WO2023109195A1 - 一种适合工业化生产的重组颗粒蛋白产品及其制备方法 - Google Patents

一种适合工业化生产的重组颗粒蛋白产品及其制备方法 Download PDF

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WO2023109195A1
WO2023109195A1 PCT/CN2022/116392 CN2022116392W WO2023109195A1 WO 2023109195 A1 WO2023109195 A1 WO 2023109195A1 CN 2022116392 W CN2022116392 W CN 2022116392W WO 2023109195 A1 WO2023109195 A1 WO 2023109195A1
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recombinant
supernatant
preparation
urea
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PCT/CN2022/116392
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French (fr)
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晋竞
周宇
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烟台派诺生物技术有限公司
广州派诺生物技术有限公司
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Priority to AU2022415852A priority Critical patent/AU2022415852B2/en
Priority to MX2024007396A priority patent/MX2024007396A/es
Priority to EP22905951.4A priority patent/EP4450513A1/en
Publication of WO2023109195A1 publication Critical patent/WO2023109195A1/zh
Priority to US18/743,724 priority patent/US20240344101A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

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  • the invention relates to the technical field of biomedicine, in particular to a recombinant granule protein product and a production and preparation method thereof.
  • Recombinant granular protein products can be prepared by a variety of preparation methods within the scope of common technical knowledge (salt precipitation, density gradient centrifugation, filtration, chromatography, dialysis, etc.), but there are still granule proteins that are not easy to form and prepare during industrial production. Low purity, high cost of industrial production and other problems, so the development of efficient, stable, simple, low-cost recombinant granule protein products and preparation methods suitable for industrial production is still a problem to be solved in this field.
  • the invention provides a recombinant granule protein product suitable for industrial production, the amino acid sequence of the protein is shown in SEQ ID NO:1.
  • the invention provides a recombinant granule protein product suitable for industrialized production, the amino acid sequence of the protein is as shown in SEQ ID NO: 1, prepared by the following steps:
  • the invention provides a recombinant granule protein product suitable for industrialized production, the amino acid sequence of the protein is as shown in SEQ ID NO: 1, prepared by the following steps:
  • the invention provides a recombinant granule protein product suitable for industrialized production, the amino acid sequence of the protein is as shown in SEQ ID NO: 1, prepared by the following steps:
  • the invention provides a recombinant granule protein product suitable for industrialized production, the amino acid sequence of the protein is as shown in SEQ ID NO: 1, prepared by the following steps:
  • the invention provides a method for industrialized preparation of recombinant granular protein products, comprising the following steps:
  • step (3) resuspends the precipitate, urea and sodium chloride are added, and then step (4) is performed.
  • the pH is 8.0-10.0, 60°C-90°C, 60°C-95°C, 60°C-100°C, 80°C-90°C or 80°C ⁇ 100°C, incubate or heat-incubate for more than 15min.
  • the pH is 9.0-10.0, more preferably the pH is 9.0;
  • the incubation temperature is 60°C-90°C, 80°C-90°C or 80°C-100°C, more preferably 80°C-90°C ;
  • the incubation time is 15-80min, more preferably 1h.
  • step (3)a the pH of the supernatant is adjusted to 8.0-10.0 or 9.0-10.0, and heated and incubated at 60°C-100°C for more than 15 minutes, or at 80°C- Heat and incubate at 100°C for more than 15 minutes, or heat and incubate at 80°C-95°C for 15-80 minutes, or heat and incubate at 60-90°C for more than 15-60 minutes, or heat and incubate at 60-95°C for more than 15-60 minutes.
  • step (3)a adjust the pH of the supernatant to 9.0, incubate at 4°C-95°C for more than 15 minutes, or heat and incubate at 80°C-95°C for 15-80 minutes, or heat and incubate at 80°C-90°C for 15-45 minutes, or heat and incubate at 60-90°C for more than 15-60 minutes.
  • the heating and incubation temperature in step (3)b is 40-90° C., and the heating and incubation treatment is 5-30 min or 5-15 min.
  • the incubation temperature is 50-60°C, more preferably 60°C; preferably, the incubation time is 5-10min, more preferably 10min.
  • the heating and incubation time in step (3)b is 5-20 minutes, preferably 15-20 minutes.
  • the chromatography in step (4) is anion exchange chromatography and hydrophobic chromatography.
  • the present invention provides a kind of preparation method of the recombinant granule protein product suitable for industrialized production, and the aminoacid sequence of described protein is as shown in SEQ ID NO: 1, and it comprises the following steps: (1) transfection contains the plasmid encoding recombinant granule protein sequence Vector to host cells, and express in the cells; (2) Harvest and crush the bacteria, and collect the supernatant; (3) a Adjust the pH of the supernatant to 9.0-10.0, and incubate at 4°C-100°C for more than 15 minutes After returning to room temperature, centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH value to pH 7.0-8.0; b.
  • the present invention provides a kind of preparation method of the recombinant granule protein product suitable for industrialized production, and the aminoacid sequence of described protein is as shown in SEQ ID NO: 1, and it comprises the following steps: (1) transfection contains the plasmid encoding recombinant granule protein sequence The vector is transferred to the host cell and expressed in the cell; (2) Harvest and crush the bacteria, and collect the supernatant; (3) a Adjust the pH of the supernatant to 9.0-10.0, and heat and incubate at 60°C-100°C for 15- After more than a minute, return to room temperature and centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH value to pH 7.0-8.0; b.
  • the present invention provides a kind of preparation method of the recombinant granule protein product suitable for industrialized production, and the aminoacid sequence of described protein is as shown in SEQ ID NO: 1, and it comprises the following steps: (1) transfection contains the plasmid encoding recombinant granule protein sequence The vector is transferred to the host cell and expressed in the cell; (2) Harvest and crush the bacteria, and collect the supernatant; (3) a Adjust the pH of the supernatant to 9.0, and heat and incubate at 80°C-95°C for 15-80 minutes After returning to room temperature, centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH value to pH 7.0-8.0; b.
  • the present invention provides a kind of preparation method of the recombinant granule protein product suitable for industrialized production, and the aminoacid sequence of described protein is as shown in SEQ ID NO: 1, and it comprises the following steps: (1) transfection contains the plasmid encoding recombinant granule protein sequence Carry the vector into the host cell and express it in the cell; (2) Harvest the bacterial cells and homogeneously crush them by high pressure; (3) a. Heat and incubate for more than 15 minutes, return to room temperature and centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH value to pH 7.0-8.0; b.
  • the present invention provides a kind of preparation method of the recombinant granule protein product suitable for industrialized production, and the aminoacid sequence of described protein is as shown in SEQ ID NO: 1, and it comprises the following steps: (1) transfection contains the plasmid encoding recombinant granule protein sequence Carry the vector into the host cell and express it in the cell; (2) Harvest the bacterial cells and homogeneously crush them by high pressure; (3) a. Heat and incubate for 15-80 minutes, return to room temperature and centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH to pH 7.0-8.0; b.
  • the invention provides a method for preparing a recombinant granule protein product suitable for industrial production, the amino acid sequence of the protein is shown in SEQ ID NO: 1, which includes the following steps: (1) transfecting the recombinant granule protein sequence containing the encoding (2) Harvest the bacterial cells and crush them by high-pressure homogeneous; (3) a. Adjust the pH of the high-pressure homogeneous supernatant to 7.0-8.0, at 4°C- Incubate at 25°C for more than 15 minutes, return to room temperature and centrifuge, discard the precipitate, and collect the supernatant; add dilution buffer to adjust the pH to pH 7.0-8.0; b.
  • the invention provides a method for preparing a recombinant granule protein product suitable for industrial production, the amino acid sequence of the protein is shown in SEQ ID NO: 1, which includes the following steps: (1) transfecting the recombinant granule protein sequence containing the encoding The plasmid vector is transferred to the host cell and expressed in the cell; (2) Harvest the bacteria and crush it by high-pressure homogeneous; (3) a.
