WO2014021604A1 - 인유두종바이러스 바이러스 유사입자의 고효율 정제방법 - Google Patents

인유두종바이러스 바이러스 유사입자의 고효율 정제방법 Download PDF

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WO2014021604A1
WO2014021604A1 PCT/KR2013/006823 KR2013006823W WO2014021604A1 WO 2014021604 A1 WO2014021604 A1 WO 2014021604A1 KR 2013006823 W KR2013006823 W KR 2013006823W WO 2014021604 A1 WO2014021604 A1 WO 2014021604A1
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protein
hpv
chromatography
heating
reducing agent
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PCT/KR2013/006823
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English (en)
French (fr)
Korean (ko)
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김홍진
김형진
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Kim Hong-Jin
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Priority to CN201380040425.9A priority Critical patent/CN104507956B/zh
Priority to EP13824788.7A priority patent/EP2881401B1/en
Priority to CA2880420A priority patent/CA2880420C/en
Priority to AU2013297306A priority patent/AU2013297306B2/en
Priority to MX2015001413A priority patent/MX361186B/es
Priority to US14/418,004 priority patent/US9994618B2/en
Priority to JP2015525348A priority patent/JP6014763B2/ja
Priority to BR112015002126-3A priority patent/BR112015002126B1/pt
Publication of WO2014021604A1 publication Critical patent/WO2014021604A1/ko

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20051—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method capable of efficiently purifying virus-like particles (VLPs) of human papillomavirus (HPV) having excellent structural and immune characteristics.
  • VLPs virus-like particles
  • HPV human papillomavirus
  • Human papillomavirus is a pathogen that accounts for nearly 100% of the causes of cervical cancer [1]. Globally, 500,000 women are diagnosed with cervical cancer each year and 250,000 women are known to die of cervical cancer [2]. Types of high-risk human papillomaviruses that cause cervical cancer include 16, 18, 45, 31, 33, 52, 58, 35, and 59, and low-risk types include 6 and 11 and the like are known [3, 4]. Human papillomavirus types 16 and 18 account for 70% of all cervical cancer causes, and are recognized as the most important type for cervical cancer prevention [5]. The types of human papillomaviruses responsible for the development of cervical cancer vary widely from region to region [5]. Infections caused by HPV type 16, 18, 31 and 45 are the leading causes of cervical cancer in Africa, Europe, North America and Latin America, whereas in Asia, type 16, 18, 58 and 33 Infection is the main cause of cervical cancer [5].
  • L1 protein The capsid of human papillomavirus consists of the major antigen L1 protein and the minor antigen L2 protein [6]. Since L1 protein is self-assembly and forms virus-like particles (VLPs), it is used as an antigen for cervical cancer vaccine and diagnostic antigen [6, 27].
  • Recombinant L1 protein includes Escherichia coli , Saccharomyces cerevisiae , Pichia Pastoris , Lactobacillus casei , Spodoptera frugiperda cells and plant cells. Produced as expressing cells [7-12, 28].
  • Commercially available vaccines for cervical cancer include Gardasil TM (Merck) and Cervarix TM (GlaxosmithKline, GSK).
  • Gardasil TM contains L1 virus analogues against human types HPV type 16, 18, 6 and 11, and Cervarix TM contains L1 virus analogues against human papilloma virus types 16 and 18 as antigen [13].
  • Gardasil TM uses Saccharomyces cerevisiae as antigen-expressing cells and Cervarix TM uses insect cell Sf ( Spodoptera frugiperda ) cells as antigen-expressing cells [13, 14]. Both vaccines are given by intramuscular injection, which costs $ 120 for a single shot and $ 360 for three shots [15].
  • the high inoculation of cervical cancer vaccines on the market is widely used in developing countries where cervical cancer is the main cause [16]. Therefore, the development of low-cost, high-efficiency cervical cancer vaccines remains an important challenge.
  • the present inventors have tried to develop a new method for purifying HPV L1 protein produced from host cells expressing L1 protein of Human Papillomavirus (HPV) with high purity and high efficiency.
