A fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein

Abstract

The present invention discloses a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein. The fusion protein includes a first peptide segment and a second peptide segment, the first peptide segment has an amino acid sequence as shown in SEQ ID NO: 1, the second peptide segment has an amino acid sequence as shown in formula (I), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment. The inclusion bodies including the fusion protein are obtained by genetic engineering bacteria construction, working seed culture, fermentation tank culture, bacterial recovery, bacterial crushing, inclusion body washing, inclusion body dissolution, and inclusion body renaturation steps, and the recombinant human growth hormone is obtained by filtration ultrafiltration, anion exchange chromatography rough purification, Kex2 enzyme digestion, weak anion exchange chromatography purification, ultrafiltration, sterilization filtration, and freeze-dried storage. The present invention can efficiently prepare recombinant human growth hormone with high purity and high activity, which is suitable for commercial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein. Background Art

[0002] Human growth hormone (hGH) is a non-glycosylated protein hormone secreted by eosinophilic cells of the anterior pituitary gland. It consists of 191 amino acid residues and has a molecular weight of about 22KDa. It has a wide range of biological functions, including protein synthesis, cell proliferation, lactation, immune regulation, and the metabolism of proteins, carbohydrates, and lipids. It is the most important hormone for promoting growth after birth. It is widely used in adults and children for growth hormone deficiency, severe burns, Tumer syndrome, and other diseases. It can also be used to treat short stature caused by chronic renal failure, short bowel syndrome, and delay aging in the elderly. In the past, human growth hormone could only be extracted from the human pituitary gland in clinical applications, and the source was limited and very expensive. Genetic engineering technology can produce recombinant human growth hormone (rhGH) on a large scale. The structure and amino acid sequence of recombinant human growth hormone are consistent with natural growth hormone.

[0003] hGH is a non-glycosylated protein with only two disulfide bonds and no post-translational modification. Therefore, Escherichia coli becomes the preferred host for production, and the Escherichia coli-mediated prokaryotic expression system becomes the mainstream expression method for recombinant human growth hormone at home and abroad. Escherichia coli reproduces quickly, has low cost, a clear genetic background, and mature technology, making it suitable for large-scale commercial production.

[0004] There are two main technical approaches to express rhGH in E. coli cells. One is to produce secretory rhGH, add a signal peptide gene to the N-terminus of the human growth hormone gene sequence, form a soluble signal peptide-rhGH fusion protein in the E. coli cytoplasm, and the signal peptide guides the growth hormone fusion protein into the periplasmic space. The signal peptide sequence is removed by the protease to obtain a recombinant human growth hormone that is completely consistent with the primary structure of natural human growth hormone. However, the secretion efficiency of E. coli is not high, the yield is not high, the downstream purification process technology route is complex, and it is not the preferred method for large-scale preparation. The other is to produce intracellular recombinant human growth hormone, which is expressed in the form of inclusion bodies. Recombinant human growth hormone with the correct spatial conformation is prepared by the inclusion body renaturation method. The expression amount is very high, but the renaturation effect of the inclusion body is often not good. The protein folding after renaturation is not good, the stability is poor, and the protein activity is low. In addition, due to the characteristics of E. coli's own expression, the N-terminus of the intracellular rhGH has one more methionine in the primary structure than the natural hGH. The presence of methionine makes this type of rhGH easy to induce antibodies and affect the efficacy. There are also patents such as CN 115947864 A that use inclusion body form to express recombinant human growth hormone fusion protein, and then use recombinant bovine enterokinase to enzymatically treat the fusion protein to form free target growth hormone and its fragments, whose N-terminus does not contain free amino acids and is consistent with the amino acid sequence of natural human growth hormone. However, the downstream purification process of this patent method is complicated. The downstream purification process of this patent includes hydrophobic chromatography I, molecular sieve chromatography I, anion exchange chromatography I, hydrophobic chromatography II, molecular sieve chromatography II, anion exchange chromatography II and molecular sieve chromatography III, 7 steps of chromatography, many chromatography steps and high cost. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the above-mentioned prior art, provide a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein, express the recombinant human growth hormone protein in the form of inclusion bodies in Escherichia coli, preferably a strong inducible promoter system (inducible promoter system: lPL, lPR, trc and T7, etc.), separate the growth and production stages, perform high-density fermentation to achieve high yield, high protein expression, overcome the defects and shortcomings of secretory expression, and have higher yield and lower cost under the same fermentation scale.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A fusion protein, comprising a first peptide segment and a second peptide segment, wherein the first peptide segment is a target growth hormone and a fragment thereof, the first peptide segment has an amino acid sequence as shown in SEQ ID NO: 1, the second peptide segment has an amino acid sequence as shown in formula (I), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment;

[0008] The amino acid sequence of the second peptide is:

[0009] MAXB(I)

[0010] In formula (I), M is methionine; A is at least one basic amino acid; X is at least one arbitrary amino acid; and B is an enzyme cleavage site formed by one of the dibasic amino acids.

[0011] Furthermore, in formula (I), A is RR.

[0012] Furthermore, in formula (I), X is TG.

[0013] Furthermore, in formula (I), B is KR or KK or PR or RR.

[0014] A method for preparing recombinant human growth hormone comprises the following steps: obtaining inclusion bodies comprising the fusion protein according to any one of claims 1 to 4 through the steps of constructing genetically engineered bacteria, culturing working seeds, culturing in a fermenter, recovering bacteria, crushing bacteria, washing inclusion bodies, dissolving inclusion bodies and renaturing inclusion bodies, obtaining inclusion body concentrate by filtering and ultrafiltration; then purifying the inclusion body concentrate by anion exchange chromatography to obtain anion exchange chromatography sample; then performing Kex2 enzyme digestion to obtain digestion products; and then purifying by weak anion exchange chromatography to obtain protein-containing eluate, and obtaining recombinant human growth hormone by ultrafiltration, sterilizing filtration and freeze-drying.

