevention and treatment research.

[0003] BMP-2 is synthesized in vivo in the form of a precursor. The gene is located in the p12 region of chromosome 20. The full length of cDNA is 1587bp, and its open reading frame is 1188bp, encoding a polypeptide consisting of 397 amino acid residues. The signal peptide and propeptide are removed by protease cleavage to obtain a mature peptide consisting of 114 amino acid residues. The mature peptide folds its conservative structure normally through 7 pairs of disulfide bonds, and has biological activity in the form of homologous or heterologous dimers. However, the content of natural BMP-2 is low in various tissues, and it is necessary to use genetic recombination methods for mass production. For example, CN117164696A discloses a method for producing a recombinant human bone morphogenetic protein-2 mature peptide dimer, comprising the following steps: (a) inclusion body denaturation pretreatment; (b) protein purification after denaturation; (c) protein renaturation; (d) BMP2 dimer protein purification after renaturation, which simplifies the production method and process of recombinant human bone morphogenetic protein-2 mature peptide dimer and improves the yield. However, current gene recombination methods are still mainly based on prokaryotic systems, and the probability of obtaining biologically active proteins is small. They require renaturation, are costly, and have a long cycle. In addition, endotoxins produced after ultrasonic fragmentation require special processes to remove, which may affect protein purity and activity, and require high purification processes.

[0004] In summary, developing a method for preparing recombinant human bone morphogenetic protein, improving the yield and renaturation efficiency, and effectively removing isomers and endotoxins are of great significance for the application of human bone morphogenetic protein-2. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for preparing recombinant human bone morphogenetic protein-2, in order to achieve rapid and efficient purification to obtain human bone morphogenetic protein-2 and promote the application of human bone morphogenetic protein-2.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing recombinant human bone morphogenetic protein-2, the preparation method comprising:

[0008] Host cells expressing recombinant human bone morphogenetic protein-2 are cultured, the host cells are induced to express recombinant human bone morphogenetic protein-2, the cells are crushed and the precipitate is collected to obtain crude protein, and the crude protein is purified to recover the recombinant human bone morphogenetic protein-2; the purification comprises affinity chromatography and ion exchange chromatography in sequence, the filler of the affinity chromatography comprises heparin agarose gel, and the filler of the ion exchange chromatography purification comprises any one of diethylamino agarose gel, Q agarose gel or QAE agarose gel, or a combination of at least two of them.

[0009] In the present invention, a new purification method is designed for the process of preparing recombinant human bone morphogenetic protein-2 by the gene recombination method, which utilizes affinity chromatography and ion exchange chromatography in coordination and controls specific chromatography fillers to achieve efficient purification to obtain high-concentration recombinant human bone morphogenetic protein-2, while effectively reducing the endotoxin content. The concentration of recombinant human bone morphogenetic protein-2 can reach 10 mg / mL, the purity can reach more than 95%, and the endotoxin content can reach less than 10 EU / mg.

[0010] It can be understood that the purification process designed by the present invention for recombinant human bone morphogenetic protein-2 can be used for any wild-type or recombinant human basic fibroblast growth factor. Host cells in the art that can express recombinant human bone morphogenetic protein-2, such as Escherichia coli, can be applied to the method of the present invention without special restrictions.

[0011] It is understood that the heparin sepharose, diethylamino sepharose (DEAE Sepharose), QAE sepharose, butyl sepharose and phenyl sepharose in the present invention are well-known Sepharose (agarose) gel chromatography fillers in the art, such as Heparin Sepharose, Cytiva, 17099803, Phenyl Sepharose TM 6FF(HS)(Cytiva, 17097303) and DEAE Sepharose TM FF (Cytiva, 17070905), etc. Those skilled in the art can know and select the same type of commercially available chromatography fillers. Heparin Sepharose TM 6FF, Heparin Sepharose TM HP and Capto TM Heparin et al.

[0012] Preferably, the affinity chromatography specifically comprises:

[0013] The chromatography column was loaded with heparin agarose gel, and the chromatography column was washed with the first elution buffer, the sample was loaded, the impurity protein was washed with the first elution buffer and the second elution buffer until the baseline was stable, and the target protein was eluted with the first elution buffer and the second elution buffer.

[0014] Preferably, the first elution buffer contains Tris hydrochloride and urea.

[0015] Preferably, the first elution buffer contains 90-110 mmol / L tris(hydroxymethyl)aminomethane hydrochloride and 3-5 mol / L urea.

[0016] Preferably, the second elution buffer contains Tris hydrochloride, urea and sodium chloride.

[0017] Preferably, the second elution buffer contains 90-110 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 3-5 mol / L urea and 1-3 mol / L sodium chloride.

[0018] Preferably, the washing of impurities comprises mixing 93% to 97% of the first elution buffer and 3% to 7% of the second elution buffer by volume of 100%; and the elution comprises mixing 75% to 85% of the first elution buffer and 15% to 25% of the second elution buffer by volume of 100%.

[0019] Preferably, the ion exchange chromatography specifically comprises:

[0020] A chromatography column is assembled using any one of diethylamino agarose gel, Q agarose gel or QAE agarose gel or a combination of at least two of them, the chromatography column is rinsed with the third elution buffer, the sample is loaded, the impurities are rinsed with a mixture of the third elution buffer and the fourth elution buffer until the baseline is stable, and then the target protein is eluted with a mixture of the third elution buffer and the fourth elution buffer.

[0021] Preferably, the third elution buffer contains tris(hydroxymethylaminomethane) and urea.

[0022] Preferably, the third elution buffer contains 40-60 mmol / L tris(hydroxymethyl)aminomethane and 5-7 mol / L urea.

[0023] Preferably, the fourth elution buffer contains Tris, urea and sodium chloride.

[0024] Preferably, the fourth elution buffer contains 40-60 mmol / L tris(hydroxymethyl)aminomethane, 5-7 mol / L urea and 0.5-1.5 mol / L sodium chloride.

[0025] Preferably, the washing of impurities comprises mixing 86% to 94% of the third elution buffer and 6% to 14% of the fourth elution buffer by volume; and the elution comprises mixing 52% to 67% of the third elution buffer and 33% to 48% of the fourth elution buffer by volume.

[0026] Preferably, the purification further includes a renaturation step.

[0027] Preferably, the renaturation comprises subjecting the purified recombinant human bone morphogenetic protein-2 to continuous tangential flow feeding renaturation.

[0028] It can be understood that the continuous tangential flow feeding operation method commonly used in the art is applicable to the present invention. Specifically, in the continuous tangential flow feeding refolding, the sample solution forms a continuous annular flow direction through the peristaltic action of the pump. After entering the fiber membrane, the protein will be retained in the loop system of the device and flow, while the waste liquid will be discharged from the system through the membrane, and the protein will return to the refolding system along the loop. On the other hand, fresh refolding liquid will be continuously added in equal amounts to form a balance.

[0029] Preferably, the refolding buffer used for the refolding contains Tween, EDTA, oxidized glutathione, reduced glutathione, tris(hydroxymethyl)aminomethane hydrochloride), polyethylene glycol, arginine, cysteine ​​and NaCl.

[0030] In the present invention, a refolding method is further designed. The refolding method using a continuous tangential flow feeding refolding process combined with a specific refolding buffer system has obvious advantages over the traditional dilution refolding method. It can renature recombinant human bone morphogenetic protein-2 at a higher concentration, which can not only promote the correct folding of the protein, but also improve the folding efficiency and reduce protein aggregation to form inactive protein aggregates.

[0031] Preferably, the refolding buffer used for the refolding contains 0.5-1.5 mL / L Tween, 4-6 mmol / L EDTA, 9-11 mmol / L oxidized glutathione, 1-3 mmol / L reduced glutathione, 90-110 mM tris(hydroxymethyl)aminomethane hydrochloride, 45-55 g / L polyethylene glycol, 90-110 mmol / L arginine, 90-110 mmol / L cysteine ​​and 900-1100 mmol / L NaCl.

[0032] Preferably, the renaturation further includes a fine purification step.