  • HPV Human Papillomavirus
  • an object of the present invention is to provide a method for purifying human papillomavirus (HPV) L1 protein with high purity and high efficiency.
  • HPV human papillomavirus
  • the present invention provides a method for purifying a Human Papillomavirus (HPV) L1 protein, comprising the following steps: (a) culturing a transgenic host cell expressing the L1 protein of HPV Recovering and crushing the cultured host cells; (b) adding a reducing agent to the lysate of the host cell; And (c) purifying the HPV L1 protein by chromatography from the lysate of the host cell to which the reducing agent is added.
  • HPV Human Papillomavirus
  • the present invention provides a method for purifying Human Papillomavirus (HPV) L1 protein, comprising the following steps: (i) transforming a host cell expressing L1 protein of HPV Recovering and culturing the cultured host cells after culturing; (ii) adding a reducing agent to the lysate of the host cell; (iii) heating and then cooling the lysate of the host cell to which the reducing agent is added; And (iv) purifying the HPV L1 protein by chromatography from the lysate of the heated and cooled host cells.
  • HPV Human Papillomavirus
  • the present inventors have endeavored to develop a new method for purifying HPV L1 protein produced from host cells made to express L1 protein of human papillomavirus (HPV) with high purity and high efficiency.
  • Treatment, or treatment with a reducing agent, followed by heating and chilling, and purification via chromatography not only dramatically improves the purity of the HPV L1 protein, but also resembles a virus-like assembly from the L1 protein.
  • the present invention was completed by experimentally confirming that the structural and immune characteristics of particles (Virus like particle, VLP) are excellent.
  • HPV L1 protein refers to a major protein constituting the capsid of HPV as expressed from the L1 gene of HPV.
  • the L1 protein has the property of self-assembling into virus-like particles (Virus-Like Particles (VLP)) under suitable conditions together with L2 protein, which is another minor protein constituting the capsid, or L1 protein alone.
  • VLP virus-Like Particles
  • Papillomavirus is a DNA genome virus of up to eight early (E) and two late (late, L) genes, 50-60 nm in size, envelope-free, and icosahedral. to be.
  • E in gene E means early
  • L in gene L means late.
  • the E gene is a gene involved in the function of viral replication and transformation.
  • L1 and L2 genes encode viral capsid proteins.
  • L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa.
  • L2 protein is a minor capsid protein with a predicted molecular weight of 55-60 kDa and an apparent molecular weight measured by PAGE of 75-100 kDa.
  • the type of human papillomavirus (HPV) from which the L1 protein is derived in the method of the present invention is not particularly limited, and preferably, HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV Type 30, HPV Type 31, HPV Type 33, HPV Type 35, HPV Type 39, HPV Type 41, HPV Type 42, HPV Type 43, HPV Type 44, HPV Type 45, HPV Type 51, HPV Type 52, HPV Type 54 , HPV type 55, HPV type 56, HPV type 58, HPV type 68 and HPV type 70, more preferably the L1 protein of the present invention is HPV type 6a, HPV type 6b, HPV type 11, HPV It is derived from HPV selected from the group consisting of Type 16, HPV Type 18, HPV Type 31, HPV Type 33, HPV Type 45 and HPV Type 58, most preferably HPV Type 16, HPV Type 18, or HPV Type 58 Is derived from.
  • Cells used as host cells in the present invention are bacteria, yeast cells, insect cells, plant cells or animal cells.
  • the yeast cells Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Saccharomyces pastorianus Saccharomyces pastorianus
  • Saccharomyces sp. Schizosaccharomyces pombe , Pichia Pastoris , or Hansenula polymorpha Saccharomyces cerevisiae
  • Saccharomyces pastorianus Saccharomyces pastorianus
  • Saccharomyces sp. Schizosaccharomyces pombe Saccharomyces pombe
  • Pichia Pastoris or Hansenula polymorpha
  • a transformed host cell expressing HPV L1 protein refers to a host cell transformed with an expression vector that successfully expresses HPV L1 protein.