[0015] Furthermore, the specific steps of the anion exchange chromatography method for crude purification of the inclusion body concentrate are: use mobile phase A to balance the chromatography column for 3CV, directly load the sample after equilibration, the loading flow rate is not more than 210cm / h, balance the chromatography column with mobile phase A for 2CV, wash with mobile phase B for 3CV, and elute the weakly bound protein; then balance the chromatography column with mobile phase A for 2CV, elute the target protein with elution buffer, start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, and then combine the samples to obtain anion exchange chromatography samples, which are temporarily stored at 4°C.

[0016] Furthermore, mobile phase A is a solution of 10-50 mM Tris, pH 7-10; mobile phase B is a solution containing 10-50 mM Tris, 30-80 mM NaCl, pH 7-10; and the elution buffer is a solution containing 10-50 mM Tris, 100-200 mM NaCl, pH 7-10.

[0017] Furthermore, the specific method of Kex2 enzyme digestion is: slowly adjust the pH of the anion exchange chromatography sample to 8.0 with 2M HCl, and then slowly add a protein diluent accounting for 1 / 100 of the total volume of the sample; calculate the total protein amount of the anion exchange chromatography sample according to the Lowry and RP-HPLC results, and calculate the required Kex2 enzyme mass according to the ratio of total protein: Kex2 enzyme amount of 100:1 to 2500:1; add Kex2 protease dilution buffer to the calculated required amount of Kex2 enzyme to make the Kex2 enzyme concentration of 1 mg / ml; then add the obtained Kex2 enzyme solution to the mixture of the anion exchange chromatography sample and the protein diluent, and place it at room temperature for digestion.

[0018] Furthermore, the protein diluent is a solution containing 10-100 mM Tris, 200-500 mM CaCl2, pH 7.0-9.0; the Kex2 protease dilution buffer is a solution containing 10-100 mM Tris, 2-5 mM CaCl2, pH 8.0-9.0.

[0019] Further, the specific steps of purification by weak anion exchange chromatography are:

[0020] The enzyme cleavage product is pumped into a container of suitable size by a peristaltic pump, and then PB is pumped into the enzyme cleavage product to dilute the enzyme cleavage product to a conductivity of <3ms / cm; the mobile phase A is used to balance the chromatography column for 3CV, and the sample is loaded, and the loading flow rate is not greater than 130cm / h, and the mobile phase A is used to wash for 3CV, and 10% mobile phase B is used to elute for 4CV, and then gradient elution is performed: at the beginning, the mobile phase A is 90%, and the mobile phase B is 10%, and at the end, the mobile phase A is 50%, and the mobile phase B is 500%, and the operation is performed for 4CV; when the UV is not less than 50mAU, the collection is started, and when the UV is less than 100mAU, the collection is stopped, and the samples are combined to collect the protein-containing eluate;

[0021] Mobile phase A is a solution of 20-50 mM PB, pH 7-10;

[0022] Mobile phase B is a solution containing 20-50 mM PB, 100-500 mM NaCl, pH 7-10.

[0023] The beneficial effects of the present invention are:

[0024] A method for preparing high-purity growth hormone is proposed: obtaining inclusion bodies, renaturing the inclusion bodies, performing crude purification, removing the second peptide segment by enzyme cleavage, and performing fine purification to obtain recombinant human growth hormone that is completely consistent with the sequence of natural human growth hormone; the method increases the yield of inclusion bodies, reduces the fermentation scale at the same yield, increases the efficiency of inclusion body renaturation, has simple purification process steps, fewer chromatography steps, shortens working hours, reduces costs, is easy to scale up and control the process, and the obtained recombinant human growth hormone has high purity and high activity, and is suitable for commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SDS-PAGE electrophoresis diagram (reduced) of the secondary fermentation sample in step 3 of Example 1 (in the diagram, M: marker; lane 1: 1 h before induction; lane 2: 1 h after induction; lane 3: 2 h after induction; lane 4: 3 h after induction; lane 5: 4 h after induction; lane 6: 5 h after induction; lane 7: 6 h after induction; the target recombinant human growth hormone is at 22 KD);

[0026] Figure 2 The SDS-PAGE electrophoresis diagram (reduced) of the recombinant human growth hormone inclusion body protein after washing in step 6 of Example 1 (in the diagram, M: marker; lane 1: inclusion body precipitate after the first washing; lane 2: inclusion body precipitate after the second washing; lane 3: inclusion body precipitate after the third washing);

[0027] Figure 3 The anion exchange chromatography (crude purity) of step 10 of Example 1, the SDS-PAGE electrophoretogram of each elution fraction, and the SDS-PAGE electrophoretogram (reduced) before and after the enzyme digestion of the sample in step 11 (M: marker; lane 1: concentrate after ultrafiltration in step 9; lane 2: anion exchange chromatography flow-through; lane 3: target protein elution; lane 4: protein before enzyme digestion in step 11; lane 5: protein after enzyme digestion in step 11);

[0028] Figure 4 This is the RP-HPLC test chromatogram of the sample after enzyme digestion in step 11 of Example 1;

[0029] Figure 5 The SEC-HPLC test chromatogram of the sample after enzyme digestion in Example 1;

[0030] Figure 6 This is the RP-HPLC test chromatogram of the sample after weak anion exchange chromatography (purified) in step 12 of Example 1;

[0031] Figure 7 This is the SEC-HPLC test chromatogram of the sample after weak anion exchange chromatography (purified) in step 12 of Example 1. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention provides a recombinant human growth hormone fusion protein (i.e., a fusion protein constructed in the molecular biology stage), comprising a first peptide segment and a second peptide segment, wherein the first peptide segment is a target growth hormone and a fragment thereof, the first peptide segment has an amino acid sequence as shown in SEQ ID NO: 1, the second peptide segment has an amino acid sequence as shown in formula (1), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment.

[0034] The amino acid sequence of the first peptide is:

[0035] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:1)

[0036] The amino acid sequence of the second peptide is:

[0037] MAXB(I)

[0038] In formula (I), M is methionine; A is at least one of basic amino acids, generally including 1 to 2 amino acids; X is at least one arbitrary amino acid, generally including 1 to 4 amino acids; B is an enzyme cleavage site formed by one of dibasic amino acids (such as KR or KK or PR or RR, etc.). The enzyme cleavage site of dibasic amino acids can be specifically recognized and cut by recombinant dibasic amino acid endopeptidase (Kex2).