  • the present invention also provides a recombinant granule protein product, which is prepared by any of the above-mentioned preparation methods.
  • the dilution buffer contains Tris-hydrochloric acid, acetic acid-sodium acetate, citric acid or phosphoric acid, the pH of which is 6.5-8.0, and the concentration is 50- 100mM, preferably 70mM, 80mM, 90mM, 100mM.
  • the dilution buffer may also contain ethylenediaminetetraacetic acid (EDTA) at a concentration of 5mM-20mM.
  • EDTA ethylenediaminetetraacetic acid
  • the dilution buffer may also contain a detergent, for example: polyethylene glycol octylphenyl ether, Tween (Tween20, Tween80), SDS, Triton X-100 or NP-40, etc.
  • the dilution buffer preferably contains Tris-HCl, EDTA and Triton X-100.
  • the concentration of the buffer is 0.1%-8%, preferably 2%, 3%, 4% or 5%.
  • the resuspension buffer contains Tris-hydrochloric acid, acetic acid-sodium acetate, citric acid or phosphoric acid, the pH of which is 2.5-10.0, and the concentration is 20 - 100 mM, preferably 70 mM, 80 mM, 90 mM, 100 mM.
  • the dilution buffer may also contain ethylenediaminetetraacetic acid (EDTA) at a concentration of 5mM-20mM; preferably Tris-hydrochloric acid and EDTA.
  • EDTA ethylenediaminetetraacetic acid
  • the host cell used to express the recombinant granule protein can be any routinely used host cell for the production of recombinant protein by genetic engineering methods, including but not limited to: Human embryonic kidney cells (e.g. HEK293), Chinese hamster ovary cells (CHO and its various subtypes, such as CHO-K1, CHO-S, CHO-GS cells with glutamine synthetase system), African green monkey Kidney fibroblasts (such as COS-7), Escherichia coli (DH5a, BL21, DH10B) and other cells.
  • Human embryonic kidney cells e.g. HEK293
  • Chinese hamster ovary cells CHO and its various subtypes, such as CHO-K1, CHO-S, CHO-GS cells with glutamine synthetase system
  • African green monkey Kidney fibroblasts such as COS-7
  • Escherichia coli DH5a, BL21, DH10B
  • the cell culture medium used can be any medium known in the art that is suitable for CHO, HEK, and Escherichia coli to express foreign proteins, including but not Limited to: CD CHO, Dynamis, CD02, CD04, CD05, ExpiCHO, DMEM, FreeStyle 293, Luria Broth, Terrific Broth, etc.
  • the concentration of urea used is 0.5-10M, preferably 4M, 4.5M, 5M, 5.5M, 6M, 6.5M, 7M, 7.5M , 8M, 8.5M, more preferably 6M-8M, the final concentration of sodium chloride is 10mM-200mM, preferably 50-200mM.
  • the anion exchange chromatography medium can use any feasible medium, including but not limited to: DEAE Sepharose FF, Q Sepharose FF, Capto DEAE, Capto Q Impres , POROS HQ, POROS 50D, POROS PI, Fractogel DEAE or Fractogel TMAE; preferably Fractogel DEAE.
  • the hydrophobic chromatography medium can use any feasible medium, including but not limited to: Butyl-S Sepharose 6FF, Butyl Sepharose 4FF, Octyl Bestarose 4FF, Phenyl Sepharose 6FF, Capto Butyl, Capto Phenyl, Capto Phenyl Impres or Capto Octyl; preferably Octyl Bestarose 4FF.
  • a stabilizer is added to the elution collection obtained by anion exchange chromatography and the hydrophobic chromatography buffer, and the stabilizer is selected from amino acids, polyols Or sugar, the amino acid is preferably arginine, glycine or histidine, the sugar is preferably sucrose, trehalose, the polyol is preferably glycerin, sorbitol, and the concentration is more than 20% (w/v).
  • the purity of the sample obtained before chromatography by the method of the invention can reach more than 85%, which reduces chromatography steps and pressure, improves production efficiency, and saves the cost of large-scale production.
  • the use of high-concentration urea and specific concentration of sodium chloride can significantly reduce impurities and improve the effect of impurity removal.
  • the method of the invention can obtain products with uniform particle size, good batch-to-batch consistency, low impurity residue and no solvent residue.
  • the preparation method of the recombinant granule protein product provided by the present invention is suitable for industrial production, can reduce the cost of large-scale industrial production, is simple to operate, and reduces the amount of organic solvent in subsequent chromatographic purification; use the recombinant granule protein product provided by the present invention to prepare The products produced effectively reduce the side effects caused by residues of impurities in the particles, host proteins, organic solvents, exogenous DNA, antibiotics, bacterial endotoxins, etc., and improve safety.
  • Fig. 1 is the SDS-PAGE picture of the supernatant after centrifugation after treating the broken supernatant of bacterial cells under different temperature and pH conditions in Example 1.
  • Fig. 2 is the SDS-PAGE picture of the supernatant after centrifugation of the broken supernatant of bacterial cells treated with different heating times in Example 1.
  • Example 3 is an SDS-PAGE image of the centrifuged supernatant at different temperatures and heating times in the second step of heating to remove impurities in Example 2.
  • Fig. 4 is the SDS-PAGE figure of the purity after two-step heating in Example 3.
  • Figure 5 is a molecular sieve chromatogram of 6M urea soaking treatment in Example 4.
  • Fig. 6 is the SDS-PAGE detection result of the molecular sieve elution peak after 6M urea treatment in Example 4.
  • FIG. 7 is a molecular sieve chromatogram of 4M urea soaking treatment in Example 4.
  • Fig. 8 is the SDS-PAGE detection result of the molecular sieve elution peak after 4M urea treatment in Example 4.
  • Figure 9 is a molecular sieve chromatogram of 1M urea soaking treatment in Example 4.
  • Fig. 10 is the SDS-PAGE detection result of the molecular sieve elution peak after 1M urea treatment in Example 4.
  • Fig. 11 is a molecular sieve chromatogram of the urea-free control group and 8M urea soaking treatment in Example 4.
  • Fig. 12 is the SDS-PAGE detection result of the molecular sieve elution peak after no urea control group and 8M urea treatment in Example 4.
  • Figure 13 is the SDS-PAGE analysis of the chromatographic peaks after treatment with four concentrations of sodium chloride in Example 4.
  • Fig. 14 is the F-DEAE chromatography SDS-PAGE analysis of KCl, Na2SO4 soaking treatment in embodiment 4.
  • Example 1 Determination of the first step heating process conditions for recombinant granule protein in Escherichia coli broken supernatant
  • the plasmid encoding the target recombinant granule protein sequence is transfected into host cells by conventional methods and expressed in the cells, and the cells are harvested, and then the target protein is released by high-pressure homogeneous crushing.
  • the amino acid sequence of the target recombinant granule protein is shown in SEQ ID NO: 1.
  • the recombinant granule protein in the supernatant after incubation for 1 hour at 4°C, 25, 40, 50, 60, 70, 80, and 90°C was significantly higher than that in other pH groups, indicating that the recombinant granule protein was at pH 9.0
  • the concentration of recombinant granule protein in the supernatant treated at 80°C and 90°C for 1 hour was higher than that of other temperature treatment groups, and the results of electrophoresis showed that the impurity content in the samples was also lower.
  • the solubility of recombinant granule protein has an inflection point between 50°C and 60°C, and between 4°C and the inflection point temperature, the solubility of recombinant granule protein decreases with the increase of temperature; between the inflection point temperature and 90°C During this period, the solubility of recombinant granule protein increased with the increase of temperature.