[0033] Preferably, the fine purification comprises subjecting the renatured recombinant human bone morphogenetic protein-2 to hydrophobic chromatography, and the filler of the hydrophobic chromatography comprises any one of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel, or a combination of at least two thereof.

[0034] In the present invention, fine purification after renaturation is further designed, which is suitable for purifying the dimer after renaturation, removing homodimers, ensuring the purity of a single target protein, improving the quality, and ensuring safety.

[0035] Preferably, the hydrophobic chromatography specifically comprises:

[0036] A chromatography column is assembled using any one of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel, or a combination of at least two of them, the chromatography column is rinsed with the fifth elution buffer, the sample is loaded, the impurities are rinsed with a mixture of the fifth elution buffer and the sixth elution buffer until the baseline is stable, and then the target protein is eluted with a mixture of the fifth elution buffer and the sixth elution buffer.

[0037] Preferably, the fifth elution buffer contains Tris-HCl, EDTA, NaCl and ammonium sulfate.

[0038] Preferably, the fifth elution buffer contains 40-60 mmol / L Tris-HCl, 4-6 mmol / L EDTA, 1000-2000 mmol / L NaCl and 1500-2500 mmol / L ammonium sulfate.

[0039] Preferably, the sixth elution buffer contains Tris-HCl, EDTA and arginine.

[0040] Preferably, the sixth elution buffer contains 40-60 mmol / L Tris-HCl, 4-6 mmol / L EDTA and 150-250 mmol / L arginine.

[0041] Preferably, the washing of impurities includes mixing 75% to 85% of the fifth elution buffer and 15% to 25% of the sixth elution buffer by volume as 100%; and the elution includes mixing 20% ​​to 40% of the fifth elution buffer and 60% to 80% of the sixth elution buffer by volume as 100%.

[0042] Preferably, the culture includes strain recovery and fermentation culture.

[0043] Preferably, the strain recovery comprises sequentially performing primary seed culture and secondary seed culture.

[0044] Preferably, the culture medium for the secondary seed culture contains tryptone, yeast powder, NaCl, KCl, MgCl2, MgSO4 and glycerol.

[0045] In the present invention, a specific secondary seed culture medium is designed to promote rapid production of strains, further accelerate the culture cycle, and improve production capacity.

[0046] Preferably, the culture medium for the secondary seed culture contains 15-25 g / L tryptone, 4-6 g / L yeast powder, 0.5-1.5 g / L NaCl, 0.4-0.6 g / L KCl, 0.5-1.5 g / L MgCl2, 2-3 g / L MgSO4 and 1-3 mL / L glycerol.

[0047] Preferably, the basic culture medium for the fermentation culture contains tryptone, yeast powder, NaCl, glucose, KH2PO4, K2HPO4, Na2HPO4·12H2O, (NH4)2SO4, NH4Cl, MgSO4·7H2O and trace elements.

[0048] Preferably, the feed medium for the fermentation culture contains glycerol, yeast, tryptone, MgSO4·7H2O and trace elements.

[0049] Preferably, the trace elements include MnSO4·5H2O, CoCl2·6H2O, Na2MoO4·2H2O, ZnCl2, CuSO4·5H2O, H3BO4, FeSO4·7H2O and CaCl2·2H2O.

[0050] In the present invention, the basic culture medium and feed culture medium of the fermentation process are further designed, and glucose and glycerol are selected as carbon sources in different growth periods. In addition, trace elements that can promote the growth of recombinant bacteria are added, which is conducive to high-density fermentation.

[0051] Preferably, the basic culture medium contains 10-20 g / L tryptone, 8-12 g / L yeast powder, 5-7 g / L NaCl, 25-35 g / L glucose, 1-3 g / L KH2PO4, 3-5 g / L K2HPO4, 5-8 g / L Na2HPO4·12H2O, 1-2 g / L (NH4)2SO4, 0.1-0.3 g / L NH4Cl, 0.5-1.5 g / L MgSO4·7H2O, and 0.5-2 mL of trace elements.

[0052] Preferably, the feed medium contains 25-35 g / L glycerol, 80-90 g / L yeast, 80-90 g / L tryptone, 6-7 g / L MgSO4·7H2O, and 25-35 mL trace elements.

[0053] Preferably, the trace elements contain 0.5-1.5 g / L MnSO4·5H2O, 3-5 g CoCl2·6H2O, 1-3 g / L Na2MoO4·2H2O, 1-3 g / L ZnCl2, 0.5-1.5 g / L CuSO4·5H2O, 0.3-0.6 g / L H3BO4, 15-25 g / L FeSO4·7H2O and 15-25 g / L CaCl2·2H2O.

[0054] Preferably, the fermentation culture method specifically comprises:

[0055] The secondary seed bacteria are aseptically transferred into a fermentation tank at an inoculation rate of 6-8%; the rotation speed is 150-250 rpm, 35-38° C., the ventilation volume is 5-10 L / min, and the pH is adjusted to 6.80±0.05; feeding begins after 4.5-5.5 hours of fermentation, the flow rate is adjusted to 100-200 mL / h, the ventilation volume is automatically adjusted to maintain 30-40% of dissolved oxygen, 0.8-1.2 mM IPTG is added at 16.5-17.5 hours of fermentation to induce protein expression, and the fermentation is terminated after 3.5-4.5 hours of induction.

[0056] Preferably, the step of collecting the precipitate further comprises extracting the inclusion bodies.

[0057] The extraction of inclusion bodies comprises mixing the collected precipitate with the inclusion body dissolving solution, collecting the supernatant, and obtaining the inclusion body solution.

[0058] The inclusion body dissolving solution contains tris(hydroxymethylaminomethane), NaCl, EDTA and guanidine hydrochloride.

[0059] As a preferred technical solution, the method for preparing recombinant human bone morphogenetic protein-2 comprises the following steps:

[0060] (1) culturing host cells expressing recombinant human bone morphogenetic protein-2, inducing the host cells to express recombinant human bone morphogenetic protein-2, disrupting the cells and collecting precipitates, mixing the collected precipitates with inclusion body solubilization solution, collecting the supernatant, and obtaining an inclusion body solution;

[0061] (2) using heparin agarose gel to pack the chromatography column, washing the chromatography column with a first elution buffer, loading the inclusion body solution for affinity chromatography, washing the impurity protein with a mixture of the first elution buffer and the second elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the first elution buffer and the second elution buffer;

[0062] Using any one or a combination of at least two of diethylamino agarose gel, Q agarose gel or QAE agarose gel, washing the chromatography column with a third elution buffer, loading the target protein obtained by the affinity chromatography for ion exchange chromatography, washing the impurities with a mixture of the third elution buffer and the fourth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the third elution buffer and the fourth elution buffer;

[0063] (3) subjecting the target protein obtained by the ion exchange chromatography to continuous tangential flow fed-batch renaturation, wherein the renaturation buffer used for the renaturation contains Tween, EDTA, oxidized glutathione, reduced glutathione, tris(hydroxymethyl)aminomethane hydrochloride), polyethylene glycol, arginine, cysteine ​​and NaCl;

[0064] (4) using a chromatography column composed of any one or a combination of at least two of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel, washing the chromatography column with the fifth elution buffer, loading the renatured target protein, washing the impurities with a mixture of the fifth elution buffer and the sixth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the fifth elution buffer and the sixth elution buffer.