[0039] The amino acid sequence of recombinant human growth hormone fusion protein is:

[0040] MAXBFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(II)

[0041] The present invention is based on preparing high-purity recombinant human growth hormone from a fusion protein in the form of intracellular inclusion bodies expressed in Escherichia coli, comprising the following steps:

[0042] Step 1: Construction of genetically engineered bacteria

[0043] The N-terminus of the first peptide segment (amino acid sequence as shown in SEQ ID NO: 1) is connected to the C-terminus of the second peptide segment (amino acid sequence as shown in formula (I)) to obtain a recombinant human growth hormone fusion protein (abbreviated as fusion protein, MAXB+hGH, amino acid sequence as shown in formula (II)), and the nucleotide sequence encoding the recombinant human growth hormone fusion protein is designed based on this, the start codon is ATG, the stop codon is TAA, the sequence between the start codon ATG and the stop codon TAA is the gene sequence of the fusion protein, the front end sequence CATATG of the fusion protein is an Nde I restriction site, and CTCGAG is an Xho I restriction site, which respectively correspond to the Nde I restriction site and the Xho I restriction site in the MCS region of the target gene cloning vector pET-30a(+); Shanghai Jierui Bioengineering Co., Ltd. is commissioned to synthesize the target gene into the cloning vector pET-30a(+), and the host cell E. coli is transformed. BL21 (DE3), clone screening, plasmid extraction, enzyme digestion identification, obtain the plasmid vector carrying the growth hormone fusion protein gene sequence and the genetic engineering bacteria expressing the growth hormone fusion protein transformed with the plasmid vector.

[0044] The specific steps of transforming host cells E.coli BL21 (DE3) are as follows: 1. Take out BL21 competent cells from the -80℃ refrigerator and place them on ice to melt. Add 5μL of plasmid to the competent cells and mix them. Place them on ice for 30 minutes. 2. Put the EP tube containing the plasmid-cell mixture into a water bath, heat shock at 42℃ for 90s, then quickly take it out and place it on ice for 2 minutes. 3. Add 1mL LB liquid culture medium (without antibiotics), shake and culture at 37℃ for 2h to revive the bacteria. 4. Spread 50μL of the transformation solution on a solid LB culture medium containing 50mg / L kanamycin sulfate, and culture at 37℃ overnight for 16h.

[0045] The specific steps of clone screening are as follows: a single colony of genetically engineered bacteria grown on a solid LB medium containing 50 mg / L kanamycin sulfate is selected, inoculated into an LB medium, and cultured at 37° C. for 16 h.

[0046] The specific steps of plasmid extraction are: extract the plasmid according to the instructions of the SanPrep column plasmid DNA small-scale extraction kit produced by Sangon Biotechnology (Shanghai) Co., Ltd. to extract the plasmid vector of the growth hormone fusion protein in the genetically engineered bacteria.

[0047] Enzyme digestion identification: According to the instructions of restriction enzymes Nde I and Xho I produced by TaKaRa, the plasmid vector for extracting growth hormone fusion protein was double-digested. After agarose gel electrophoresis, the size of the double-digested fragments was consistent with expectations, and the genetically engineered bacteria expressing growth hormone fusion protein were successfully constructed.

[0048] Step 2: Cultivate working seeds

[0049] Take 1 mL of the working seed of the genetically engineered bacteria and inoculate all of it into LB shake flask medium (180 mL) containing kanamycin monosulfate (50 μg / mL) for shake flask culture at 37.0°C and 220 rpm. When the bacterial concentration OD600 is ≥ 2, and the bacterial morphology detected by optical microscopy is a typical Escherichia coli morphology and there is no other microbial contamination, the working seed culture is terminated.

[0050] Step 3: Fermentation tank culture

[0051] The cultivation scale is further expanded through a two-stage gradually enlarged bacterial fermentation system. The first-stage fermentation cultivation step is: the working seeds of the shake flask culture are inoculated at a 1% inoculation amount into a first-stage fermentation tank (5L) filled with 1.8L sterile culture medium for first-stage fermentation cultivation until the bacterial concentration OD600 is ≥20, the bacterial morphology detected by optical microscope is a typical Escherichia coli morphology, and there is no other microbial contamination, and the first-stage fermentation cultivation is terminated; wherein the culture temperature is 30.0°C, the rotation speed is 120-600rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation volume is 10-100L / min, the dissolved oxygen is ≥20.0%, and the culture time is 6-12h. The secondary fermentation culture step is: transferring all the fermentation liquid in the primary fermentation tank to a secondary fermentation tank (50L) filled with 18L sterile culture medium for secondary fermentation culture, the culture temperature is 30.0°C, the rotation speed is 120-800rpm, the pH value is 7.0 (adjusted by ammonia water), the ventilation volume is 200.0-1000.0L / min, and the dissolved oxygen is 20.0%-80.0%. During the culture process, when the carbon source (glycerol) is consumed, the pH value is on an upward trend, and the dissolved oxygen is increased, feeding is performed and the feeding rate is adjusted to make the secondary fermentation culture. The carbon source, pH value and dissolved oxygen in the first fermentation tank meet the conditions for secondary fermentation culture; after culturing for 7 to 9 hours and the bacterial concentration OD600 ≥ 100.0, add 100 to 400 g of lactose inducer or IPTG inducer (control the concentration of IPTG inducer in the secondary fermentation system to be 0.1 to 1 mM) and induce by cooling. The induction temperature is 26°C to 30°C and the induction time is 4 to 15 hours. Then cool down to 15°C and place in the tank. At the same time, take samples for SDS-PAGE electrophoresis test to detect the expression of recombinant human growth hormone fusion protein.

[0052] The components of the sterile culture medium and feed culture medium used in the primary fermentation culture and the secondary fermentation culture are:

[0053] Primary fermentation tank culture medium: peptone 5g / L, yeast powder 5g / L, disodium hydrogen phosphate 4g / L, sodium dihydrogen phosphate 3.4g / L, magnesium sulfate 1g / L, glycerol 21g / L.