  • the solubility of the recombinant granule protein in the supersuspension of E.coli bacteria under different pH and temperature conditions is as follows:
  • the recombinant granule protein exhibits higher solubility at 4°C to 90°C than at low pH, and the solubility is the highest at 80°C to 90°C, and the effect of removing impurities is the best; recombinant granule protein
  • the solubility at pH 9.0 has an inflection point between 50°C and 60°C;
  • the recombinant granule protein has extremely high solubility between 4°C and 90°C, and the solubility of impurities is relatively higher, and the impurity removal effect is slightly worse than that at pH 9.0, but not Affect the overall purification effect.
  • the purity of the recombinant protein can reach more than 60%.
  • Example 2 Determination of the second step heating process conditions of recombinant granule protein in Escherichia coli broken supernatant
  • the E.Coli thallus liquid is subjected to the first heat treatment to separate impurities and recombinant granule proteins, and then the sample is subjected to the second heat treatment to further separate impurities and recombinant granule proteins.
  • the influence of treatment temperature and time on the solubility of recombinant granule protein and impurities was explored.
  • the first step of heating take the crushed supernatant of the bacteria and adjust it to pH 9.0, heat at 80°C for 1h, centrifuge at 12000g at 4°C for 30min, and take the supernatant.
  • An equal volume of 100 mM Tris-HCl, 5 mM EDTA, 4% Triton X-100, pH 7.4 buffer and 10% of the total volume of 1M Tris-HCl pH 7.4 buffer were added to the supernatant.
  • the second step of heating is set to 6 inspection points: 40°C, 50°C, 60°C, 70°C, 80°C, 90°C.
  • Five inspection points are set for heating time: 1min, 3min, 5min, 10min, 15min.
  • the pretreated samples were heated at 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 1, 3, 5, 10, and 15 minutes, respectively, and then immediately centrifuged at 15,000g for 60s to obtain the supernatant. The supernatant was then tested by SDS-PAGE, and the test results are shown in Figure 3.
  • the purity of the recombinant protein can reach more than 85%.
  • the recombinant granule protein has the lowest solubility at 50°C, and electrophoresis shows that the impurity protein still exists in the supernatant;
  • Embodiment 3 Two-step heating preparation process of recombinant granule protein product
  • the recombinant granule protein is expressed in Escherichia coli. After harvesting the cells, the target protein needs to be released by high-pressure homogeneous crushing and the feed liquid is clarified. The main purpose is to remove cell fragments and impurity proteins. Feed liquid clarification is mainly completed by heat treatment. Use the method of embodiment 1 and embodiment 2 to carry out heat treatment, carry out the first step of heating and the second step of heating (i.e. "two-step heating") to the E.coli broken supernatant, and remove the two-step heating step The hybrid effect and the purity of the recombinant granule protein were measured.
  • the granule protein can maintain high stability and high solubility under high temperature conditions, and can also form a resolvable precipitate under specific pH conditions.
  • the purity of the recombinant granule protein can be increased to more than 85% through a two-step heating process, which can reduce the pressure of subsequent chromatographic purification and reduce the required chromatographic steps and production costs.
  • the molecular sieve chromatogram of the 6M urea soaking treatment is shown in Figure 5
  • the SDS-PAGE detection result of the molecular sieve elution peak after the 6M urea treatment is shown in Figure 6
  • the molecular sieve chromatogram of the 4M urea soaking treatment is shown in Figure 7
  • the SDS-PAGE detection result of the molecular sieve elution peak after 4M urea treatment is shown in Figure 8
  • Figure 10 shows.
  • the molecular sieves can separate other substances (peak1-5) whose molecular weight is smaller than the main peak of recombinant granule protein, and the peak time is basically the same.
  • Fig. 12 that only peak No. 5 (soaked in 8M urea for 16h) has an obvious band between 25-35kDa; peak No. 3 (soaked in 8M urea for 1h) has a relatively shallow band between 25-35kDa. There are no visible bands for No. 1 and No. 2 peaks soaked in urea. Experiments have shown that soaking in 8M high-concentration urea helps to remove some small molecular impurities, and the preferred soaking time is more than 12 hours.
  • the recombinant granule protein samples were soaked for 16 hours with 8M urea and four concentrations of sodium chloride, 50mM, 100mM, 150mM and 200mM, respectively. Then it was chromatographically purified using Fractogel DEAEM packing material, and its flow-through and elution peaks were detected by SDS-PAGE (as shown in Figure 13).
  • Fig. 13a it can be observed that after soaking in 8M urea and 50mM sodium chloride for 16h, the content of the 25-35kDa substance in the main elution peak was significantly reduced, and the substance appeared in the chromatographic sample flow-through. It can be observed from Fig. 13b that after increasing the concentration of sodium chloride, the band of 25-35 kDa was not significantly reduced compared with the treatment with 50 mM sodium chloride.
  • the optimal process conditions for pretreatment of recombinant granule protein samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.
  • the salts for comparison include potassium chloride, sodium sulfate, and magnesium chloride.
  • the salts for parallel comparison include potassium chloride, sodium sulfate, and magnesium chloride. Observe different types and concentrations of salts The effect on the removal of impurities:
  • Figure 14(a) shows the SDS-PAGE gel image of the KCl-treated sample after F-DEAE chromatography. It can be seen from the figure that there are no 25-35kDa particles in the flow-through of the two concentrations of KCl soaking treatment groups. Impurities, the impurity bands are not obvious due to the low concentration in the elution, but the impurity distribution of 25-35kDa can still be seen. Compared with the soaking treatment effect of NaCl, KCl has no significant advantage.
  • Figure 14(b) shows the SDS-PAGE analysis chart of the Na 2 SO 4 treated sample after F-DEAE chromatography.
  • the recombinant granule protein product of the present application appeared in the flow-through liquid, but there was no recombinant granule protein product of the present application in the elution, and the chromatography also showed that the UV absorption value was extremely low during elution.
  • NaCl is the most suitable pretreatment salt in terms of impurity removal effect, process compatibility, and pH stability.
  • the recombinant granule protein sample liquid that has been pretreated in Example 3 and Example 4 was refined by ion exchange and hydrophobic chromatography, and the first step of chromatographic purification was chromatographic purification using Fractogel DEAE M chromatography process, specific steps and parameters See Table 4.
  • the Fractogel DEAE M elution collection solution sample was first diluted with buffer, and 50% (w/v) sucrose stabilizer was added to prevent the precipitation of recombinant granule protein during the next step of chromatography. For specific parameters, see Table 5. Then use the hydrophobic chromatography Octyl Bestarose 4FF chromatography process for purification (the second step of chromatography purification). See Table 6 for specific steps and parameters.
  • the first step of chromatography method chromatography packing material - Fractogel DEAE M, retention time - 12.5min
  • the second step chromatography method chromatography packing material-Octyl Bestarose 4FF, retention time-12.5min
  • the purity of the obtained product can reach more than 99.0% after further refining through the combination of the above chromatographic media.
  • sucrose, sorbitol or trehalose at a concentration of ⁇ 20% as a stabilizer can effectively protect the recombinant granule protein. Since the indoor temperature control range of the laboratory and production workshop is between 18-26°C, the addition of stabilizers can provide sufficient guarantees for protein stability and process robustness.