  • the expression vector may include transcriptional or translational regulatory elements and other marker genes known in the art.
  • Transgenic host cells expressing the HPV L1 protein of the present invention can be readily prepared using methods known in the art.
  • reducing agent refers to an element or compound that donates electrons to other species in a reduction-oxidation reaction, preferably in a peptide or protein. It means a compound used for the reduction of disulfide bonds or the stabilization of free sulfhydryl groups.
  • the reducing agent is, for example, ⁇ -mercaptoethanol ( ⁇ -mercaptoethanol), DTT (dithiothreitol), 2-mercaptoethylamine-HCl (2-mercaptoethylamine-HCl), TCEP [tris (2-carboxyethyl) phosphine] , Cysteine-HCl (cystein-HCl), more preferably ⁇ -mercaptoethanol or DTT.
  • the final concentration of the reducing agent in the cell lysate in the present invention is at least 0.1% by weight, preferably 0.1-20% by weight, more preferably 0.1-10% by weight, even more preferably 1-8% by weight, even more It is preferably 3-7% by weight, most preferably 4-6% by weight.
  • reaction time after the addition of the reducing agent to the cell lysate is not limited to a specific time range, and those skilled in the art can select and use a reaction time suitable for the method of the present invention.
  • the heating and cooling treatment of the cell lysate with the reducing agent not only greatly increases the removal efficiency of impurities in the lysate of the transformed host cell, but also the VLP assembled from the L1 protein. Has desirable structural and immune properties.
  • the heating temperature of the cell lysate in the present invention means a temperature above room temperature, preferably above 25 ° C, more preferably above 25 ° C and below 80 ° C, even more preferably It is a temperature of 30-65 degreeC, More preferably, it is 35-65 degreeC, Most preferably, it is 35-60 degreeC,
  • the optimal heating conditions are the temperature of 35-55 degreeC.
  • the heating time of the cell lysate may vary depending on the heating temperature, for example, but may be appropriately selected in the range of 10 minutes to 72 hours, but is not limited thereto. Preferably it is the range of 30 minutes-72 hours, More preferably, it is the range of 30 minutes-48 hours, More preferably, it is the range of 30 minutes-24 hours, Most preferably, it is the range of 30 minutes-12 hours. .
  • the temperature of the cooling means a temperature such that the sample of the cell debris does not freeze
  • the cooling temperature is a temperature of 0-10 °C, preferably more than 0 °C less than 8 °C, more preferably Preferably it is cooled to a temperature above 0 ° C and below 7 ° C, even more preferably above 0 ° C and below 6 ° C, most preferably above 0 ° C and below 5 ° C.
  • the cooling time can be used by selecting a suitable cooling time to fit the method of the present invention.
  • the embodiment it can be maintained at room temperature without heating and cooling the debris of the host cell to which the reducing agent is added.
  • room temperature refers to a temperature of 15 ° C or more and 25 ° C or less as it is, without being heated or cooled.
  • the HPV L1 protein is purified by chromatographic purification of the host cell lysate to which the reducing agent is added, or by chromatographic purification after adding the reducing agent to the lysate of the host cell, heating and cooling.
  • Chromatography usable in the present invention is known in the art, but is not limited to, for example, ion exchange chromatography (ion exchange chromatography), such as cation exchange chromatography or anion exchange chromatography, size exclusion chromatography ( size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography can be used. Since the substance to be separated and purified in the present invention is a protein, ion exchange chromatography that is most suitable for the separation of proteins or peptides is preferred. In a specific embodiment of the present invention, HPV L1 protein was successfully isolated and purified using heparin resin chromatography and cation exchange chromatography, which are one of cation exchange chromatography.
  • the present invention provides a method for purifying Human Papillomavirus (HPV) L1 protein with high purity and high efficiency.
  • the purification purity of HPV L1 protein can be improved significantly.
  • VLP of the HPV L1 protein purified by the purification method of the present invention forms a good structure and is very excellent in immunogenicity.