[0054] Secondary fermentation tank culture medium: yeast powder 5.8g / L, dipotassium hydrogen phosphate 4g / L, potassium dihydrogen phosphate 4g / L, disodium hydrogen phosphate 7g / L, magnesium sulfate 1.2g / L, ammonium chloride 0.2g / L, ammonium sulfate 1.2g / L, calcium chloride 0.02g / L.

[0055] Feed medium: peptone 30g / L, yeast powder 15g / L, dipotassium hydrogen phosphate 7g / L, potassium dihydrogen phosphate 7g / L, disodium hydrogen phosphate 10g / L, magnesium sulfate 2.5g / L, ammonium chloride 10g / L.

[0056] Step 4: Bacteria recovery

[0057] The secondary fermentation culture fluid was centrifuged to obtain bacterial cells, and the centrifugation parameters were 15000 rpm / min and 1 hour.

[0058] Step 5: Broken cells to obtain inclusion bodies

[0059] The cells were added to a lysis buffer (containing 10-50 mM Tris (tromethamine), 0.1-5 mM metal ion chelator EDTA or EGTA, 0.5-2 M denaturant urea or 0.1-0.5 M denaturant guanidine hydrochloride, 1-5% non-ionic surfactant (Triton X-100 or Tween-20), pH 7.0-8.5) at a mass volume ratio of 1:10 between the cells and the lysis buffer, and the cells were dispersed using a high-speed disperser at a speed of 700-1000 bar, and the homogenate was broken 1-5 times, followed by centrifugation at 4°C and 9000 rpm for 30 minutes to collect the inclusion body precipitate.

[0060] Step 6: Inclusion body washing

[0061] Add inclusion body washing buffer I (containing 10-50 mM Tris, 0.1-5 mM metal ion chelator EDTA or EGTA, 1-5% non-ionic surfactant (Triton X-100 or Tween-20), pH 7.0-8.5) to the inclusion body precipitate, the inclusion body washing buffer I is added in a ratio of 1:3 in mass volume ratio of the bacteria added in step 5 to the inclusion body washing buffer I, use a high-speed disperser to disperse the inclusion body precipitate for the first washing, and collect the inclusion body precipitate after centrifugation at 4°C and 9000 rpm for 30 min. Then, according to the mass volume ratio of the added bacteria to the inclusion body washing buffer II of 1:3 in step 5, the inclusion body washing buffer II (10-50mM Tris, 0.1-5mM metal ion chelator EDTA or EGTA, pH value 7.0-8.5) was added, and the inclusion body precipitate was dispersed by a high-speed disperser to wash the inclusion body precipitate for the second time, and the inclusion body precipitate was collected after centrifugation at 4°C and 9000rpm for 30min. Then, according to the mass volume ratio of the added bacteria to the pure water of 1:3 in step 5, pure water was added, and the inclusion body precipitate was dispersed by a high-speed disperser to wash the inclusion body precipitate for the third time, and the inclusion body precipitate was collected after centrifugation at 4°C and 9000rpm for 30min, and the inclusion body was weighed.

[0062] Step 7: Dissolution of inclusion bodies

[0063] The inclusion body precipitate harvested in step 6 is broken into small pieces, and the volume of the inclusion body dissolution buffer (containing 10-50mM glycine, 7-8M urea or 5-6M guanidine hydrochloride, 0.1-5mM metal ion chelator EDTA or EGTA, pH 9-11) is calculated according to the ratio of the mass volume ratio of the inclusion body precipitate to the inclusion body dissolution buffer of 1:15; first, β-mercaptoethanol is added to the inclusion body dissolution buffer to make its final concentration 30-60mM, and then the inclusion body fragments are added, and a high-speed disperser is used to accelerate the dissolution of the inclusion body, and the dissolution temperature needs to be controlled at 4°C. After the inclusion body is completely dissolved, the inclusion body dissolution buffer is continued to be added and slowly diluted. At this time, the inclusion body dissolution buffer is added according to the ratio of the dissolution system obtained after the inclusion body is completely dissolved to the inclusion body dissolution buffer of 1:3 to obtain the inclusion body mixture I.

[0064] Step 8: Inclusion body renaturation

[0065] According to the volume ratio of the inclusion body mixture I obtained in step 7 to the inclusion body dilution buffer of 1:3, the inclusion body mixture I was added to the freshly prepared 4°C inclusion body dilution buffer (containing 10-50mM glycine, 0.1-5mM metal ion chelator EDTA or EGTA, pH 9-11) by a peristaltic pump, the peristaltic pump controlled the flow rate of the inclusion body mixture I to be 5ml / min, and the mixture was stirred at a low speed (50-200rpm) with a magnetic stirrer during the addition process. The entire dilution process was carried out in an ice bath (temperature of 0°C to 4°C). After the dilution was completed, the mixture was added to the freshly prepared 4°C inclusion body renaturation buffer (containing 10-50mM glycine, 0.1-5mM metal ion chelator EDTA or EGTA, pH 9-11) at a volume ratio of 1:1, placed in a 4°C refrigerator and renatured overnight at a low speed of 100rpm for 8-16h to obtain an inclusion body mixture II.

[0066] Step 9: Filtration, ultrafiltration

[0067] The inclusion body mixture II was filtered through a 0.45 μm capsule filter, and then ultrafiltered through a 10KD ultrafiltration membrane package to obtain a concentrate; an ice bath was required before and after filtration.

[0068] Step 10: Purification by anion exchange chromatography

[0069] Mobile phase A: 10-50 mM Tris, pH 7-10.

[0070] Mobile phase B: 10-50 mM Tris + 30-80 mM NaCl, pH 7-10.

[0071] Elution buffer: 10-50 mM Tris + 100-200 mM NaCl, pH 7-10.

[0072] Use mobile phase A to balance the column for 3CV, load the sample directly after balancing, the sample flow rate is no more than 210cm / h, balance the column with mobile phase A for 2CV, wash with mobile phase B for 3CV, elute weakly bound proteins; then balance the column with mobile phase A for 2CV, elute the target protein with elution buffer, start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, then combine the samples to obtain anion exchange chromatography samples, and store them at 4℃. Send for inspection to RP-HPLC, Lowry.