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Abstract

涉及一种适合工业化生产的重组颗粒蛋白及其制备方法，该产品通过如下方法制备：转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞并在细胞内进行表达、收获并破碎菌体、加热处理、尿素和氯化钠孵育、层析。产品粒径均匀，粒径大小批间一致性好。通过该方法制备产品可减少工业化大生产的成本，操作简单，产品杂质残留低，安全性好。

Description

一种适合工业化生产的重组颗粒蛋白产品及其制备方法 技术领域
本发明涉及生物医药技术领域，特别涉及重组颗粒蛋白产品及其生产制备方法。
背景技术
重组颗粒蛋白产品可通过多种普通技术知识范围内的制备方法(盐沉淀、密度梯度离心、过滤、层析、透析等)进行制备，但是仍然存在颗粒蛋白在工业化生产制备过程中不易成型、制备纯度低，工业化生产成本高等问题，因此开发适合工业化生产的高效、稳定、简便、低成本的重组颗粒蛋白产品及其制备方法，仍是本领域需要解决的问题。
发明内容
本发明提供一种适合工业化生产的重组颗粒蛋白产品，所述蛋白的氨基酸序列如SEQ ID NO:1所示。
>SEQ ID NO:1
Figure PCTCN2022116392-appb-000001
本发明提供一种适合工业化生产的重组颗粒蛋白产品，所述蛋白的氨基酸序列如SEQ ID NO:1所示，采用如下步骤制备：
(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为7.0-9.0，在80℃-100℃加热15分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠，尿素浓度为6M-8M；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品，所述蛋白的氨基酸序列如SEQ ID NO:1所示，采用如下步骤制备：
(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为8.0-10.0，在80℃-95℃加 热15-80分钟，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-65℃条件下加热5-20分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品，所述蛋白的氨基酸序列如SEQ ID NO:1所示，采用如下步骤制备：
(1)转染含编码所述重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为7.0-8.0，在4℃-25℃孵育15分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品，所述蛋白的氨基酸序列如SEQ ID NO:1所示，采用如下步骤制备：
(1)转染含编码所述重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为9.0，在4℃-95℃孵育15分钟以上；优选在60℃-90℃加热孵育15-60分钟；更优选在80℃-90℃加热孵育15-45分钟；然后，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种重组颗粒蛋白产品的工业化制备方法，包括以下步骤：
(1)转染含编码颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获并破碎菌体，收集上清液；(3)a.调节上清液pH为7.0-8.0，在4℃-25℃孵育15分钟以上，或调节上清液pH为9.0-10.0，在4℃-100℃孵育15分钟以上；然后恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-65℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH9.0-11.0条件下重悬沉淀；(4)进行层析纯化；所述重组颗粒蛋白产品如SEQ ID NO:1所示。
在一些实施方案中，上述制备方法中，步骤(3)重悬沉淀后，加入尿素和氯化钠，再进 行步骤(4)。
在一些实施方案中，上述任一制备方法中，步骤(3)a中，pH为8.0～10.0，60℃～90℃、60℃～95℃、60℃～100℃、80℃～90℃或80℃～100℃，孵育或加热孵育15min以上。优选地，pH为9.0～10.0,更优选地为pH为9.0；优选地，孵育温度为60℃～90℃、80℃～90℃或80℃～100℃，更优选地为80℃～90℃；优选地，孵育时间为15-80min，更优选地孵育1h。
在一些实施方案中，上述任一制备方法中，步骤(3)a中调节上清液pH为8.0-10.0或9.0-10.0，在60℃-100℃加热孵育15分钟以上，或在80℃-100℃加热孵育15分钟以上，或在80℃-95℃加热孵育15-80分钟，或在60-90℃加热孵育15-60分钟以上，或在60-95℃加热孵育15-60分钟以上。
在一些实施方案中，上述任一制备方法中，步骤(3)a中调节上清液pH为9.0，在4℃-95℃孵育15分钟以上，或在80℃-95℃加热孵育15-80分钟，或在80℃-90℃加热孵育15-45分钟，或在60-90℃加热孵育15-60分钟以上。在一些实施方案中，上述任一制备方法中，步骤(3)b中加热孵育温度为40-90℃、加热孵育处理5-30min或5-15min。优选地,孵育温度为50-60℃，更优选地为60℃；优选地，孵育时间为5-10min，更优选地为10min。
在一些实施方案中，上述任一制备方法中，步骤(3)b中加热孵育时间5-20分钟，优选15-20分钟。
在一些实施方案中，上述任一制备方法中，步骤(4)的层析为阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获并破碎菌体，收集上清液；(3)a调节上清液pH为9.0-10.0，在4℃-100℃孵育15分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获并破碎菌体，收集上清液；(3)a调节上清液pH为9.0-10.0，在60℃-100℃加热孵育15-分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加 入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获并破碎菌体，收集上清液；(3)a调节上清液pH为9.0，在80℃-95℃加热孵育15-80分钟，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为7.0-9.0，在80℃-100℃加热孵育15分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠，尿素浓度为6M-8M；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为8.0-10.0，在80℃-95℃加热孵育15-80分钟，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-65℃条件下加热孵育5-20分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码所述重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为7.0-8.0，在4℃-25℃孵育15分钟以上，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明提供一种适合工业化生产的重组颗粒蛋白产品的制备方法，所述蛋白的氨基酸序列如SEQ ID NO:1所示，其包括如下步骤：(1)转染含编码所述重组颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；(2)收获菌体，并通过高压均质破碎；(3)a.调节高压均质上清液pH为9.0，在4℃-95℃孵育15分钟以上；优选在60℃-90℃加热孵育15-60分钟；更优选在80℃-90℃加热孵育15-45分钟；然后，恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；b.将溶液置于50℃-70℃条件下加热孵育5-30分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH7.0-11.0条件下重悬沉淀；(4)加入尿素和氯化钠；(5)进行阴离子交换层析和疏水层析。