  • the present invention relates to a method for purifying Human Papillomavirus (HPV) L1 protein with high purity and high efficiency. According to the purification method of the present invention, not only can greatly improve the purification purity of the HPV L1 protein, but also the VLP of the purified HPV L1 protein has an excellent immunogenicity by forming a structure more similar to the original HPV virion.
  • HPV Human Papillomavirus
  • FIG 1 shows the process of the prior art (hereinafter referred to as "T-1" method) and the process of the present invention (hereinafter also referred to as "T-5" method) for purifying virus analog particles of human papillomavirus. Shows.
  • T-1 prior art
  • T-5" method process of the present invention
  • the host cell Saccharomyces cerevisiae expressing HPV L1 protein was disrupted for the T-5 method and then a reducing agent was added. After the reducing agent was treated, impurities were removed by heating and chilling.
  • the L1 protein contained in the cell lysate from which the impurities were removed was first purified through heparin chromatography. The first purified L1 protein was purified by high purity by further performing second heparin chromatography.
  • L1 protein in the Saccharomyces cerevisiae lysate for the T-1 method was recovered via ammonium sulfate precipitation. Impurities in the recovered fractions were removed through the removal of precipitated contaminants. L1 protein in the lysate after these processes was purified by heparin chromatography.
  • Figure 2 relates to the results of purification by the T-5 method, the lysate of the L1 protein-expressing host cell is subjected to dialysis, treatment with a reducing agent, and then subjected to a heating and cooling process, and subjected to primary heparin chromatography, SDS This is the result confirmed by -PAGE.
  • SDS secondary heparin chromatography
  • LS indicates a loading sample loaded on heparin resin
  • FT indicates flow-through without binding to heparin resin.
  • W is the wash from the step of washing the heparin resin (wash)
  • E means a fraction (Elution) eluted HPV L1 protein in a buffer containing 1M NaCl.
  • Figure 3 relates to the purification results by the T-5 method, without the dialysis on the lysate of the HPV L1 protein-expressing host cell, the cell lysate is directly treated with a reducing agent, followed by heating and cooling process, the first heparin Chromatography was performed and confirmed by SDS-PAGE. Similar to the results in FIG. 2, high purity HPV L1 protein was also recovered after the first heparin chromatography. LS, FT, W and E are the same as described in FIG.
  • Figure 4a relates to the purification results by the T-5 method, after treatment with a reducing agent in the lysate of HPV L1 protein-expressing host cells undergo a heating and cooling process, the first heparin chromatography, and the second heparin chromatography This is the result confirmed by SDS-PAGE.
  • LS and FT are the same as described in FIG.
  • the numbers 3 to 18 shown on the top of the SDS-PAGE photograph indicate the number of each fraction recovered in the linear gradient elution with NaCl.
  • FIG. 4B shows the profile of the second heparin chromatography. Although no protein band was detected in FT (flow-through) of SDS-PAGE of FIG. 4A, it was confirmed that a large amount of impurities flow out of FT (flow-through) in FIG. 4B. Thus, the results in FIG. 4b show that a significant amount of impurities have been removed by secondary heparin chromatography.
  • FIG. 5 shows SDS-PAGE results of heparin chromatography of the T-1 method.
  • Treatment of the sample before heparin chromatography proceeded as shown in FIG. 1.
  • LS stands for loading sample. Proteins bound to heparin resin were eluted by increasing the gradient of NaCl. Gradient elution proceeded from 0.325 M NaCl to 2 M NaCl. The number at the top of the SDS-PAGE results indicates the number of elution fractions. It is shown that fractions 11-14 elute the L1 protein in high purity.
  • FIG. 6a is a result of comparing the purity of the VLP purified by the conventional method (hereinafter also referred to as "T-1" method) and the T-5 method of the present invention through SDS-PAGE.