[0073] Step 11: Kex2 enzyme digestion

[0074] Slowly adjust the pH of the anion exchange chromatography sample in step 10 to 8.0 with 2M HCl, and then slowly add a protein diluent (containing 10-100mM Tris, 200-500mM CaCl2, pH 7.0-9.0) accounting for 1 / 100 of the total volume of the sample. Calculate the total protein amount of the anion exchange chromatography sample according to the Lowry and RP-HPLC results, and calculate the required Kex2 enzyme mass according to the ratio of total protein: Kex2 enzyme amount of 100:1-2500:1; add Kex2 protease dilution buffer (10-100mM Tris, 2-5mM CaCl2, pH 8.0-9.0) to the calculated required amount of Kex2 enzyme to make the Kex2 enzyme concentration 1mg / ml; then gently add the obtained Kex2 enzyme solution to the mixture of anion exchange chromatography sample and protein diluent within 20-30s, and place it at room temperature (18-30°C) for enzyme digestion for 8-20h.

[0075] Step 12: Purification by weak anion exchange chromatography

[0076] Mobile phase A: 20-50 mM PB (phosphate buffer), pH 7-10.

[0077] Mobile phase B: 20-50 mM PB + 100-500 mM NaCl, pH 7-10.

[0078] Use a peristaltic pump to pump the digestion product of step 11 into a container of suitable size, then pump PB into the digestion product, and dilute the digestion product to <3ms / cm. Use mobile phase A to balance the chromatography column for 3CV, load the sample, and the loading flow rate is no more than 130cm / h. Use mobile phase A to wash for 3CV, elute with 10% mobile phase B for 4CV, and then perform gradient elution: mobile phase A 90%, mobile phase B 10% at the beginning, mobile phase A 50%, mobile phase B 500% at the end, and run for 4CV. Start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, merge the samples, and collect the protein-containing eluate.

[0079] Step 13: Ultrafiltration

[0080] The protein-containing eluate was concentrated by 10-fold ultrafiltration using a 10KDa membrane package to obtain a concentrate.

[0081] Step 14: Sterile Filtration

[0082] The concentrated solution was sterilized, filtered and packaged using a 0.22 μm filter in an ultra-clean bench to obtain a growth hormone stock solution.

[0083] Step 15: Freeze-drying

[0084] The growth hormone stock solution is freeze-dried and stored at 2-8°C. The freeze-drying steps are as follows: the product is placed in a box at 5°C, kept for 1 hour, cooled to -40°C and kept at -40°C for 1 hour, the product is heated to -18°C and kept for 1 hour, the product is cooled again and kept at -35°C for 1 hour; the plate temperature is then controlled at 10°C and maintained for 15 hours until the frozen ice in the product sublimates; the product temperature is heated to the maximum allowable temperature of 30°C and maintained until the freeze-drying is completed, high vacuum is restored, and the product is maintained at the maximum allowable temperature and high vacuum for more than 6 hours to make the residual moisture of the product reach the requirement of less than 2%, thereby completing the freeze-drying.

[0085] The 50L fermentation system of the present invention can obtain more than 75g of finished growth hormone. The purification process can remove Kex2 enzyme residues, and the removal rate reaches 99.99%. The growth hormone obtained after purification contains almost no cleavage enzyme residues. At the same time, the prepared recombinant human growth hormone stock solution is subjected to RP-HPLC and SEC-HPLC detection to test the purity of the product. After analysis and determination, the purity of the recombinant human growth hormone is obtained to be above 95%. The in vitro biological activity of the prepared recombinant human growth hormone is determined according to the Nb2-11 cell proliferation method, and the specific activity is above 3.0IU / mg.

[0086] RP-HPLC method: According to the detection method of human growth hormone related protein content for injection in the 2020 edition of the Chinese Pharmacopoeia, butylsilane bonded silica gel is used as a filler (5-10 μm); 0.05 mol / L tris(hydroxymethylaminomethane) buffer (adjust the pH value to 7.5 with 1 mol / L hydrochloric acid solution)-n-propanol (tris(hydroxymethylaminomethane) buffer and n-propanol volume ratio 71:29) is used as the mobile phase, and the proportion of n-propanol in the mobile phase is adjusted to make the retention time of the main peak of human growth hormone 30-36 minutes; the flow rate is 0.5 ml per minute; the column temperature is 45 ° C; the detection wavelength is 220 nm. Take the human growth hormone reference substance, add 0.05 mol / L tris(hydroxymethylaminomethane) buffer to dissolve and make a solution containing 2 mg per 1 ml, filter and sterilize, and place at room temperature for 24 hours as a system suitability solution. Take 20 μl of the system suitability solution and inject it into the liquid chromatograph. The separation between the main peak of human growth hormone and the deaminated human growth hormone peak is not less than 1.0, and the tailing factor of the main peak of human growth hormone is 0.9-1.8.

[0087] SEC-HPLC method: According to the detection method of high molecular weight protein content of human growth hormone for injection in the 2020 edition of the Chinese Pharmacopoeia, hydrophilic modified silica gel suitable for separating globular proteins with a molecular weight of 5000-60000Da is used as a filler; isopropanol-0.063mol / L phosphate buffer (take 5.18g of anhydrous disodium hydrogen phosphate and 3.65g of sodium dihydrogen phosphate, add 950ml of water, adjust the pH value to 7.0 with phosphoric acid or sodium hydroxide test solution, and make 1000ml with water) (volume ratio of isopropanol to phosphate buffer 3:97) is used as the mobile phase; the flow rate is 0.6ml per minute; the detection wavelength is 214nm. Take the human growth hormone monomer and dimer mixture reference substance, add 0.025mol / L phosphate buffer (pH 7.0) to make a solution containing 1.0mg per 1ml, take 20μl and inject it into the liquid chromatograph, and the separation between the human growth hormone monomer peak and the dimer peak meets the requirements.