[0065] Compared with the prior art, the present invention has at least the following beneficial effects:

[0066] (1) The present invention designs a new purification method, which utilizes affinity chromatography and ion exchange chromatography in coordination, and controls specific chromatography fillers at the same time, so as to achieve efficient purification to obtain high-concentration recombinant human bone morphogenetic protein-2, while effectively reducing the endotoxin content. The concentration of recombinant human bone morphogenetic protein-2 can reach 10 mg / mL, the purity can reach more than 95%, and the endotoxin content can reach less than 10 EU / mg;

[0067] (2) The present invention further designs a refolding method, which utilizes a continuous tangential flow feeding refolding process combined with a specific refolding buffer system, which has obvious advantages over the traditional dilution refolding method, and can refold recombinant human bone morphogenetic protein-2 at a higher concentration, which can not only promote the correct folding of the protein, but also improve the folding efficiency and reduce protein aggregation to form inactive protein aggregates;

[0068] (3) The present invention further designs a specific secondary seed culture medium, which can promote the rapid production of strains, further accelerate the culture cycle, and improve production capacity; designs the basic culture medium and feed culture medium for the fermentation process, selects glucose and glycerol as carbon sources at different growth stages, and further adds trace elements that can promote the growth of recombinant bacteria, which is conducive to high-density fermentation;

[0069] (4) The present invention further designs fine purification after renaturation, which is suitable for purifying the dimer after renaturation, removing homodimers, ensuring the purity of a single target protein, improving quality, and ensuring safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The wet weight results of the bacteria obtained by culturing on different secondary seed media;

[0071] Figure 2 It is the fermentation growth curve diagram of different fermentation processes;

[0072] Figure 3 This is the result diagram of fermentation strain verification;

[0073] Figure 4 The SDS-PAGE images of the inclusion body solution and the bacterial cells after ultrasonic disruption, lane 1 is the bacterial cells after ultrasonic disruption; lane 2 is the inclusion body solution;

[0074] Figure 5 This is the SDS-PAGE image of the target protein purified by affinity chromatography in Example 2;

[0075] Figure 6 is a process diagram of ion exchange chromatography purification in Example 2;

[0076] Figure 7 This is the SDS-PAGE image of the target protein purified by ion exchange chromatography in Example 2;

[0077] Figure 8 This is a graph showing the target protein and endotoxin content after affinity chromatography purification and ion exchange chromatography purification in Example 2;

[0078] Fig. 9 It is a process diagram of ion exchange chromatography purification in Comparative Example 1;

[0079] Fig.10This is the SDS-PAGE image of the target protein purified by ion exchange chromatography in Comparative Example 1;

[0080] Fig.11 The target protein and endotoxin content after final purification in Example 2 and Comparative Example 1 are shown;

[0081] Fig.12 This is the SDS-PAGE image of the target protein after renaturation in Example 3-6;

[0082] Fig.13 This is a graph showing the refolding efficiency results in Examples 3-6;

[0083] Fig.14 The figure is a comparison chart of the renaturation time, cost and efficiency of Example 3 and Examples 4, 5 and 6;

[0084] Fig.15 This is a diagram of the fine purification process after renaturation in Example 7. DETAILED DESCRIPTION

[0085] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0086] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0087] In the specific embodiment of the present invention, Escherichia coli expressing recombinant human bone morphogenetic protein-2 is used as an example to verify the preparation method of the present invention, including fermentation, purification before renaturation, renaturation and purification after renaturation.

[0088] The amino acid sequence of recombinant human bone morphogenetic protein-2 is as follows (theoretical molecular weight 14865.97, theoretical isoelectric point 8.375):

[0089] TKKTLRTGTGSAGSAAGSGGVDQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVA PPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENE KVVLKNYQDMVVEGCGCR.

[0090] Example 1

[0091] This embodiment provides a method for fermenting recombinant human bone morphogenetic protein-2.

[0092] 1. Seed preparation

[0093] 1. Glycerol culture: Thaw the -80°C BL21 (DE3) glycerol culture expressing recombinant human bone morphogenetic protein-2 at 25°C (the seed bacteria are stored in 15% glycerol to prepare glycerol-preserved bacteria, and frozen at -80°C).

[0094] 2. Plate monocloning: Inoculate onto LB-Kana plates using the three-step streak method and culture at 37°C overnight to grow monoclones.

[0095] 3. Primary seeds: transfer the monoclonal bacteria on the plate to 10 mL of type A seed medium (Kana 50 μg / mL) and culture at 37°C, 200 rpm for 13 h.

[0096] 4. Secondary seeds: 10% of the primary seed bacteria were transferred into 100 mL of secondary seed culture medium (Kana 50 μg / mL), cultured at 37°C, 300 rpm, for 8 h.

[0097] The state of the secondary seed bacteria will directly affect the production efficiency of the subsequent amplified fermentation expression. Good culture conditions and appropriate culture medium are used to culture the seed bacteria. Four secondary seed bacteria culture medium formulas were selected for comparison and optimization, and the other conditions were the same.

[0098] The formula and dosage of 1L of type A seed culture medium are: 10g tryptone, 5g yeast powder, 10g NaCl, dilute to 1L with deionized water, and sterilize in water.

[0099] The formula and dosage of 1L of type B seed culture medium are: 20g tryptone, 5g yeast powder, 1g NaCl, 0.5g KCl, 1g MgCl2, 2.5g MgSO4, 2mL glycerol, dilute to 1L with deionized water, and sterilize in water.

[0100] The formula and dosage of 1L of type C seed culture medium are: 20g tryptone, 5g yeast powder, 4mL glycerol, dilute to 1L with deionized water, and sterilize in water.

[0101] The formula and dosage of 1L of D-type seed culture medium are: 12g tryptone, 16g yeast powder, 6g NaCl, dilute to 1L with deionized water, and sterilize in water.

[0102] OD of secondary seeds at different time points in 4 seed culture media 600 The values ​​are shown in Table 1. Note that the data at each time point are obtained from the average of three sets of parallel samples. The wet weight results of the four culture media are shown in Figure 1 shown.

[0103] Table 1

[0104] Culture medium 0.5h 2h 4h 6h 8h A 0.139 0.444 0.508 0.604 0.741 B 0.146 0.568 0.705 0.945 1.318 C 0.137 0.531 0.679 0.786 0.930 D 0.147 0.454 0.539 0.624 0.787

[0105] From Table 1 and Figure 1 The results showed that type B seed bacteria culture could promote faster growth of seed bacteria.

[0106] 2. Fermentation Tank Preparation

[0107] 1. Sterilization of fermentation tank: total volume 14L, liquid volume 8L, add 1mL defoamer before sterilization, calibrate pH electrode, polarize pH and DO electrodes for ≥6h before preparation, calibrate pH 6.86-4.01 (slope is about 1.0), and calibrate DO zero point after power off.

[0108] 2. Before preparation, power on the polarized pH and DO electrodes for ≥ 6 hours, calibrate the pH to 6.86-4.01 (the slope is about 1.0), and turn off the power to calibrate the DO zero point.

[0109] 3. Calibrate the flow rates of the 2M H2SO4 feed bottle-acid pump and the ammonia feed bottle-base pump.

[0110] 4. Dissolve each component of the basic culture medium in 7.5L of water, adjust the pH to 6.8, make up to 8L, transfer to the fermenter, and add 1mL of defoamer.

[0111] 5. Install electrodes, air filters, air supply ducts and exhaust ducts (wrap the ends with 16 layers of gauze and 4 layers of newspapers), seal the tank cover, feeding port, air supply duct, sampling tube, waterproof the electrode joint and shaft with tin foil, and extinguish the tank at 121℃×20min.

[0112] After cooling to room temperature (25°C), when installing the fermenter, supply air, condensate water, and power (motor, temperature probe, pH electrode, and DO electrode) in sequence, set the speed to 200 rpm, maintain a constant temperature of 37°C, connect the acid / base feed bottle, and vent.

[0113] At 6.37℃, 200rpm, 5L / min ventilation, tank pressure 0.05MPa, adjust pH to 6.80±0.05 with acid / base, calibrate saturated DO, slope <5, and the smaller the slope, the smaller the deviation.