本发明还提供了一种重组颗粒蛋白产品，采用上述任一制备方法进行制备。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，所述稀释缓冲液含有Tris-盐酸、醋酸-醋酸钠、柠檬酸或磷酸，其pH为6.5-8.0，浓度为50-100mM,优选为70mM、80mM、90mM、100mM。所述稀释缓冲液还可以包含乙二胺四乙酸(EDTA)，浓度为5mM-20mM。所述稀释缓冲液还可以包含去污剂，例如：聚乙二醇辛基苯基醚、吐温(Tween20、Tween80)、SDS、Triton X-100或NP-40等。稀释缓冲液优选含有Tris-盐酸、乙二胺四乙酸和Triton X-100。缓冲液的浓度为0.1％-8％，优选浓度为2％、3％、4％或者5％。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，所述重悬缓冲液含有Tris-盐酸、醋酸-醋酸钠、柠檬酸或磷酸，其pH为2.5-10.0，浓度为20-100mM,优选为70mM、80mM、90mM、100mM。所述稀释缓冲液还可以包含乙二胺四乙酸(EDTA)，浓度为5mM-20mM；优选含有Tris-盐酸和乙二胺四乙酸。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，用于表达重组颗粒蛋白的宿主细胞可以是应用基因工程方法生产重组蛋白的任何常规使用的宿主细胞，包括但不限于：人胚胎肾细胞(例如HEK293)、中国仓鼠卵巢细胞(CHO及其各种亚型，例如CHO-K1、CHO-S、带有谷氨酞胺合成酶系统的CHO-GS细胞)、非洲绿猴肾成纤维细胞(例如COS-7)、大肠杆菌(DH5a、BL21、DH10B)等细胞。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，所用细胞培养基可以是本领域已知的任何适合于CHO、HEK、大肠杆菌表达外源蛋白的培养基，包括但不限于：CD CHO、Dynamis、CD02、CD04、CD05、ExpiCHO、DMEM、FreeStyle 293、Luria Broth、Terrific Broth等培养基。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，使用尿素浓度为0.5-10M,优选地可以为4M、4.5M、5M、5.5M、6M、6.5M、7M、7.5M、8M、8.5M，更优选地可以为6M-8M，氯化钠终浓度为10mM-200mM,优选地可以为50-200mM。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，阴离子交换层析介质可使用任何可行的介质，包括但不限于：DEAE Sepharose FF、Q Sepharose FF、Capto DEAE、Capto Q Impres、POROS HQ、POROS 50D、POROS PI、Fractogel DEAE或Fractogel TMAE；优选为Fractogel DEAE。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，疏水层析介质可使用任何可行的介质，包括但不限于：Butyl-S Sepharose 6FF、Butyl Sepharose 4FF、Octyl Bestarose 4FF、Phenyl Sepharose 6FF、Capto Butyl、Capto Phenyl、Capto Phenyl Impres或Capto Octyl；优选为Octyl Bestarose 4FF。
在一些实施方案中，上述任一重组颗粒蛋白产品或其制备方法中，在阴离子交换层析获得的洗脱收集液中和疏水层析缓冲液中添加稳定剂，稳定剂选自氨基酸、多元醇或糖，氨基酸优选为精氨酸、甘氨酸或组氨酸，糖优选蔗糖、海藻糖，多元醇优选甘油、山梨醇，浓度为20％以上(w/v)。
本发明采用的所有的试剂均可商业化购买获得。
本发明的有益效果：
采用本发明的方法在层析前获得的样品纯度在层析前可达85％以上，减少层析步骤和压力，提高生产效率，节约大规模生产的成本。采用高浓度的尿素和特定浓度的氯化钠可以显著减少杂质，提高除杂效果。本发明的方法可获得粒径均匀、粒径大小批间一致性好、杂质残留低，无溶剂残留的产品。
综上，本发明提供的重组颗粒蛋白产品的制备方法，适合工业化生产，可减少工业化大生产的成本，操作简单，减少后续层析纯化中有机溶媒的用量；使用本发明提供的重组颗粒蛋白制备出的产品，有效降低了由颗粒中杂质、宿主蛋白质、有机溶媒、外源性DNA、抗生素、细菌内毒素等物质的残留所带来的副作用，提高了安全性。
附图说明
图1为实施例1中的不同温度和pH条件处理菌体破碎上清液后的离心后上清的SDS-PAGE图。
图2为实施例1中不同加热时间处理的菌体破碎上清液离心后上清的SDS-PAGE图。
图3为实施例2中第二步加热除杂中不同温度和加热时间下离心上清的SDS-PAGE图。
图4为实施例3中两步加热后的纯度的SDS-PAGE图。
图5为实施例4中6M尿素浸泡处理的分子筛层析图谱。
图6为实施例4中6M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果。
图7为实施例4中4M尿素浸泡处理的分子筛层析图谱。
图8为实施例4中4M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果。
图9为实施例4中1M尿素浸泡处理的分子筛层析图谱。
图10为实施例4中1M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果。
图11为实施例4中无尿素对照组、8M尿素浸泡处理的分子筛层析图谱。
图12为实施例4中无尿素对照组、8M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果。
图13为实施例4中四种浓度的氯化钠处理后的层析峰SDS-PAGE分析。
图14为实施例4中KCl、Na2SO4浸泡处理的F-DEAE层析SDS-PAGE分析。
具体实施方式
以下结合实例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1确定大肠杆菌破碎上清液中重组颗粒蛋白的第一步加热工艺条件
采用常规方法将编码目标重组颗粒蛋白序列的质粒转染宿主细胞并在细胞内进行表达，并收获菌体，然后通过高压均质破碎释放目标蛋白，目标重组颗粒蛋白的氨基酸序列如SEQ ID NO:1所示。
1、不同pH和温度对菌体破碎上清液中重组颗粒蛋白溶解度的影响
对于pH设置8个考察点：pH3.0、pH4.0、pH5.0、pH6.0、pH7.0、pH8.0、pH9.0、pH10.0。对于温度设置8个考察点：4℃、25℃、40℃、50℃、60℃、70℃、80℃、90℃。样品为E.coli菌体破碎上清液，调节pH的情况如表1和表2所示。将上述不同pH的样品分别置于4℃、25℃、40℃、50℃、60℃、70℃、80℃、90℃孵育60min后，立即15000g离心15min，取上清液进行SDS-PAGE检测，结果如图1所示。
表1孵育温度为4℃～70℃的样品pH调节情况
Figure PCTCN2022116392-appb-000002
Figure PCTCN2022116392-appb-000003
表2孵育温度为80℃和90℃的样品pH调节情况
Figure PCTCN2022116392-appb-000004
结果与分析：
由图1可见，当pH处于3.0、4.0、5.0、6.0时，所有温度条件下处理1小时的离心上清液中，均不能分辨出明显的目标重组颗粒蛋白条带，且在pH分别调节至3.0、4.0、5.0、6.0的过程中，菌体破碎上清中出现大量淡黄色沉淀且不能复溶的现象，说明在pH3.0～6.0时，无论温度在4℃至90℃如何变化，重组颗粒蛋白溶解度均极低。
在pH7.0、8.0时，当处理温度为4、25、40或50℃时，上清液中出现重组颗粒蛋白条带，但条带灰度低于同温度下pH9.0或10.0的样品，推测在pH7.0或8.0，温度在4、25、40或50℃处理1小时条件下，重组颗粒蛋白在菌泥破碎液中具有一定的溶解度，但溶解度未达到最大，且杂质蛋白很多。由图1e/f/g/h可观察到，在温度分别为60、70、80、90℃处理1小时后，pH＝8.0条件相对于pH＝7.0条件下杂质蛋白更少。