  • T-1 HPV 16 VLPs is a well-known method [Kim et al. (2010) Protein Expr Purif 70: 68-74; Kim et al. (2009) Arch Pharm Res 32: 1759-1766, and "T-5 HPV 16 VLP” is the result purified according to the method of the present invention. Protein quantification was performed for SDS-PAGE analysis of HPV L1 protein and then 500 ng, 250 ng, 125 ng, 62 ng of protein were loaded per well. Two replicates were independently performed and presented in Panels A and B, respectively. In both experiments, the intensity of the L1 protein band of T-5 HPV 16 VLP was significantly higher than that of T-1 HPV 16 VLP.
  • FIG. 6B is a graph showing the L1 protein band intensities of the T-1 HPV 16 VLPs and the T-5 HPV 16 VLPs confirmed in the two experiments of FIG. 6A. Results were expressed as mean ⁇ standard deviation and calculated as 100% of the intensity of the L1 protein band of T-5 HPV 16 VLPs loaded with 500 ng per well. This result shows that the purity of VLP purified by T-5 method is much better than that of VLP purified by T-1 method.
  • Figure 7 shows the results of SDS-PAGE by loading so that the intensity of the L1 protein band of T-1 HPV 16 VLP is the same as the intensity of the L1 protein band of T-5 HPV 16 VLP.
  • T-1 HPV 16 VLPs were identified with a size of 20-50 nm, while T-5 HPV16 VLPs were identified with a size of 40-65 nm. This means that the size of the T-5 HPV 16 VLPs is closer to the size of the original HPV (50-60 nm) [29, 30].
  • FIGS. 9 and 10 show the results of dynamic light scattering (DLS) analysis.
  • Dynamic light scattering analysis measures the size of VLPs present in solution. In-solution size distribution of VLPs was analyzed using the DLS-700 (FIG. 9) and ELS-Z2 (FIG. 10) systems. As can be seen from the results of FIGS. 9 and 10, the size distribution of the two VLP particles was found to be different. These results show that the physical properties of T-5 HPV 16 VLPs are different from T-1 HPV16 VLPs.
  • FIG. 11 shows the reactivity of monoclonal antibodies against T-1 HPV 16 L1 VLPs and T-5 HPV16 L1 VLPs.
  • the known monoclonal antibodies H16.V5 and H16.E70 were used. Increased reactivity of VLPs against both antibodies is known to be closely associated with increased immunogenicity [26, 32-34].
  • the reactivity of T-5 HPV 16 VLPs with H16.V5 and H16.E70 antibodies was found to be significantly higher than that of T-1 HPV 16 VLPs with both antibodies.
  • T-1 HPV16 L1 VLP T-5 HPV16 L1 VLP.
  • each 1 ng of VLP was administered subcutaneously to mice with 200 ⁇ g of aluminum hydroxide. Immunization was performed four times every two weeks. Anti-HPV16 L1 IgG antibody titers were measured from serum 10 days after the third and fourth immunization. As a result of anti-HPV16 L1 IgG antibody titer, it was confirmed that T-5 HPV16 L1 VLP induced anti-HPV16 L1 IgG at a level 10 times higher than T-1 HPV16 L1 VLP.
  • Figure 13 shows the results of measuring the anti-HPV16 neutralizing antibody activity of the serum collected after the fourth immunity in the immunization carried out in Figure 12.
  • the neutralizing activity of mouse serum immunized with T-5 HPV16 L1 VLP was 78%, whereas the neutralization activity of mouse serum immunized with T-1 HPV16 L1 VLP was 33%.
  • FIG. 14 shows the conditions in which the cell lysate is not treated (non-treatment), a reducing agent treated condition ( ⁇ -ME), and a reducing and treating process followed by heating and cooling ( ⁇ -ME + heating and chilling). This is the result of comparing the purity of the purified HPV L1 protein. Samples subjected to each of the above conditions were eluted by primary heparin chromatography, and the same volume (5, 2.5, 1.2 ⁇ L) of the protein was taken from the eluted fractions, and loaded into gels, followed by SDS-PAGE and western blotting. Was performed. From the results of FIG. 14, it was confirmed that the purity of the HPV L1 protein under the conditions of heating and cooling after the reducing agent treatment (T-5, ⁇ -ME + heating and chilling) was much higher than other conditions.