[0114] 3. High-density fermentation

[0115] 1. Inoculation: Inoculate the secondary seed bacteria into the basic culture medium at an inoculation volume of 8%, add 10mL of Kana (final concentration 50μg / mL) before inoculation, and perform the whole process aseptically;

[0116] 2. Fermentation: The culture is set at 37°C, the stirring speed is 200rpm, and the ventilation is automatically adjusted at about 30L / min. The system automatically adds ammonia water or 2M (mol / L) H2SO4 to adjust the pH to maintain the pH value of the culture system at about 7.0. 0~5h temperature step control 37.00±0.05℃, pH 7.0±0.05, tank pressure 0.05MPa, ventilation 20~40L / min. DO series speed (200-600rpm) maintains DO ≥ 30%, until the speed reaches 600rpm and then the speed is constant until the end of fermentation. DO is about 30% in the first 5h of fermentation, and OD is about 20% after 5h. 600 At this time, the dissolved oxygen and pH are above 7, and the increase is ≥0.1 unit / min. Feeding begins. At this time, the carbon source in the culture medium is basically exhausted. The flow rate is 100mL / h in the first hour, and then the flow rate is adjusted to 100-200mL / h according to the growth status of the bacteria. The ventilation volume is automatically adjusted to maintain 30-40% dissolved oxygen, and the defoaming agent is added regularly. The logarithmic phase is from about 6h to the end of the 18th hour. During this period, DO is adjusted to about 40% until the speed reaches 600rpm and then a constant speed. Choose to add 1mM (mmol / L) IPTG at the 16th hour to induce protein expression, and stop harvesting after 4h of induction. After 18h, the growth rate of the bacteria entering the period of induced protein expression slows down, and the feeding rate is reduced accordingly. Samples are taken every hour during the fermentation process to detect the biomass (OD 600 , wet weight, residual sugar content), the fermentation broth was diluted 10 times to prepare electrophoresis samples and the expression level was detected by electrophoresis after the fermentation. 600 The fermentation process can be completed within 20 hours, and the biomass accumulates to OD 600 At around 70, wet weight ≥ 100g / L. The tail gas produced by fermentation is filtered through a 0.22μm membrane and then discharged. There is no environmental pollution problem in the entire fermentation process.

[0117] Experimental Group A:

[0118] Basic medium (1 L): 16 g tryptone, 10 g yeast powder, 8 g NaCl, 20 g glucose, 2 g KH2PO4, 4 g K2HPO4 and 1 g KCl, dilute to 1 L with deionized water;

[0119] Feed medium 1 L: 70 g glucose, 83 g yeast, 83 g tryptone, 6.7 g MgSO4·7H2O, and deionized water to 1 L.

[0120] Experimental Group B:

[0121] Basic culture medium (1L): 16g tryptone, 10g yeast powder, 6g NaCl, 30g glucose, 2g KH2PO4, 4g K2HPO4, 7g Na2HPO4·12H2O, 1.2g (NH4)2SO4, 0.2g NH4Cl, 1g MgSO4·7H2O, mixed trace elements (1000×) 1mL, dilute to 1L with deionized water;

[0122] Feed medium 1L: 30g glycerol, 83g yeast, 83g tryptone, 6.7g MgSO4·7H2O, 30mL trace elements (1000×), and deionized water to 1L.

[0123] The formula composition and dosage of trace elements (1000×) 1L are: weigh 1g MnSO4·5H2O, 4g CoCl2·6H2O, 2g Na2MoO4·2H2O, 2g ZnCl2, 1g CuSO4·5H2O, 0.5g H3BO4, 20g FeSO4·7H2O, and 20g CaCl2·2H2O, and make up to 1L with deionized water.

[0124] Formulas of other reagents used in the fermentation process:

[0125] (1) A 500 mL sterile feeding bottle is filled with 300 mL of 25%-28% ammonia water.

[0126] (2) 50 mL of dilute sulfuric acid: Slowly dissolve concentrated sulfuric acid in 100 mL of water, make up to 200 mL to prepare 2M (mol / L) H2SO4, and place in a 500 mL sterile feeding bottle.

[0127] (3) Kanamycin (concentration: 50 mg / mL): Filter and sterilize, aseptically dispense into 5 mL / 15 mL centrifuge tubes, and freeze at -20°C.

[0128] (4) IPTG (concentration of 1 M): 23.8 g was dissolved in 100 mL of water, filtered to sterilize, aseptically divided into 5 mL / 15 mL centrifuge tubes, and stored at -20°C.

[0129] Figure 2 The results of high-density fermentation experiments were carried out by Figure 2It can be seen that the fermentation results of the method using glucose as the carbon source in the early stage and supplementing glycerol in the later stage are significantly better than those of the ordinary culture medium containing only glucose. This is because in the high-density culture process of Escherichia coli, glucose is a conventionally used sugar in the carbon source, but it is also easy to produce acetic acid. The accumulation of acetic acid not only inhibits the growth of the bacteria, but also reduces the expression efficiency of its exogenous genes. Therefore, in the fermentation process, when the feed stream is added, the selection of a suitable carbon source is the key to successful fermentation. In practical applications, according to the needs and metabolic characteristics of the recombinant human bone morphogenetic protein growth factor Escherichia coli BL21 (DE3), glucose and glycerol are selected as carbon sources in different growth periods of Escherichia coli BL21 (DE3). In addition, trace elements that can promote the growth of recombinant bacteria are added. The addition of trace elements is conducive to the high-density fermentation of Escherichia coli BL21 (DE3). The trace element mixture improves the growth of the bacteria, especially the concentration of the bacteria in the logarithmic growth period is greatly increased.

[0130] 3. Collect bacteria in tanks: After fermentation is complete, release the fermentation liquid by sterile operation, and collect the bacteria by centrifugation (8000rpm, 4℃, 30min). The bacteria need to be washed once with PBS buffer and then centrifuged (8000rpm, 4℃, 30min).

[0131] 4. Seed Testing

[0132] 1. Shake flask: Inoculate the secondary seed bacteria into 200mL 2YT medium in a 1000mL Erlenmeyer flask at a ratio of 1:1000, and culture at 37℃ and 200rpm for 10h. Then lower the culture temperature to 30℃ and continue to culture for 10h.

[0133] 2. Sample preparation: Sample 30 μL of the shake flask culture liquid and mix it with an equal volume of 2×Loading Buffer to prepare the electrophoresis sample before induction; sample 30 μL of the fermentation liquid in the fermenter before centrifugation and mix it with an equal volume of 2×Loading Buffer to prepare the electrophoresis sample after induction.

[0134] 3. Electrophoresis: 15% separation gel, 5 μL sample per lane, electrophoresis until the bromophenol blue front comes out of the gel. Rinse the gel with deionized water, stain with quick staining solution until the bands are clear, rinse with deionized water to remove the light blue background, and take photos to record the electrophoresis results. Figure 3 As shown, SDS-PAGE detection showed that the sample after induction expressed a clear new band at about 15kD compared with the sample before induction, indicating that the strain was qualified.

[0135] 5. Ultrasonic fragmentation

[0136] A certain amount of bacteria was taken and suspended with ultrasonic lysis buffer at a dilution of 1:100 (m / v). The mixture was homogenized with a high-pressure homogenizer for 5 min in an ice-water bath. The bacteria were then broken with an ultrasonic crusher in an ice-water bath with ultrasonic crushing parameters (5 s working, 5 s rest, 120 min, 1000 w) until the bacterial solution was no longer viscous. After staining and smearing, and microscopic examination, when there were basically no intact bacteria, the precipitate was collected by centrifugation (12000 rpm, 4°C, 30 min).

[0137] After dissolving the precipitate with 200 mL of inclusion body solubilization solution, centrifuge (12000 rpm 16°C, 30 min) to obtain inclusion body lysate, which was then filtered through 0.45 μm and 0.22 μm in turn. The results of SDS-PAGE electrophoresis analysis are as follows Figure 4 The target protein expression level is about 10% of the total protein, and it can be stably present in the lysis buffer. After high-speed centrifugation, the protein expression level of the inclusion body solution obtained by dissolving with 6M guanidine hydrochloride is about 70% of the total protein. The purity has been greatly improved after a simple centrifugation, indicating that the early fermentation process is very suitable for the expression of recombinant human bone morphogenetic protein cell growth factor, and the entire process is easy to scale up to large-scale production.

[0138] Ultrasonic lysis buffer: contains 100 mmol / L (mM) Tris (tris(hydroxymethyl)aminomethane), 100 mM cysteine, 50 mM trehalose, 5 mM EDTA (ethylenediaminetetraacetic acid) and 1 mM PMSF (phenylmethylsulfonyl fluoride); pH 8.3.