[0088] In the present invention, the fusion protein is cut by recombinant dibasic amino acid endopeptidase (Kex2 enzyme) to form free target recombinant human growth hormone and fragments thereof, the target recombinant human growth hormone and fragments thereof are not cut by recombinant Kex2 enzyme, the N-terminus of the free target growth hormone and fragments thereof does not contain additional amino acid residues, the products before and after cutting are significantly different, the products before and after enzyme cutting can be distinguished, which is beneficial to subsequent purification.

[0089] Preferred embodiment 1 is as follows:

[0090] A recombinant human growth hormone fusion protein comprises a first peptide segment and a second peptide segment, wherein the first peptide segment is a target growth hormone and a fragment thereof, the first peptide segment has an amino acid sequence as shown in SEQ ID NO:1, the second peptide segment amino acid sequence is MRRTGPR (M is methionine, that is, A is RR, X is TG, and B is PR in formula (I)), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment.

[0091] The amino acid sequence of the recombinant human growth hormone fusion protein is:

[0092] MRRTGPRFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:2).

[0093] This example is based on the preparation of high-purity recombinant human growth hormone from a fusion protein in the form of intracellular inclusion bodies expressed in Escherichia coli, comprising the following steps:

[0094] Step 1: Prepare genetically engineered bacteria plasmid vector and genetically engineered bacteria

[0095] The nucleotide sequence encoding the recombinant human growth hormone fusion protein was designed based on the amino acid sequence shown in SEQ ID NO:2, the start codon was ATG (positions 4-6 in the SEQ ID NO:3 sequence), the stop codon was TAA (positions 598-600 in the SEQ ID NO:3 sequence), the sequence between the start codon ATG and the stop codon TAA was the gene sequence of the fusion protein (as shown in SEQ ID NO:3), the front end sequence CATATG of the fusion protein was an Nde I restriction site, and CTCGAG was an Xho I restriction site, which respectively corresponded to the Nde I restriction site and the Xho I restriction site in the MCS region of the target gene cloning vector pET-30a(+); Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the target gene into the cloning vector pET-30a(+), and after transformation of the host cell E.coli BL21(DE3), clone screening, plasmid extraction, and enzyme digestion identification, a plasmid vector carrying the growth hormone fusion protein gene sequence and a genetically engineered bacterium expressing the growth hormone fusion protein transformed with the plasmid vector were obtained.

[0096] The nucleotide sequence of the recombinant human growth hormone fusion protein is:

[0097] (SEQ ID NO:3)

[0098] Step 2: Cultivate working seeds

[0099] Take 1 mL of the working seed of the genetically engineered bacteria and inoculate all of it into LB shake flask medium (180 mL) containing kanamycin monosulfate (50 μg / mL) for shake flask culture at 37.0°C and 220 rpm. When the bacterial concentration OD600 is ≥ 2, and the bacterial morphology detected by optical microscopy is a typical Escherichia coli morphology and there is no other microbial contamination, the working seed culture is terminated.

[0100] Step 3: Fermentation tank culture

[0101] The cultivation scale is further expanded through a two-stage gradually enlarged bacterial fermentation system. The first-stage fermentation cultivation steps are: the working seeds of the shake flask culture are inoculated at a 1% inoculation amount into a first-stage fermentation tank (5L) filled with 1.8L sterile culture medium for first-stage fermentation cultivation until the bacterial concentration OD600 is ≥20, the bacterial morphology detected by optical microscope is a typical Escherichia coli morphology, and there is no other microbial contamination, and the first-stage fermentation cultivation is terminated; wherein the culture temperature is 30.0°C, the rotation speed is 400rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation volume is 40L / min, the dissolved oxygen is ≥20.0%, and the culture time is 6h. The secondary fermentation culture step is: transferring all the fermentation liquid in the primary fermentation tank to a secondary fermentation tank (50L) filled with 18L sterile culture medium for secondary fermentation culture, the culture temperature is 30.0°C, the rotation speed is 600rpm, the pH value is 7.0 (ammonia adjustment), the ventilation volume is 500.0L / min, and the dissolved oxygen is 60.0%. During the culture process, when the carbon source (glycerol) is consumed, the pH value is on an upward trend, and the dissolved oxygen increases, feeding is performed and the feeding rate is adjusted so that the carbon source, pH value, and dissolved oxygen in the secondary fermentation tank meet the secondary fermentation culture conditions; after culturing for 7h and the bacterial concentration OD600 ≥100.0, 300g of lactose inducer is added for induction, the induction temperature is 26°C, the induction time is 6h, the temperature is lowered to 15°C and the tank is lowered, and samples are taken for SDS-PAGE electrophoresis test to detect the expression of the recombinant human growth hormone fusion protein.

[0102] The components of the sterile culture medium and feed culture medium used in the primary fermentation culture and the secondary fermentation culture are:

[0103] Primary fermentation tank culture medium: peptone 5g / L, yeast powder 5g / L, disodium hydrogen phosphate 4g / L, sodium dihydrogen phosphate 3.4g / L, magnesium sulfate 1g / L, glycerol 21g / L.

[0104] Secondary fermentation tank culture medium: yeast powder 5.8g / L, dipotassium hydrogen phosphate 4g / L, potassium dihydrogen phosphate 4g / L, disodium hydrogen phosphate 7g / L, magnesium sulfate 1.2g / L, ammonium chloride 0.2g / L, ammonium sulfate 1.2g / L, calcium chloride 0.02g / L.

[0105] Feed medium: peptone 30g / L, yeast powder 15g / L, dipotassium hydrogen phosphate 7g / L, potassium dihydrogen phosphate 7g / L, disodium hydrogen phosphate 10g / L, magnesium sulfate 2.5g / L, ammonium chloride 10g / L.

[0106] Step 4: Bacteria recovery

[0107] The secondary fermentation culture fluid was centrifuged to obtain bacterial cells, and the centrifugation parameters were 15000 rpm / min and 1 hour.

[0108] Step 5: Broken cells to obtain inclusion bodies

[0109] The bacteria were added to a lysis buffer (containing 20 mM Tris, 3 mM metal ion chelator EDTA, 1.2 M denaturant urea, 2.6% non-ionic surfactant Triton X-100), pH 7.0) at a mass volume ratio of 1:10 between the bacteria and the lysis buffer. The bacteria were dispersed using a high-speed disperser at a speed of 850 bar, and the homogenate was broken three times. The cells were then centrifuged at 4°C and 9000 rpm for 30 min to collect the inclusion body precipitate.