  • FIG. 15 is a condition in which the cell lysate is not treated (non-treatment), a reducing agent treated condition ( ⁇ -ME), a reducing agent treatment and heating and cooling process (T-5, ⁇ -ME + heating) and chilling) to compare the purity of the purified HPV L1 protein.
  • Samples subjected to each of the above conditions were eluted by primary heparin chromatography, and the same amount of protein (1, 0.5, 0.25 ⁇ g) was taken from the eluted fractions and loaded on gels, followed by SDS-PAGE and western blotting. Was performed. From the results of FIG. 15, it was confirmed that the purity of HPV L1 protein under the conditions of heating and cooling after the reducing agent treatment (T-5, ⁇ -ME + heating and chilling) was much higher than other conditions.
  • FIG. 16 shows the conditions in which the cell lysate is not treated (non-treatment), the heating and cooling without the reducing agent (heating and chilling), the heating and cooling after the reducing agent (T- 5, ⁇ -ME + heating and chilling) to compare the purity of the purified HPV L1 protein.
  • Samples subjected to each of the above conditions were subjected to SDS-PAGE and western blotting by taking the same volume of protein (5, 2.5, 1.2 ⁇ L) from the fraction eluted in primary heparin chromatography and loading it on a gel. From the results of FIG. 16, it was confirmed that the recovery and purity of the HPV L1 protein under the heating and cooling processes (T-5, ⁇ -ME + heating and chilling) after the reducing agent treatment were significantly higher than other conditions.
  • FIG. 17 is a method for purifying by size-exclusion chromatography SEC, a method of purifying by ammonium sulfate precipitation, a method of heating / cooling after treatment with a reducing agent (T-5, ⁇ -ME + The process of the purification method by heating & chilling) is shown.
  • the step of heating / cooling after reducing agent treatment in T-5 purification method is size exclusion chromatography or ammonium sulfate Replaced by precipitation method.
  • the method by size exclusion chromatography (SEC) and ammonium sulfate precipitation is based on previously known methods of the prior art.
  • Figure 18 shows the difference between the ammonium sulphate precipitation method, the method of treating only the reducing agent ( ⁇ -ME), the purification method of heating / cooling after the reducing agent treatment (T-5, ⁇ -ME + heating & chilling).
  • the purity of the first chromatographic elution fraction for each purification condition was analyzed by SDS-PAGE and Western blotting. As a result, it was confirmed that the purity of L1 protein recovered from the method of heating / cooling after ⁇ -ME treatment was the best.
  • FIG. 19 shows the primary heparin chromatography results of the purification method by ammonium sulfate precipitation and the purification method (T-5, ⁇ -ME + heating & chilling) after treatment with reducing agent.
  • the L1 protein eluted in the first heparin chromatography showed high purity while the first heparin chromatography was performed after the ammonium sulfate precipitation. It was confirmed that contains a large number of impurity proteins.
  • FIG. 21 is a first step of size exclusion chromatography method (SEC), a method of ammonium sulfate precipitation, and a purification method of heating / cooling after reducing agent treatment (T-5, ⁇ -ME + heating & chilling).
  • SEC size exclusion chromatography method
  • T-5 ⁇ -ME + heating & chilling
  • the L1 protein elution fraction of the chromatography was analyzed by SDS-PAGE and Western blot. Samples of each condition were prepared for 1 st chromatography as shown in FIG. 17.
  • SEC size exclusion chromatography method
  • the cell lysate was subjected to ammonium sulfate precipitation and subjected to size exclusion chromatography (primary chromatography).
  • the cell debris was precipitated by ammonium sulfate, followed by sediment removal, followed by heparin chromatography (primary chromatography).
  • the cell lysate was subjected to heat and cooling after the reducing agent treatment and subjected to heparin chromatography (primary chromatography).
  • heparin chromatography primary chromatography
  • fractions of each condition were loaded in equal volumes (0.35, 0.17, 0.08 ⁇ L). As a result, the L1 protein purity of the method of heating / cooling after the reducing agent treatment was the best.