在pH＝9.0时，4℃、25、40、50、60、70、80、90℃孵育1小时后上清液中重组颗粒蛋白均显著高于其他pH组，说明重组颗粒蛋白在pH9.0时溶解性较其他pH更好。80℃和90℃处理1小时的上清液中重组颗粒蛋白浓度高于其他温度处理组，且电泳结果表明样品中杂质含量也较低。在pH9.0时，经过40℃和50℃处理后的离心上清中重组颗粒蛋白含量明显低于4℃和25℃处理后的含量，但在60℃和70℃处理组中上清液重组颗粒蛋白含量重新上升，80℃和90℃时溶解度最高。说明在pH9.0时，重组颗粒蛋白的溶解度在50℃～60℃之间存在一个拐点，在4℃至拐点温度之间，重组颗粒蛋白溶解度随温度上升而下降；在拐点温度至90℃之间，重组颗粒蛋白溶解度随温度上升而上升。
当pH为10.0时，在4℃～90℃的不同温度处理1小时的上清液中均存在大量重组颗粒蛋白，说明重组颗粒蛋白在此pH下溶解度较高，上清中同时观察到存在相对而言量稍多的杂质蛋白。
综上，经过第一步加热处理时，E.coli菌体重悬液中的重组颗粒蛋白在不同pH和温度条件下的溶解性表现为：
a)在偏酸性(pH3.0～6.0)条件下，无论采用何种温度处理，重组颗粒蛋白溶解度均较低；
b)在pH＝7.0条件下，重组颗粒蛋白在4℃-90℃孵育下虽呈现出一定的溶解度，但杂质较多，除杂效果较差；
c)在pH＝8.0条件下，60℃～90℃加热处理时，杂质去除效果相对明显，重组颗粒蛋白纯度较高；
d)在pH9.0时，重组颗粒蛋白在4℃～90℃均呈现相较于低pH时的较高溶解度，在80℃～90℃时溶解度最高，且除杂效果最好；重组颗粒蛋白在pH9.0时的溶解度在50℃～60℃之间存在拐点；
e)在pH10.0时，重组颗粒蛋白在4℃～90℃之间均有极高的溶解性，同时杂质溶解性也相对稍高，相对于pH9.0时除杂效果略差，但不影响整体纯化效果。
2、不同加热时间对菌体破碎上清液中重组颗粒蛋白溶解度的影响
进一步对加热时间进行研究，确定重组颗粒蛋白溶解度较高时的加热时间限度：
样品为E.coli菌体破碎上清液，将pH调节为pH＝9.0，分装后分别置于80℃和90℃加热孵育，设置15min，30min，45min三个加热时间考察点，孵育后立即15000g离心15min，取上清液进行SDS-PAGE检测，检测结果如图2。
结果与分析：
由图2可见，在pH9.0,80℃～90℃孵育15min,30min,45min后的离心上清液中，存在显著的重组颗粒蛋白条带。与加热孵育1小时条件对比后发现，15min-45min加热后上清的条带灰度略小于加热1小时，其除杂效果与60-90℃加热1小时的效果接近。可见，在80～90℃，pH9.0，加热15min,30min,45min时重组颗粒蛋白同样可以呈现较高的溶解度，同时仍能保持较好的除杂效果。
经过第一步加热处理(例如：pH＝9.0，80℃～90℃加热孵育处理1h)，可使重组蛋白纯度达到60％以上。
综上，利用第一步加热工艺处理重组颗粒蛋白产品可提高重组颗粒蛋白纯度。综合重组颗粒蛋白在不同条件下的溶解性和纯度状态，获得第一步加热工艺条件为60℃～90℃、加热孵育15min以上、pH＝8.0～10.0；优选为80℃～90℃、加热孵育1h、pH＝9.0。
实施例2确定大肠杆菌破碎上清液中重组颗粒蛋白的第二步加热工艺条件
E.Coli菌体破碎液经过第一步加热处理分离杂质和重组颗粒蛋白，再对样品进行第二步加热处理，进一步分离杂质和重组颗粒蛋白。在第一步加热的基础上，在pH7.4条件下，摸索处理温度与时间对重组颗粒蛋白和杂质的溶解度的影响。
第一步加热：取菌体破碎上清液调整至pH9.0后，80℃加热1h，12000g 4℃离心30min取上清液。上清液中加入等体积的100mM Tris-HCl、5mM EDTA、4％Triton X-100、pH 7.4缓冲液和总体积的10％的1M Tris-HCl pH7.4缓冲液。
第二步加热：加热温度设置6个考察点：40℃、50℃、60℃、70℃、80℃、90℃。加热时间设置5个考察点：1min,3min,5min,10min,15min。将前处理样品分别置于40℃、50℃、60℃、70℃、80℃、90℃加热1、3、5、10、15min后，立即进行15000g离心60s取上清液。随后对上清液进行SDS-PAGE检测，检测结果如图3。
结果与分析：
由图3a/b可知，在40℃和50℃的加热条件下，重组颗粒蛋白在加热3min后全部沉淀，上清液中无重组颗粒蛋白，在加热的前3min时看到上清液中仍有重组颗粒蛋白条带，推测可能是加热时间较短样品温度尚未升高至设定温度所致。由图3c/d/e/f可知，在60℃、70℃、80℃、90℃加热条件下，上清液中的重组颗粒蛋白含量在前3min随加热时间延长增加，且随着加热温度的升高，重组颗粒蛋白溶解度增加，至5min溶解度不再变化。
经过第二步加热处理(例如：pH＝7.4，60℃加热孵育处理10min)，可使重组蛋白纯度达到85％以上。
综上，经过第一步加热处理后的样品，再进行第二步加热时，表现为：
a)重组颗粒蛋白在50℃条件下溶解度最低，电泳显示杂质蛋白仍然存在于上清液中；
b)60～90℃加热时，温度越高重组颗粒蛋白溶解度越高。
综上，重组颗粒蛋白产品的第二步加热工艺条件为40-90℃、加热孵育处理5-15min、pH＝7.0-8.0；优选为50-60℃，加热孵育处理5-10min，pH7.4。
实施例3重组颗粒蛋白产品的两步加热制备工艺
重组颗粒蛋白在大肠杆菌内进行表达，收获菌体后需要通过高压均质破碎释放目标蛋白并进行料液澄清，主要目的是去除菌体碎片与杂质蛋白。料液澄清主要通过加热处理完成。使用实施例1和实施例2的方法进行加热处理，对E.coli破碎后上清液进行第一步加热和第二步加热(即“两步加热”)，并对两步加热步骤的除杂效果和重组颗粒蛋白的纯度进行测算。
取离心收集的E.coli湿菌体60g，重悬于240ml的缓冲液(20mM Tris-HCl、2mM PMSF、pH＝9.0)，使用高压匀质机1000bar压力下破碎，离心后收集到280ml上清液，取其中40ml进行两步加热操作。对破碎后的上清液、第一步加热离心上清液，第二步加热离心沉淀重悬液进行SDS-PAGE分析(如图4所示)。图4显示了E.coli的破碎后上清液，第一步加热后上清液，第二步加热后沉淀重悬液之间的SDS-PAGE检测结果。
结果与分析：
通过对重组颗粒蛋白在不同温度、pH条件下进行实验，发现该颗粒蛋白可在高温条件下保持高稳定性和高溶解度，亦可在特定pH条件下形成可复溶的沉淀。
具体地，如表3所示，第一步加热中，调节pH＝9.0，80℃水浴加热1小时，恢复至室温后离心收集到约35ml上清液。第二步加热中，向其中加入100mM Tris-HCl、5mM EDTA、4％Triton X-100、pH 7.4缓冲液35ml，再加入7ml的1M Tris-HCl pH7.4，混匀。置于60℃水浴加热10min，立即离心收集沉淀，使用20mM Tris-HCl 5mM EDTA pH＝9.0的缓冲液复溶沉淀。
表3 重组颗粒蛋白产品两步加热提取方法
Figure PCTCN2022116392-appb-000005
综上，在层析操作之前，经过两步加热工艺，可将重组颗粒蛋白纯度提升至85％以上，从而可减轻后续层析纯化的压力，减少所需的层析步骤和生产成本。
实施例4重组颗粒蛋白产品的尿素和氯化钠联合制备工艺
在两步加热工艺之后，层析纯化之前加入不同浓度的尿素和氯化钠可以显著减少目标重组颗粒蛋白条带附近存在的不明物质，并通过实验确定了最适宜的尿素处理时间和氯化钠浓度。
1、尿素处理：
(1)设置6M尿素短时浸泡组(1h)、6M尿素过夜浸泡组(17h)、4M尿素短时浸泡组(1h)、4M尿素过夜浸泡组(17h)、1M尿素短时浸泡组(1h)、1M尿素过夜浸泡组(17h)六种处理条件进行尿素处理：
取菌体破碎上清液调整至pH9.0后，80℃加热1h，12000g，4℃离心30min取上清液。上清液中加入等体积的100mM Tris-HCl,5mM EDTA,4％Triton X-100,pH 7.4缓冲液和总体积的10％的1M Tris-HCl pH7.4缓冲液。60℃加热10min，立即6000g，10min离心去除上清液。将沉淀重溶于20mM Tris-HCl,5mM EDTA,pH 9.0缓冲液中。
设置6M尿素短时浸泡组(1h)、6M尿素过夜浸泡组(17h)、4M尿素短时浸泡组(1h)、4M尿素过夜浸泡组(17h)、1M尿素短时浸泡组(1h)、1M尿素过夜浸泡组(17h)六个组别。对尿素处理后的本申请的重组颗粒蛋白样品进行分子筛层析分析，对蛋白样品主峰后的小分子杂质峰进行SDS-PAGE检测以确定其成分，进而明确各个组别对于杂质去除的效果。