[0139] Inclusion body solubilization solution: 100 mM Tris, 500 mM NaCl, 5 mM EDTA, 6 M guanidine hydrochloride, pH 8.3.

[0140] Example 2

[0141] This example performs purification before renaturation.

[0142] Equipment model: GE AKTApilot, UNICORN control system.

[0143] Affinity chromatography and anion exchange chromatography were performed sequentially before protein renaturation.

[0144] 1. Affinity chromatography purification

[0145] The filling specifications of the chromatography column 1 are: column bed volume 1 L, column inner diameter 100 mm.

[0146] Chromatographic medium: Heparin Sepharose (Cytiva, 17099803).

[0147] Elution buffer 1A: final concentration of 100 mmol / L Tris-HCl and final concentration of 4 mol / L urea (Urea); pH 8.0.

[0148] Elution buffer 1B: final concentration of 100mmol / L Tris-HCl and final concentration of 4mol / L urea, final concentration of 2mol / L NaCl; pH 8.0.

[0149] Wash buffer 1C: final concentration 50mmol / L Tris, final concentration 2.0mol / L NaCl and final concentration 8mol / L Urea; pH 9.0.

[0150] Wash buffer 1D: 50 mM sodium acetate buffer, final concentration of 2.0 M NaCl and final concentration of 8 mol / LUrea; pH 4.0.

[0151] Specific operations

[0152] 1.1 Equilibrate the column with elution buffer 1A at a flow rate of 200 mL / min until the baseline of the effluent is stable.

[0153] 1.2 The inclusion body lysate obtained in Example 1 was directly loaded onto the sample, with a loading volume of 200 mL. The chromatography column 1 was then flushed with a mixture of 95% (volume fraction) equilibration buffer 1A + 5% (volume fraction) elution buffer 1B at a flow rate of 100 mL / min for 3 column volumes to wash away foreign proteins.

[0154] 1.3 Elution: Use a mixture of 80% (volume fraction) elution buffer 1A + 20% elution buffer 1B for one-step elution, flow rate 100mL / min, elution volume 3L, UV detection, start collecting when the peak appears, and stop collecting when the detected peak falls back to near the baseline. The results of SDS-PAGE electrophoresis analysis are as follows: Figure 5 The purity of the target protein is 87%; and the endotoxin of the collected protein is less than 200EU / mg as detected by the Limulus amebocyte lysate gel method.

[0155] 1.4 Column post-maintenance process: Use a flow rate of 100 mL / min to alternately rinse the chromatography column 1 with washing buffer 1C and washing buffer 1D for a total of 3 times (washing buffer 1C 3 column volumes, water wash 3 column volumes, washing buffer 1D 3 column volumes; washing buffer 1C 3 column volumes, water wash 3 column volumes washing buffer 1D 3 column volumes; washing buffer 1C 3 column volumes, water wash 3 column volumes, washing buffer 1D 3 column volumes). Wash with water for 3 column volumes, wash with 20% ethanol for 1.5 column volumes, and the flow rate is 100 mL / min to ensure stable and reliable column efficiency.

[0156] 2. Ion exchange chromatography purification

[0157] Chromatographic medium 2: DEAE Sepharose TM FF, Cytiva, 17070905).

[0158] The specifications of the anion exchange chromatography column used are: column bed volume 1.0 L, column inner diameter 100 mm.

[0159] Elution buffer 2A: final concentration of 50 mmol / L Tris and final concentration of 6 mol / L Urea; pH 8.3.

[0160] Elution buffer 2B: final concentration 50mmol / L Tris, final concentration 6mol / L Urea, final concentration 1mol / LNaCl; pH 8.3.

[0161] Washing buffer 2A: final concentration of 1.0 mol / L NaOH aqueous solution.

[0162] Wash buffer 2B: 50mmol / L sodium acetate buffer, final concentration 1.0mol / L NaCl, final concentration 8mol / L Urea; pH 4.0.

[0163] Specific operations:

[0164] 2.1 Use elution buffer 2A to flush the column for 2000 mL at a flow rate of 100 mL / min to level the baseline.

[0165] 2.2 The sample collected in step 1.3 was diluted 1:3 (V / V) using elution buffer 2A, that is, 300 mL of the sample obtained by affinity chromatography purification in purification step 1 was fully mixed with 900 mL of elution buffer 2A, and the sample was loaded at a flow rate of 100 mL / min.

[0166] 2.3 Use 10% (volume fraction) elution buffer 2B + 90% (volume fraction) elution buffer 2A at a flow rate of 20 mL / min to flush impurities, and 40% (volume fraction) elution buffer 2B + 60% (volume fraction) elution buffer 2A to flush 1 column volume to elute the target protein. Start collecting when the absorbance at 280 nm of the UV peak graph exceeds 50 mAU, and stop collecting when the detected peak drops 50 mAU to near the baseline. The elution time is 10 minutes. The manually collected eluate is the target protein, with a volume of about 200 mL. The chromatography process is as follows: Figure 6 As shown, the arrow indicates the target protein peak. The results of SDS-PAGE electrophoresis analysis are as follows: Figure 7The purity of the target protein is above 97%, and the endotoxin of the collected protein is less than 10EU / mg as detected by the horseshoe crab reagent gel method.

[0167] 2.4 Column post-maintenance process: Use a flow rate of 100 mL / min to wash the anion exchange column with wash buffer 2A for 3 column volumes, wash with water for 3 column volumes, wash with wash buffer 2B for 3 column volumes, wash with water for 3 column volumes, and wash with 20% ethanol for 1.5 column volumes at a flow rate of 100 mL / min to ensure stable and reliable column efficiency;

[0168] 2.5 Finally, use 50L of sterile water at a low flow rate of 20mL / min to flush the affinity column and all the channels of the entire system, and finally fill the affinity column and the entire equipment piping system with 20% ethanol.

[0169] Before renaturation, the sample after affinity chromatography only needs to be passed through anion chromatography DEAE Sepharose TM FF, no need to adjust the pH value of the sample buffer solution, which not only reduces the endotoxin content, but also increases the concentration of sample protein ( Figure 8 ), the production process connection is more reasonable and effective.

[0170] Comparative Example 1

[0171] This comparative example performs purification before renaturation, and the only difference from Example 2 is the ion exchange chromatography purification step.

[0172] Affinity chromatography purification was performed by referring to the affinity chromatography purification method in Example 2.

[0173] The affinity chromatography purified product is subjected to ion exchange chromatography purification, specifically comprising:

[0174] Chromatographic medium 3: SP Sepharose TM FF, Cytiva, 17072904);

[0175] The specifications of the cation exchange chromatography column used were: column bed volume 1.0 L, column inner diameter 100 mm;

[0176] Elution buffer 3A: final concentration of 50mmol / L sodium acetate buffer, final concentration of 6mol / L Urea; pH 4.5;

[0177] Elution buffer 3B: final concentration of 50mmol / L sodium acetate buffer, final concentration of 6mol / L Urea, final concentration of 1mol / L NaCl; pH 4.5;

[0178] Washing buffer 3A: final concentration of 1.0 mol / L NaOH aqueous solution;

[0179] Washing buffer 3B: 50mmol / L sodium acetate buffer, final concentration of 2mol / L NaCl, final concentration of 6mol / L guanidine hydrochloride; pH 4.0;

[0180] Specific operations:

[0181] 1. Use elution buffer 3A to wash the column for 2000 mL at a flow rate of 100 mL / min to level the baseline;

[0182] 2. The sample collected by affinity chromatography purification was diluted with elution buffer 3A at 1:10 (V / V), that is, 300 mL of the sample purified by affinity chromatography was fully mixed with 3000 mL of elution buffer 3A, and the sample was loaded at a flow rate of 100 mL / min;

[0183] 3. Use 20% (volume fraction) elution buffer 3B + 80% (volume fraction) elution buffer 3A at a flow rate of 20 mL / min to wash impurities, and 50% (volume fraction) elution buffer 3B + 50% (volume fraction) elution buffer 3A to wash 1 column volume to elute the target protein. Start collecting when the absorbance at 280 nm of the UV peak graph exceeds 50 mAU, and stop collecting when the detected peak drops 50 mAU to near the baseline. The elution time is 10 minutes. The manually collected eluate is the target protein, with a volume of about 1200 mL. The chromatography process is as follows: Fig. 9 The results were analyzed by SDS-PAGE electrophoresis, as shown in Fig.10 The purity of the target protein is above 87%; and the endotoxin of the collected protein is less than 20EU / mg as detected by the horseshoe crab reagent gel method;

[0184] 4. Post-maintenance process of the chromatography column: Use the flow rate of 100mL / min to flush the cation exchange chromatography column SP Sepharose with flushing buffer 3A. TM FF, 3 column volumes, 3 column volumes of water wash, 3 column volumes of wash buffer 3B, 3 column volumes of water wash, 1.5 column volumes of 20% ethanol wash, flow rate 100 mL / min to ensure stable and reliable column efficiency;

[0185] 5. Finally, use 50L of sterile water at a low flow rate of 20mL / min to flush the affinity column and all passages of the entire system. Finally, fill the affinity column and the entire equipment piping system with 20% ethanol.