[0110] Step 6: Inclusion body washing

[0111] Add inclusion body washing buffer I (containing 20mM Tris, 3mM metal ion chelator EDTA, 2.6% non-ionic surfactant Triton X-100, pH value is 7.0) to the inclusion body precipitate, the inclusion body washing buffer I is added in a ratio of 1:3 between the added bacteria and the inclusion body washing buffer I in step 5, use a high-speed disperser to disperse the inclusion body precipitate for the first washing, and collect the inclusion body precipitate after centrifugation at 4°C and 9000rpm for 30min. Then add inclusion body washing buffer II (20mMTris, 3mM metal ion chelator EDTA, pH value is 7.0) in a ratio of 1:3 between the added bacteria and the inclusion body washing buffer II in step 5, use a high-speed disperser to disperse the inclusion body precipitate for the second washing, and collect the inclusion body precipitate after centrifugation at 4°C and 9000rpm for 30min. Then, pure water was added according to the mass volume ratio of the added bacteria to pure water of 1:3 in step 5, and the inclusion body precipitate was dispersed using a high-speed disperser to wash the inclusion body precipitate for the third time. The inclusion body precipitate was collected after centrifugation at 4°C and 9000rpm for 30min, and the inclusion bodies were weighed.

[0112] Step 7: Dissolution of inclusion bodies

[0113] The inclusion body precipitate harvested in step 6 was broken into small pieces, and the volume of the inclusion body dissolution buffer (containing 25mM glycine, 7M urea, 1mM metal ion chelator EDTA, pH 10.2) was calculated according to the mass volume ratio of the inclusion body precipitate to the inclusion body dissolution buffer of 1:15; β-mercaptoethanol was first added to the inclusion body dissolution buffer to make its final concentration 50mM, and then the inclusion body fragments were added, and a high-speed disperser was used to accelerate the dissolution of the inclusion bodies, and the dissolution temperature was controlled at 4°C. After the inclusion bodies were completely dissolved, the inclusion body dissolution buffer was continued to be added and slowly diluted. At this time, the inclusion body dissolution buffer was added according to the ratio of the dissolution system obtained after the inclusion bodies were completely dissolved to the inclusion body dissolution buffer of 1:3 to obtain the inclusion body mixture I.

[0114] Step 8: Inclusion body renaturation

[0115] The inclusion body mixture I obtained in step 7 and the inclusion body dilution buffer were in a volume ratio of 1:3, and the inclusion body mixture I was added to the freshly prepared 4°C inclusion body dilution buffer (containing 25mM glycine, 1mM metal ion chelator EDTA, pH 10.2) using a peristaltic pump. The peristaltic pump controlled the flow rate of the inclusion body mixture I to be 5ml / min. During the addition process, the mixture was stirred at a low speed (80rpm) with a magnetic stirrer. The entire dilution process was carried out in an ice bath (temperature of 0-4°C). After the dilution was completed, the mixture was added to the freshly prepared 4°C inclusion body renaturation buffer (containing 25mM glycine, 1mM metal ion chelator EDTA, pH 10.2) at a volume ratio of 1:1, placed in a 4°C refrigerator and renatured overnight at a low speed of 100rpm for 12h to obtain an inclusion body mixture II.

[0116] Step 9: Filtration, ultrafiltration

[0117] The inclusion body mixture II was filtered through a 0.45 μm capsule filter, and then ultrafiltered through a 10KD ultrafiltration membrane package to obtain a concentrate; an ice bath was required before and after filtration.

[0118] Step 10: Purification by anion exchange chromatography

[0119] Mobile phase A: 20 mM Tris, pH 8.

[0120] Mobile phase B: 20 ​​mM Tris + 50 mM NaCl, pH 8.

[0121] Elution buffer: 20 mM Tris + 150 mM NaCl, pH 8.

[0122] Use mobile phase A to balance the column for 3CV, load the sample directly after balancing, the sample flow rate is no more than 210cm / h, balance the column with mobile phase A for 2CV, wash with mobile phase B for 3CV, elute weakly bound proteins; then balance the column with mobile phase A for 2CV, elute the target protein with elution buffer, start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, then combine the samples to obtain anion exchange chromatography samples, and store them at 4℃. Send for inspection to RP-HPLC, Lowry.

[0123] Step 11: Kex2 enzyme digestion

[0124] Slowly adjust the pH of the anion exchange chromatography sample in step 10 to 8.0 with 2M HCl, and then slowly add a protein diluent (containing 50mM Tris, 500mM CaCl2, pH 7.5) accounting for 1 / 100 of the total volume of the sample. Calculate the total protein amount of the anion exchange chromatography sample according to the results of Lowry and RP-HPLC, and calculate the required Kex2 enzyme mass according to the ratio of total protein: Kex2 enzyme amount of 1000:1; add Kex2 protease dilution buffer (20mM Tris, 5mMCaCl2, pH 8.5) to the calculated required amount of Kex2 enzyme to make the Kex2 enzyme concentration 1mg / ml; then gently add the obtained Kex2 enzyme solution to the mixture of anion exchange chromatography sample and protein diluent within 30s, and place it at room temperature (23℃) for enzyme digestion for 10h. According to RP-HPLC detection, the content of related proteins in the sample after enzyme digestion is 8.17%; according to SEC-HPLC detection, the content of high molecular weight protein in the sample after enzyme digestion is 10.44%.

[0125] Step 12: Purification by weak anion exchange chromatography

[0126] Mobile phase A: 50 mM PB (phosphate buffer), pH 7.5 (adjusted with NaOH).

[0127] Mobile phase B: 20 ​​mM PB + 220 mM NaCl, pH 7.5.