结果与分析：
6M尿素浸泡处理的分子筛层析图谱如图5所示，6M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果如图6所示；4M尿素浸泡处理的分子筛层析图谱如图7所示，4M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果如图8所示；1M尿素浸泡处理的分子筛层析图谱如图9所示，1M尿素处理后分子筛洗脱峰的SDS-PAGE检测结果如图10所示。
由层析图谱可知，三种不同浓度的尿素在处理1h和17h之后，在分子筛中均能够分离出分子量小于本申请重组颗粒蛋白主峰的其他物质(peak7-18)。另外，发现在所有三种17h浸泡处理组中，6M尿素处理的杂质峰峰高为1.13mAU，4M尿素处理的杂质峰峰高为0.54mAU，1M尿素处理的杂质峰峰高为0.30mAU。该情况表明在相同处理时间下，尿素浓度越高，对该杂质的分离去除效果越强。综上，可以得出结论，在相同处理时间情况下，对杂质的分离去除效果随着尿素浓度升高而增强。
(2)进一步地，设置无尿素对照组、8M尿素短时浸泡(1h)、8M尿素过夜浸泡(16-18h) 三种处理条件：
取菌体破碎上清液调整至pH9.0后，80℃加热1h，12000g,4℃离心30min取上清液。上清液中加入等体积的100mM Tris-HCl,5mM EDTA,4％Triton X-100,pH 7.4缓冲液和总体积的10％的1M Tris-HCl pH7.4缓冲液。60℃加热10min，立即6000g，10min离心去除上清液。将沉淀重溶于20mM Tris-HCl,5mM EDTA,pH 9.0缓冲液中。
设置无尿素对照组、8M尿素短时浸泡(1h)、8M尿素过夜浸泡(16-18h)三种处理条件。对处理后的样品使用分子筛层析进行物质分离(如图11所示)，并对重组颗粒蛋白主峰后的小分子峰进行SDS-PAGE检测以确定其分子量(如图12所示)。
结果与分析：
由图11可知，三种不同时间的高浓度尿素处理之后，分子筛均能分离出分子量小于重组颗粒蛋白主峰的其他物质(peak1-5)，且出峰时间基本相同。由图12可知，只有5号峰(8M尿素浸泡16h)出现明显的25-35kDa之间的条带；3号峰(8M尿素浸泡1h)出现较浅的25-35kDa之间的条带。无尿素浸泡的1号和2号峰则无可见条带。实验表明，8M高浓度尿素浸泡有助于去除部分小分子杂质，优选的浸泡时间12h以上。
2、氯化钠处理
(1)设置四个氯化钠浓度考察点—50mM、100mM、150mM、200mM，观察不同浓度的氯化钠对杂质去除的效果:实验样品与尿素实验样品相同。在加入8M尿素的基础上，再加入氯化钠对样品进行处理。设置四个氯化钠浓度考察点：50mM、100mM、150mM、200mM。
分别使用8M尿素和50mM、100mM、150mM、200mM四种浓度的氯化钠对重组颗粒蛋白样品浸泡16h。随后使用Fractogel DEAE M填料对其进行层析纯化，并对其流穿液和洗脱峰各部分进行SDS-PAGE检测(如图13所示)。
结果与分析：
根据图13a可观察到，在8M尿素和50mM氯化钠浸泡16h后，主洗脱峰中的25-35kDa的物质含量明显降低，同时在层析上样流穿液中出现该物质。由图13b可以观察到，提高氯化钠浓度后，25-35kDa的条带相比于50mM氯化钠处理并未明显减少。
a)8M高浓度尿素浸泡重组颗粒蛋白的前处理步骤有助于去除25-35kDa的不明物质。
b)在8M尿素基础上添加氯化钠浸泡同样有助于去除该不明物质，且50mM-200mM氯化钠的除杂效果并无显著差别。
综上，在Fractogel DEAE M层析前对重组颗粒蛋白样品进行预处理的优选工艺条件为8M尿素和50-200mM氯化钠浸泡。
(2)验证试验：
设置不同的盐进行对比，供比较选择的盐类包括氯化钾、硫酸钠、氯化镁，进行对比实验，并行比较的盐类包括氯化钾、硫酸钠、氯化镁，观察不同种类、不同浓度盐类对杂质去除的效果:
①实验方法：
取菌体破碎上清液调整至pH9后，80℃加热1h，12000g,4℃离心30min取上清液。上清液中加入等体积的100mM Tris-HCl,5mM EDTA,4％Triton X-100,pH 7.4缓冲液和总体积的10％的1M Tris-HCl pH7.4缓冲液。60℃加热10min，立即6000g，10min离心去除上清液。将沉淀重溶于20mM Tris-HCl,5mM EDTA,pH 9缓冲液中。
为探寻确认常用盐类的效果，选取KCl、Na 2SO 4、MgCl 2三种不同价型不同酸根的盐类，根据NaCl处理实验的经验，设置50mM KCl、100mM KCl、50mM Na 2SO 4、100mM Na 2SO 4、50mM MgCl 2、100mM MgCl 2共6个组别，过夜(16-18h)浸泡处理后，使用Fractogel DEAE M填料进行层析纯化，纯化时将层析平衡液中的NaCl改为相对应的三种盐，层析结束后，对各个组别的流穿物和洗脱峰进行SDS-PAGE分析。
②实验结果：
a.KCl处理本申请重组颗粒蛋白的实验结果
图14(a)所示为KCl处理后的样品经过F-DEAE层析后的SDS-PAGE胶图，由图可知，两种浓度的KCl浸泡处理组的流穿中都没有出现25-35kDa的杂质，洗脱中因浓度较低杂质条带不明显，但依然可见25-35kDa的杂质分布，与NaCl的浸泡处理效果对比来看，KCl并无显著优势。
b.Na 2SO 4处理本申请重组颗粒蛋白的实验结果
图14(b)所示为Na 2SO 4处理后的样品通过F-DEAE层析后的SDS-PAGE分析图，可以观察到两种浓度的Na 2SO 4处理后通过F-DEAE层析时，流穿液中都出现本申请重组颗粒蛋白产物，而洗脱中没有本申请重组颗粒蛋白产物，层析同样提示洗脱时UV吸收值极低。这表明Na 2SO 4添加后增大了样品的电离性，使得本申请重组颗粒蛋白无法结合到F-DEAE柱子上。因此，Na 2SO 4不能被采用作预处理样品的盐。
c.MgCl 2处理本申请重组颗粒蛋白的实验结果
在使用MgCl 2处理本申请重组颗粒蛋白的过程中，发现缓冲液在加入50mM或100mMMgCl 2后,pH值由8.9降低至8.2或8.0左右，已经低于F-DEAE层析工艺所规定的pH范围，若使用NaOH将pH进行回调，则容易产生Mg(OH) 2难溶性沉淀物。因此，MgCl 2不能被采用作预处理样品的盐。
③实验分析
通过对比NaCl、KCl、Na 2SO4、MgCl 2四种盐类的实验，发现KCl预处理后的流穿液中目标杂质少，对比NaCl没有明显优势；Na 2SO 4因其本身离子强度高，会导致本申请重组颗粒蛋白在层析过程中流穿；MgCl 2会导致体系pH降低且不宜用NaOH回调。
综合考虑，NaCl从除杂效果、工艺兼容性、pH稳定性等方面是最适用的预处理盐类。实施例5重组颗粒蛋白产品的两步层析纯化工艺
将先后经过实施例3和实施例4预处理的重组颗粒蛋白样液使用离子交换和疏水层析进行精制，第一步层析纯化使用Fractogel DEAE M层析工艺进行层析纯化，具体步骤和参数参见表4。Fractogel DEAE M洗脱收集液样品先进行缓冲液稀释，添加50％(w/v)蔗糖稳定剂防止下一步层析过程中产生重组颗粒蛋白沉淀，具体参数参见表5。然后使用疏水层析Octyl Bestarose 4FF层析工艺进行精纯(第二步层析纯化)，具体步骤和参数参见表6。
1、第一步层析方法：层析填料-Fractogel DEAE M，保留时间-12.5min
表4：第一步层析方法
Figure PCTCN2022116392-appb-000006
表5：第二步层析前样品稀释方法
Figure PCTCN2022116392-appb-000007
2、第二步层析方法：层析填料-Octyl Bestarose 4FF，保留时间-12.5min
表6：第二步层析方法
Figure PCTCN2022116392-appb-000008
结果与分析：
通过纯度检测发现，进一步经过以上层析介质组合进行精制后，获得产品的纯度可达到99.0％以上。
实施例6稳定剂浓度与种类对重组颗粒蛋白产品的保护作用