[0186] By comparing with the comparative example, it was found that DEAE Sepharose was selected in ion exchange chromatography purification. TM FF (anion chromatography), compared to SP Sepharose TMFF (cation chromatography) has four advantages: 1. No need to adjust the pH of the sample buffer to avoid protein precipitation. 2. Save reagent costs and improve efficiency. 3. Reduce the endotoxin content of the protein from more than 200EU / mg to 10EU / mg. 4. Without affecting the purity, it plays a role in concentrating the protein, increasing the concentration from 1mg / mL to 10mg / mL. Fig.11 .

[0187] Example 3

[0188] This example performs renaturation.

[0189] Equipment model: CentramateTM (PALL FS013K05DLV), chromatography refrigerator (10°C), 2 LangerPumpBT600-2J.

[0190] Renaturation buffer system A: final concentrations of 1 mL / L Tween X-100, 5 mmol / L EDTA, 10 mmol / L oxidized glutathione, 2 mmol / L reduced glutathione, 100 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 50 g / L polyethylene glycol 6000, 100 mmol / L arginine, 100 mmol / L cysteine ​​and 1000 mmol / L NaCl; pH 8.7.

[0191] Washing buffer 3A: sodium acetate buffer with a final concentration of 100 mmol / L acetic acid and a final concentration of 2.0 mol / L NaCl; pH 5.0.

[0192] Washing buffer 3B: final concentration of 100 mmol / L Tris hydrochloride, final concentration of 2 mol / L NaCl, and final concentration of 8 mol / L Urea; pH 9.0.

[0193] Washing buffer 3C: NaOH solution with a final concentration of 1.0 M.

[0194] The calculation method of protein refolding efficiency includes:

[0195] Protein renaturation efficiency = total protein concentration after renaturation × total volume × percentage of dimers ÷ total protein amount before renaturation.

[0196] A continuous tangential flow fed-batch refolding method was used.

[0197] (1) Peristaltic pump 2 pipe: 100 mL of 10 mg / mL ion exchange chromatography purified sample in Example 2 was added to a 900 mL sample cup containing refolding buffer system A at a flow rate of 1 mL / min, diluted to 1 mg / mL, a total of 1000 mL of sample, and placed in the sample cup for rapid mixing. The entire operation process was completed in a 10°C chromatography refrigerator.

[0198] (2) Use peristaltic pump 1 pipeline A at a flow rate of 100 mL / min, use flushing buffer 3C, 2000 mL, to clean all pipelines and tangential flow system equipment, peristaltic pump pipeline B to circulate, and peristaltic pump pipeline C to flow out.

[0199] (3) Use 100 mL / min flow rate in peristaltic pump 1 pipeline A, use 2000 mL of injection water to clean all pipelines and tangential flow system equipment, peristaltic pump pipeline B circulates, and peristaltic pump pipeline C flows out.

[0200] (4) Use 500 mL of refolding buffer solution at a flow rate of 100 mL / min in peristaltic pump line A to clean all pipelines and tangential flow system equipment, circulate in peristaltic pump line B, and flow out in peristaltic pump line C.

[0201] (5) The peristaltic pump 1 pipeline A uses a flow rate of 100 mL / min, and the sample 1000 mL is circulated through the peristaltic pump pipeline B. The peristaltic pump pipeline C is set to flow out 6 mL / min. The sample cup is automatically replenished with 6 mL / min of renaturation buffer system to the sample cup every minute through the peristaltic pump 2. The whole process takes 12 hours, and the total sample volume remains unchanged at 1000 mL. After 12 hours, 1000 mL of sample is collected. The results are analyzed by SDS-PAGE electrophoresis ( Fig.12 Lane 2), the dimer content of the target protein is above 41%; and the endotoxin of the collected protein detected by the Limulus amebocyte lysate gel method is less than 10EU / mg.

[0202] (6) Peristaltic pump 1 pipeline A4 uses a flow rate of 100 mL / min, and uses injection water, 2000 mL, to clean all pipelines and tangential flow system equipment. Peristaltic pump pipeline B4 circulates, and peristaltic pump pipeline C4 flows out.

[0203] (7) Peristaltic pump line A4 uses a flow rate of 50 mL / min, and uses flushing buffer 3A, 2000 mL, to clean all lines and tangential flow system equipment. Peristaltic pump line B4 circulates, and peristaltic pump line C4 flows out.

[0204] (8) Peristaltic pump line A4 uses a flow rate of 50 mL / min, and uses flushing buffer 3B, 2000 mL, to clean all lines and tangential flow system equipment. Peristaltic pump line B4 circulates, and peristaltic pump line C4 flows out.

[0205] (9) Peristaltic pump line A4 uses a flow rate of 50 mL / min, and uses flushing buffer 3C, 2000 mL, to clean all pipelines and tangential flow system equipment. Peristaltic pump line B4 circulates, and peristaltic pump line C4 flows out.

[0206] (10) Peristaltic pump line A4 uses a flow rate of 100 mL / min, and uses injection water, 2000 mL, to clean all pipelines and tangential flow system equipment. Peristaltic pump line B4 circulates, and peristaltic pump line C4 flows out.

[0207] (11) The peristaltic pump line A4 uses a flow rate of 100 mL / min, and uses 800 mL of 20% ethanol solution to clean all the lines and tangential flow system equipment. The peristaltic pump line B4 circulates, and the peristaltic pump line C4 flows out.

[0208] Example 4

[0209] This example performs renaturation.

[0210] Compared with Example 3, the only difference is that the refolding buffer system B (final concentration of 1 mM EDTA, 100 mM Tris hydrochloride, 100 mM NaCl, 200 mM urea, 200 mM 2-cyclohexylaminoethanesulfonic acid and 300 mM arginine, 10 mM oxidized glutathione and 2 mM reduced glutathione, pH 8.3) is used to replace the refolding buffer system A in equal amounts.

[0211] The continuous tangential flow feeding refolding method (see Example 3) was used to analyze the results by SDS-PAGE electrophoresis. Fig.12 (Lane 1) shows that the refolding efficiency of the target protein is less than 3%.

[0212] Example 5

[0213] This example performs renaturation.

[0214] Renaturation buffer system B: final concentrations of 1 mM EDTA, 100 mM Tris hydrochloride, 100 mM NaCl, 200 mM urea, 200 mM 2-cyclohexylaminoethanesulfonic acid and 300 mM arginine, 10 mM oxidized glutathione and 2 mM reduced glutathione, pH 8.3.

[0215] Using the dilution and renaturation method, 10 mL of the ion exchange chromatography purified sample in Example 2 at a concentration of 10 mg / mL was added to a 990 mL sample cup containing the renaturation buffer system B at a flow rate of 1 mL / min using peristaltic pump 2, diluted to 0.1 mg / mL, a total of 1000 mL of sample, and placed in the sample cup and mixed quickly. The entire operation process was completed in a 10°C chromatography refrigerator. After 96 hours, the results were analyzed by SDS-PAGE electrophoresis, as shown in Fig.12(Lane 3) shows that the refolding efficiency of the target protein is relatively low, about 15%.