[0128] Use a peristaltic pump to pump the digestion product of step 11 into a container of suitable size, then pump PB into the digestion product, and dilute the digestion product to <3ms / cm. Use mobile phase A to balance the chromatography column for 3CV, load the sample, and the loading flow rate is not more than 130cm / h. Use mobile phase A to wash for 3CV, elute with 10% mobile phase B for 4CV, and then perform gradient elution: start with mobile phase A 90%, mobile phase B 10%, end with mobile phase A 50%, mobile phase B 500%, run for 4CV. Start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, merge samples, and collect protein-containing eluent. According to RP-HPLC detection, the content of related proteins in the sample after weak anion exchange chromatography is 2.65%; according to SEC-HPLC detection, the content of high molecular weight protein in the sample after weak anion exchange chromatography is 0.04%.

[0129] Step 13: Ultrafiltration

[0130] The protein-containing eluate was concentrated by 10-fold ultrafiltration using a 10KDa membrane package to obtain a concentrate.

[0131] Step 14: Sterile Filtration

[0132] The concentrated solution was sterilized, filtered and packaged using a 0.22 μm filter in an ultra-clean bench to obtain a growth hormone stock solution.

[0133] Step 15: Freeze-drying

[0134] After freeze-drying, it is stored at 4°C. The freeze-drying steps are as follows: the product is put into the box at 5°C, kept for 1 hour after entering the box, cooled to -40°C, and kept at -40°C for 1 hour, the product is heated to -18°C and kept for 1 hour, the product is cooled again and kept at -35°C for 1 hour; the plate temperature is then controlled at 10°C and maintained for 15 hours until the frozen ice in the product sublimates; the product temperature is heated to the maximum allowable temperature of 30°C and maintained until the freeze-drying is completed, high vacuum is restored, and it is maintained at the maximum allowable temperature and high vacuum for more than 6 hours to make the residual moisture of the product reach the requirement of less than 2%, and the freeze-drying is completed.

[0135] After testing, the 50L fermentation system of this embodiment obtained 100.2g of finished growth hormone; the purification process can remove Kex2 enzyme residues, and the removal rate is 99.99%; the purity of recombinant human growth hormone is 99.6%; the in vitro biological activity of the prepared recombinant human growth hormone is determined according to the Nb2-11 cell proliferation method, and the specific activity is 3.98IU / mg.

[0136] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing recombinant human growth hormone, characterized in that: The following steps are involved: The inclusion bodies including the fusion protein are obtained through the steps of constructing genetically engineered bacteria, culturing working seeds, culturing in a fermenter, recovering bacteria, crushing bacteria, washing inclusion bodies, dissolving inclusion bodies, and renaturing inclusion bodies, and the inclusion body concentrate is obtained through filtering and ultrafiltration operation; then the inclusion body concentrate is crudely purified by anion exchange chromatography to obtain an anion exchange chromatography sample; then Kex2 enzyme digestion is performed to obtain digestion products; Then, it is purified by weak anion exchange chromatography to obtain a protein-containing eluate, which is then ultrafiltered, sterilized and filtered, and freeze-dried to obtain recombinant human growth hormone; The fusion protein consists of a first peptide segment and a second peptide segment, the first peptide segment is a target growth hormone, the amino acid sequence of the first peptide segment is shown in SEQ ID NO: 1, the amino acid sequence of the second peptide segment is shown in formula (I), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment; The amino acid sequence of the second peptide is: MAXB(I) In formula (I), M is methionine; A is RR; X is TG; B is PR; The specific method of Kex2 enzyme digestion is as follows: slowly adjust the pH of the anion exchange chromatography sample to 8.0 with 2M HCl, and then slowly add a protein diluent accounting for 1 / 100 of the total volume of the sample; calculate the total protein amount of the anion exchange chromatography sample according to the Lowry and RP-HPLC results, and calculate the required Kex2 enzyme mass according to the ratio of total protein: Kex2 enzyme amount of 100:1 to 2500:1; add Kex2 protease dilution buffer to the calculated required amount of Kex2 enzyme to make the Kex2 enzyme concentration 1 mg / ml; then add the obtained Kex2 enzyme solution to the mixture of the anion exchange chromatography sample and the protein diluent, and place it at room temperature for digestion; The protein diluent is a solution containing 10~100mM Tris, 200~500mM CaCl2, pH 7.0~9.0; the Kex2 protease dilution buffer is a solution containing 10~100mM Tris, 2~5mM CaCl2, pH 8.0~9.

0.

2. A method for preparing recombinant human growth hormone according to claim 1, characterized in that: The specific steps of the anion exchange chromatography method for crude purification of the inclusion body concentrate are as follows: use mobile phase A to balance the chromatography column for 3CV, directly load the sample after equilibration, the loading flow rate is not more than 210cm / h, balance the chromatography column with mobile phase A for 2CV, wash with mobile phase B for 3CV, elute the weakly bound protein; then balance the chromatography column with mobile phase A for 2CV, elute the target protein with elution buffer, start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, then combine the samples to obtain anion exchange chromatography samples, and store them at 4°C.

3. A method for preparing recombinant human growth hormone according to claim 2, characterized in that: Mobile phase A is a solution of 10~50mM Tris, pH 7~10; mobile phase B is a solution containing 10~50mM Tris, 30~80mM NaCl, pH 7~10; elution buffer is a solution containing 10~50mM Tris, 100~200mM NaCl, pH 7~10.

4. A method for preparing recombinant human growth hormone according to claim 1, characterized in that: The specific steps of purification by weak anion exchange chromatography are: Use a peristaltic pump to pump the digestion product into a container of suitable size, then pump PB into the digestion product to dilute the conductivity of the digestion product to <3ms / cm; balance the chromatography column with mobile phase A for 3CV, load the sample, and the loading flow rate is no more than 130cm / h. Wash with mobile phase A for 3CV, elute with 10% mobile phase B for 4CV, and then perform gradient elution: at the beginning, mobile phase A 90%, mobile phase B 10%, at the end, mobile phase A 50%, mobile phase B 500%, run for 4CV; Start collecting when UV is not less than 50mAU, stop collecting when UV is less than 100mAU, combine samples, and collect protein-containing eluate; Mobile phase A is a solution of 20-50 mM PB, pH 7-10; Mobile phase B is a solution containing 20~50mM PB, 100~500mM NaCl, pH 7~10.

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