在不同层析步骤的间隔中，观察到重组颗粒蛋白因浓度较高出现的沉淀聚集现象，且沉淀现象显示出随温度升高而增加的趋势。需要加入稳定剂提高蛋白稳定性和工艺稳健性。为摸索加入稳定剂的浓度与种类对重组颗粒蛋白的保护作用的影响，本实验选取蔗糖(Sucrose)、山梨糖醇(Sorbitol)、海藻糖(Trehalose)三种稳定剂，根据工艺需要和常规经验，考察下表7所示浓度的稳定剂对重组颗粒蛋白的保护作用。取Fractogel DEAE M的洗脱液，按照表8体积和顺序混合样品、稳定剂与缓冲液，此外，设置将稳定剂替换为纯化水的阴性对照组。将混匀的样品置于30℃下孵育30min，测量并记录孵育后的UV320nm和UV280nm吸光值。UV320nm的吸光值越高，表明蛋白产生聚集沉淀的程度更高。具体结果如表9所示。
结果与分析：
根据表9可知，蔗糖、山梨糖醇和海藻糖均在浓度≧20％时，可以使重组颗粒蛋白溶液在30℃加热30min后不出现沉淀，且三者的效果较为接近。
可见，在Fractogel DEAE M层析之后，使用≧20％浓度的蔗糖、山梨糖醇或海藻糖作为稳定剂，可以对重组颗粒蛋白起到有效的保护作用。由于实验室和生产车间对室内温度控制范围在18-26℃之间，加入稳定剂后可提供足够的蛋白稳定性和工艺稳健性保证。
表7：稳定剂种类及其考察浓度
Figure PCTCN2022116392-appb-000009
表8：稳定剂实验样品稀释方法
Figure PCTCN2022116392-appb-000010
Figure PCTCN2022116392-appb-000011
表9：不同稳定剂和浓度30℃孵育后吸光度
Figure PCTCN2022116392-appb-000012
综上所述，上述各实施例及附图仅为本发明的较佳实施例而已，并不用以限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，皆应包含在本发明的保护范围内。

Claims (10)

  1. 一种重组颗粒蛋白产品的制备方法，包括以下步骤：
    (1)转染含编码颗粒蛋白序列的质粒载体至宿主细胞，并在细胞内进行表达；
    (2)收获并破碎菌体，收集上清液；
    (3)a.调节上清液pH为9.0-10.0，在80℃-100℃加热孵育15分钟以上；优选地，调节上清液pH为9.0-10.0，在80℃-95℃加热孵育15-80分钟；更优选地，调节上清液pH为9.0，在80℃-90℃加热孵育15-80分钟；然后恢复至室温后离心，弃沉淀，收集上清液；加入稀释缓冲液，调节pH值为pH7.0-8.0；
    b.将溶液置于50℃-65℃条件下加热孵育5-20分钟；优选地在50℃-60℃条件下加热孵育5-10分钟，立即离心，弃上清，收集沉淀；使用重悬缓冲液在pH9.0-11.0条件下重悬沉淀；
    (4)进行层析纯化；
    所述重组颗粒蛋白产品如SEQ ID NO:1所示。
  2. 根据权利要求1所述的制备方法，其中，步骤(3)重悬沉淀后，加入尿素和氯化钠，再进行步骤(4)；优选尿素的浓度为6M-8M。
  3. 根据权利要求1至2任一所述的制备方法，其中，步骤(3)a中稀释缓冲液含有Tris-盐酸、醋酸-醋酸钠、柠檬酸或磷酸；优选进一步含有乙二胺四乙酸或/和Triton X-100。
  4. 根据权利要求1至3任一所述的制备方法，其中，步骤(3)b中重悬缓冲液含有Tris-盐酸、醋酸-醋酸钠、柠檬酸或磷酸；优选进一步含有乙二胺四乙酸。
  5. 根据权利要求1至4任一所述的制备方法，其中，步骤(4)的层析为阴离子交换层析和疏水层析。
  6. 根据权利要求5所述的制备方法，其中，阴离子交换层析介质选自DEAE Sepharose FF、Q Sepharose FF、Capto DEAE、Capto Q Impres、POROS HQ、POROS 50D、POROS PI、Fractogel DEAE或Fractogel TMAE，优选Fractogel DEAE；
    疏水层析介质选自Butyl-S Sepharose 6FF、Butyl Sepharose 4 FF、Octyl Bestarose 4FF、Phenyl Sepharose 6FF、Capto Butyl、Capto Phenyl、Capto Phenyl Impres或Capto Octyl，优选Octyl Bestarose 4FF。
  7. 根据权利要求5至6任一所述的制备方法，其中，在阴离子交换层析获得的洗脱收集液中和疏水层析缓冲液中添加稳定剂，稳定剂选自氨基酸、多元醇或糖。
  8. 根据权利要求7所述的制备方法，其中，所述氨基酸选自精氨酸、甘氨酸或组氨酸，多元醇选自甘油或山梨糖醇，糖选自蔗糖或海藻糖。
  9. 根据权利要求7至8任一所述的制备方法，其中，所述稳定剂的浓度为20％以上(w/v)。
  10. 一种重组颗粒蛋白产品，采用权利要求1-9任一所述的制备方法制备。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109134620A (zh) * 2018-09-21 2019-01-04 中国科学院过程工程研究所 一种纯化HBc-VLPs或HBc-VLPs衍生物的方法
CN111991556A (zh) * 2020-10-29 2020-11-27 中山大学 SARS-CoV-2 RBD共轭纳米颗粒疫苗
CN114395015A (zh) * 2021-12-17 2022-04-26 烟台派诺生物技术有限公司 一种适合工业化生产的重组颗粒蛋白产品及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104098700B (zh) * 2014-05-05 2017-06-16 兰州大学 结核分枝杆菌融合蛋白eammh、其构建、表达和纯化方法及其应用
CN109134621A (zh) * 2018-09-21 2019-01-04 中国科学院过程工程研究所 一种HBcAg-VLP或HBcAg-VLP衍生物的纯化方法
CN109966483B (zh) * 2019-04-08 2021-07-30 中国科学院过程工程研究所 一种基于铁蛋白的多抗原通用流感疫苗及其制备方法和应用
CN112089831B (zh) * 2020-11-04 2021-04-02 浙江普康生物技术股份有限公司 重组新型冠状病毒亚单位疫苗的制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109134620A (zh) * 2018-09-21 2019-01-04 中国科学院过程工程研究所 一种纯化HBc-VLPs或HBc-VLPs衍生物的方法
CN111991556A (zh) * 2020-10-29 2020-11-27 中山大学 SARS-CoV-2 RBD共轭纳米颗粒疫苗
WO2022088953A1 (zh) * 2020-10-29 2022-05-05 中山大学 SARS-CoV-2 RBD共轭纳米颗粒疫苗
CN114395015A (zh) * 2021-12-17 2022-04-26 烟台派诺生物技术有限公司 一种适合工业化生产的重组颗粒蛋白产品及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRUUN, T.U.J. ET AL.: "Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination", ACS NANO, vol. 12, 20 July 2018 (2018-07-20), pages 8855 - 8866, XP055751545, DOI: 10.1021/acsnano.8b02805 *
NG, M.Y.T. ET AL.: "Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen", JOURNAL OF VIROLOGICAL METHODS, vol. 137, 24 July 2006 (2006-07-24), pages 134 - 139, XP025030198, DOI: 10.1016/j.jviromet.2006.06.016 *
XIE, HANGHANG ET AL.: "The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation", CHINA BIOTECHNOLOGY, vol. 40, no. 5, 31 December 2020 (2020-12-31), pages 40 - 47, XP009547237 *

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