[0216] Example 6

[0217] This example performs renaturation.

[0218] Using the dilution and renaturation method, 10 mL of 10 mg / mL growth factor was added to the 990 mL sample cup containing the renaturation buffer system A at a flow rate of 1 mL / min using the peristaltic pump 2 line to dilute to 0.1 mg / mL, a total of 1000 mL of sample, and quickly mixed in the sample cup. All operations were completed in a 10°C chromatography refrigerator. After 96 hours, the results were analyzed by SDS-PAGE electrophoresis, as shown in Fig.12 (Lane 4) shows that the refolding efficiency of the target protein is relatively low, about 10%.

[0219] Inclusion body proteins are in a soluble extended state under the action of denaturants. When the denaturants are removed or the concentration is reduced, they will spontaneously change from the denatured state to the natural stable state, forming a natural structure with biological activity. However, when the denaturants are removed, the recombinant protein folds in vitro, and there are a lot of misfolding and aggregation between molecules. The refolding efficiency is often very low. The efficiency of inclusion body protein folding and refolding actually depends on the competition between the correct folding process and the aggregation process. The requirements for the reagent formula of the refolding solution and the refolding time method are very demanding. Figure 12-14 The results show that the conventional dilution refolding method can achieve a refolding efficiency of up to 15%, which still contains mismatched isomers, a long refolding period, a large refolding volume, and expensive refolding reagents, resulting in high production costs. The present invention adopts a continuous tangential flow feed refolding method, combined with a specific refolding buffer system, which can refold recombinant human bone morphogenetic protein-2 at a higher concentration, which can not only promote the correct folding of the protein, but also improve the folding efficiency and reduce protein aggregation to form inactive protein aggregates.

[0220] That is to say, the renaturation method of the present invention using a continuous tangential flow feeding renaturation process combined with a specific renaturation buffer system has obvious advantages over the traditional dilution renaturation method, which is specifically manifested in: the renaturation time of the traditional dilution renaturation method requires 4-7 days, which is a long time; the renaturation efficiency is low, generally around 15%; and the reagent 2-cyclohexylaminoethanesulfonic acid in the renaturation buffer system B is expensive, and the renaturation cost remains high; while the present application uses a continuous tangential flow feeding renaturation process combined with a specific renaturation buffer system, with a short production cycle, a small total processing volume, a high renaturation efficiency, a reduced reagent cost, a reasonable process connection, a high protein yield, and a low cost, and is very suitable for industrial-scale production.

[0221] Example 7

[0222] This example performs purification after renaturation.

[0223] Since BMP2 protein has the typical characteristics of TGF-β family proteins: 7 cysteines, 6 of which form intramolecular disulfide bonds. Disulfide bonds are chemical bonds that connect the sulfhydryl groups of two different cysteine ​​residues in different peptide chains or the same peptide chain. Disulfide bonds are relatively stable covalent bonds. In protein molecules, they play a role in stabilizing the spatial structure of peptide chains. The more disulfide bonds there are, the greater the stability of protein molecules against external factors. However, artificial renaturation is required in prokaryotic expression, and the probability of mismatching is greatly increased. Although experiments have shown that mismatched isomers also have biological activity, there are also unknown safety risks. Therefore, purification after renaturation is required.

[0224] Purification Chromatography system.

[0225] The filling specifications of the chromatography column 4 are as follows: the column bed volume is 1 L, and the column inner diameter is 100 mm.

[0226] Chromatographic medium 4: Butyl Sepharose TM 4FF(HS), Cytiva, 17098004).

[0227] Elution buffer 4A: Final concentrations: 50 mM Tris-HCl, 5 mM EDTA, 1500 mM NaCl, and 2000 mM ammonium sulfate, pH 8.3.

[0228] Elution buffer 4B: Final concentrations are 200 mM arginine, 50 mM Tris-HCl, and 5 mM EDTA, pH 8.3.

[0229] Elution buffer 4C: Final concentrations are 200 mM arginine, 50 mM Tris-HCl, 5 mM EDTA, and 1000 mM ammonium sulfate, pH 8.3.

[0230] Wash buffer 4A: final concentration 50 mM Tris-HCl, final concentration 1.0 M NaCl, and final concentration 8 M Urea; pH 9.0.

[0231] Wash buffer 4AB: 50 mM sodium acetate buffer, 1.0 M NaCl final concentration, and 8 M Urea final concentration; pH 4.0.

[0232] Wash Buffer 4C: final concentration 0.5 M NaOH and final concentration 0.5 M NaCl.

[0233] 1. Rinse the column with 3000 mL of elution buffer 4A to balance the column in an appropriate stable buffer system at a flow rate of 100 mL / min.

[0234] 2. After mixing 1000 mL of refolding solution with 250 mL of elution buffer 4A, the sample buffer solution first passes through the chromatography column at a flow rate of 100 mL / min and the sample volume is 1250 mL.

[0235] 3. Use 80% (volume fraction) elution buffer 4A + 20% (volume fraction) elution buffer 4B at a flow rate of 20 mL / min to rinse the impurities for 3 column volumes until the baseline of the effluent is stable. Use a flow rate of 20 mL / min, starting from 80% (volume fraction) elution buffer 4A + 20% (volume fraction) elution buffer 4B, ending with 30% (volume fraction) elution buffer 4A + 70% (volume fraction) elution buffer 4B, linearly rinse 1 column volume, elute the target protein, manually collect the absorbance at 280 nm of the UV peak graph from 850 min, stop collecting when the peak to be detected falls back 100 mAU to near the baseline, the elution time is 75 minutes, and the manually collected eluate is the target protein, with a volume of about 1500 mL.

[0236] 4. Post-maintenance process of the chromatography column: Use the flow rate of 100mL / min to flush the cation exchange chromatography column SP Sepharose with flushing buffer 3A. TM FF, 3 column volumes, 3 column volumes of water wash, 3 column volumes of wash buffer 3B, 3 column volumes of water wash, 1.5 column volumes of 20% ethanol wash, flow rate 100 mL / min to ensure stable and reliable column efficiency;

[0237] 5. Finally, use 50L of sterile water at a low flow rate of 20mL / min to flush the affinity column and all passages of the entire system. Finally, fill the affinity column and the entire equipment piping system with 20% ethanol.

[0238] The present invention adopts Butyl Sepharose TM The purpose of the 4FF method is to extract pure protein. The advantage is that the Butyl chromatography filler has moderate hydrophobicity. 1000mL of the refolding solution can be directly mixed with 250mL of elution buffer 4A and loaded onto the chromatography column at a flow rate of 100mL / min. The target protein quickly binds to the chromatography column and is eluted with elution buffer 4B and elution buffer 4C in a linear salt gradient to separate the isomers of the target protein produced during the refolding process. The target protein is eluted with a linear salt gradient. The separation is monitored by measuring the absorbance at 280nm and 490nm ( Fig.15). It is suitable for the treatment of dimers after purification and refolding, removing homodimers, ensuring the purity of a single target protein, improving quality, and ensuring safety.

[0239] The purified protein was freeze-dried and stored.

[0240] Take the purified target protein, detect the concentration by lowrry method, and adjust the protein concentration to 1 mg / mL appropriately, divide the protein into 3 mL vials, load into freeze dryer, and pre-freeze the freeze dryer plate at -40℃ for 5 hours.

[0241] The freeze dryer program includes:

[0242] Procedure 1: Set the shelf temperature to -40°C;

[0243] Procedure 2: The temperature of the shelf is -40°C, and the freeze-drying plate is placed on the shelf for 60 minutes;

[0244] Procedure 3: Set the shelf temperature to -40°C, the safety pressure to 0.220 mbar, the vacuum degree to 0.100 mbar, and the time to 10 min.

[0245] Procedure 4: slowly raise the temperature of the plate to -25°C, safety pressure 0.220 mbar, vacuum degree 0.100 mbar, time 5 hours;

[0246] Procedure 5: Shelf stable temperature at -25°C, safety pressure 0.220 mbar, vacuum degree 0.100 mbar, time 5 hours;

[0247] Procedure 6: Slowly heat the plate to -20°C, safety pressure 0.220 mbar, vacuum 0.100 mbar, time 5 hours;

[0248] Procedure 7: Shelf stabilization temperature at -20°C, safety pressure 0.220 mbar, vacuum degree 0.100 mbar, time 5 hours;

[0249] Procedure 8: Shelf stable temperature at 0°C, safety pressure 0.220 mbar, vacuum degree 0.100 mbar, time 10 h;

[0250] Procedure 9: The shelf temperature is stabilized at 0°C, the safety pressure is 0.220 mbar, the vacuum degree is 0.010 mbar, and the rubber plug is pressed.

[0251] In summary, the present invention designs a new purification process for human bone morphogenetic protein cell growth factor, reduces the endotoxin content, and the purity can reach more than 95%, and the endotoxin content is lower than 10EU / mg. The fermentation and renaturation process is further designed to effectively improve the expression amount and renaturation efficiency of the target protein, remove the isomers of the dimer protein, and is suitable for large-scale industrial production.

[0252] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing recombinant human bone morphogenetic protein-2, characterized in that: The preparation method comprises: Cultivating host cells expressing recombinant human bone morphogenetic protein-2, inducing the host cells to express recombinant human bone morphogenetic protein-2, crushing the cells and collecting the precipitate to obtain crude protein, and purifying the crude protein to recover the recombinant human bone morphogenetic protein-2; The purification comprises affinity chromatography and ion exchange chromatography in sequence, the filler of the affinity chromatography comprises heparin agarose gel, and the filler of the ion exchange chromatography purification comprises any one of diethylamino agarose gel, Q agarose gel or QAE agarose gel or a combination of at least two thereof.

2. The method for preparing recombinant human bone morphogenetic protein-2 according to claim 1, characterized in that: The affinity chromatography specifically comprises: A chromatography column is loaded with heparin agarose gel, the chromatography column is washed with a first elution buffer, a sample is loaded, the impurity protein is washed with a mixture of the first elution buffer and the second elution buffer until the baseline is stable, and then the target protein is eluted with a mixture of the first elution buffer and the second elution buffer; Preferably, the first elution buffer contains tris(hydroxymethylaminomethane) hydrochloride and urea; Preferably, the second elution buffer contains Tris hydrochloride, urea and sodium chloride.

3. The method for preparing recombinant human bone morphogenetic protein-2 according to claim 1 or 2, characterized in that: The ion exchange chromatography specifically comprises: Using any one or a combination of at least two of diethylamino agarose gel, Q agarose gel or QAE agarose gel, washing the chromatography column with a third elution buffer, loading the sample, washing the impurities with a mixture of the third elution buffer and the fourth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the third elution buffer and the fourth elution buffer; Preferably, the third elution buffer contains tris(hydroxymethylaminomethane) and urea; Preferably, the fourth elution buffer contains Tris, urea and sodium chloride.

4. The method for preparing recombinant human bone morphogenetic protein-2 according to any one of claims 1 to 3, characterized in that: The purification further includes a renaturation step; The refolding comprises subjecting the purified recombinant human bone morphogenetic protein-2 to continuous tangential flow feeding refolding.

5. The method for preparing recombinant human bone morphogenetic protein-2 according to claim 4, characterized in that: The refolding buffer used in the refolding contains Tween, EDTA, oxidized glutathione, reduced glutathione, tris(hydroxymethyl)aminomethane hydrochloride), polyethylene glycol, arginine, cysteine ​​and NaCl; Preferably, the refolding buffer used for the refolding contains 0.5-1.5 mL / L Tween, 4-6 mM EDTA, 9-11 mM oxidized glutathione, 1-3 mM reduced glutathione, 90-110 mM tris(hydroxymethyl)aminomethane hydrochloride, 45-55 g / L polyethylene glycol, 90-110 mM arginine, 90-110 mM cysteine ​​and 900-1100 mM NaCl.

6. The method for preparing recombinant human bone morphogenetic protein-2 according to claim 4, characterized in that: The renaturation also includes a fine purification step; The fine purification comprises subjecting the renatured recombinant human bone morphogenetic protein-2 to hydrophobic chromatography, wherein the filler of the hydrophobic chromatography comprises any one of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel, or a combination of at least two thereof.

7. The method for preparing recombinant human bone morphogenetic protein-2 according to claim 6, characterized in that: The hydrophobic chromatography specifically comprises: Using any one or a combination of at least two of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel as a chromatography column, washing the chromatography column with a fifth elution buffer, loading the sample, washing the impurities with a mixture of the fifth elution buffer and the sixth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the fifth elution buffer and the sixth elution buffer; Preferably, the fifth elution buffer contains Tris-HCl, EDTA, NaCl and ammonium sulfate; Preferably, the sixth elution buffer contains Tris-HCl, EDTA and arginine.

8. The method for preparing recombinant human bone morphogenetic protein-2 according to any one of claims 1 to 7, characterized in that: The culture includes bacterial strain recovery and fermentation culture; Preferably, the strain recovery comprises sequentially performing primary seed culture and secondary seed culture; Preferably, the culture medium for the secondary seed culture contains tryptone, yeast powder, NaCl, KCl, MgCl2, MgSO4 and glycerol; Preferably, the basic medium for the fermentation culture contains tryptone, yeast powder, NaCl, glucose, KH2PO4, K2HPO4, Na2HPO4·12H2O, (NH4)2SO4, NH4Cl, MgSO4·7H2O and trace elements; Preferably, the feed medium for the fermentation culture contains glycerol, yeast, tryptone, MgSO4·7H2O and trace elements; Preferably, the trace elements include MnSO4·5H2O, CoCl2·6H2O, Na2MoO4·2H2O, ZnCl2, CuSO4·5H2O, H3BO4, FeSO4·7H2O and CaCl2·2H2O.

9. The method for preparing recombinant human bone morphogenetic protein-2 according to any one of claims 1 to 8, characterized in that: The step of collecting the precipitate also includes extracting the inclusion bodies; The extraction of inclusion bodies comprises mixing the collected precipitate with the inclusion body dissolving solution, collecting the supernatant, and obtaining the inclusion body solution; The inclusion body dissolving solution contains tris(hydroxymethylaminomethane), NaCl, EDTA and guanidine hydrochloride.

10. The method for preparing recombinant human bone morphogenetic protein-2 according to any one of claims 1 to 9, characterized in that: The preparation method comprises the following steps: (1) culturing host cells expressing recombinant human bone morphogenetic protein-2, inducing the host cells to express recombinant human bone morphogenetic protein-2, disrupting the cells and collecting precipitates, mixing the collected precipitates with inclusion body solubilization solution, collecting the supernatant, and obtaining an inclusion body solution; (2) using heparin agarose gel to pack the chromatography column, washing the chromatography column with a first elution buffer, loading the inclusion body solution for affinity chromatography, washing the impurity protein with a mixture of the first elution buffer and the second elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the first elution buffer and the second elution buffer; Using any one or a combination of at least two of diethylamino agarose gel, Q agarose gel or QAE agarose gel, washing the chromatography column with a third elution buffer, loading the target protein obtained by the affinity chromatography for ion exchange chromatography, washing the impurities with a mixture of the third elution buffer and the fourth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the third elution buffer and the fourth elution buffer; (3) subjecting the target protein obtained by the ion exchange chromatography to continuous tangential flow fed-batch renaturation, wherein the renaturation buffer used for the renaturation contains Tween, EDTA, oxidized glutathione, reduced glutathione, tris(hydroxymethyl)aminomethane hydrochloride), polyethylene glycol, arginine, cysteine ​​and NaCl; (4) using a chromatography column composed of any one or a combination of at least two of phenyl agarose gel, butyl agarose gel, n-butyl agarose gel or n-octyl agarose gel, washing the chromatography column with the fifth elution buffer, loading the renatured target protein, washing the impurities with a mixture of the fifth elution buffer and the sixth elution buffer until the baseline is stable, and then eluting the target protein with a mixture of the fifth elution buffer and the sixth elution buffer.

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