  • FIG. 22 shows the results of further purification of the primary chromatography L1 protein elution fraction of FIG. 21 by secondary chromatography.
  • the difference between the L1 protein elution fractions after the second chromatography was analyzed by SDS-PAGE and Western blot.
  • Each purification condition elution fraction was loaded in equal volumes (2, 1, 0.5 ⁇ L) for analysis. As a result, the purification method of heating / cooling after reducing agent treatment was the best in terms of L1 protein recovery.
  • FIG. 23 shows the results of analyzing the reactivity of the monoclonal antibody (H16.E70) of the final purified L1 proteins by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 24 shows the results of analyzing immunogenicity of the purification method by size exclusion chromatography (SEC), the purification method by ammonium sulfate precipitation, and the purification method by heating / cooling after reducing agent treatment.
  • SEC size exclusion chromatography
  • the amount of L1 protein prior to immunization of each HPV16 L1 VLPs with mice was matched identically as shown in FIG. 23A.
  • 1 ng of HPV16 L1 VLPs were mixed with 200 ⁇ g of aluminum hydroxide and then administered subcutaneously to mice.
  • Mouse immunization progressed four times every two weeks. After four immunizations, mouse sera were taken and neutralizing activity of each mouse group was measured by a known virus-based neutralizing antibody activity assay [26].
  • mice immunized with HPV16 L1 VLP obtained through purification by size exclusion chromatography was 16%
  • the neutralization activity of HPV16 L1 VLP immunized mice obtained by purification by ammonia sulfate precipitation was 28%
  • Neutralizing activity of mice immunized with HPV16 L1 VLP obtained through purification of heating / cooling after reducing agent treatment was 50%. Therefore, the neutralizing antibody inducing ability of HPV16 L1 VLP obtained through the purification method of heating / cooling after reducing agent treatment was the best.
  • Figure 25 shows the effect of the heating temperature in the process of heating and cooling after reducing agent treatment in the purification of HPV16 L1 protein by T-5 method.
  • Cell debris was heated for 15 minutes at each heating temperature, cooled, and centrifuged to remove precipitates.
  • Panel A shows the result of measuring protein concentration with heating temperature.
  • Panel B is the result of SDS-PAGE analysis of samples with the same volume of each heating temperature condition.
  • Panel C shows the results of analyzing the L1 protein by Western blot by loading the same volume of samples under each heating temperature condition.
  • Panel D is the result of analyzing the L1 protein by Western blot by loading the same amount of protein after protein quantification of the sample under each heating temperature condition.
  • panel D shows the purity of the L1 protein.
  • Figure 26 shows the effect of the heating temperature in the process of heating and cooling after reducing agent treatment in the purification of HPV18 L1 protein by T-5 method. Details of each panel are the same as in FIG. 25. These results confirmed that the HPV18 L1 protein remained in the cell lysate until 65 °C heating conditions, and confirmed that the purity of the L1 protein when heated to 45-55 °C.
  • Figure 27 shows the effect of the cooling process in the process of heating, cooling after reducing agent treatment in HPV16 L1 VLP purification through the T-5 method.
  • Panel A shows the difference in protein concentrations between the heated and chilled samples after heating to 45 ° C. and 50 ° C. and the unheated samples.
  • Panel B shows the results of the analysis of the amount of L1 protein on Western blot of the cooled and uncooled samples after heating to 45 ° C.
  • Panel C shows the results of analysis of the amount of L1 protein on Western blot of the cooled and uncooled samples after heating to 50 ° C.
  • Figure 28 shows the effect of the cooling process in the process of heating, cooling after reducing agent treatment in HPV16 L1 VLP purification by the T-5 method.
  • the difference between the SDS-PAGE and the Western blot of the cooled sample (HC) and the uncooled sample (H) after heating to 45 ° C is shown. SDS-PAGE and Western blots showed that after cooling, L1 protein was not reduced but impurity protein was decreased.
  • FIG. 29 shows the band intensities of impurity proteins protein 1, protein 2 and protein 3 shown on the SDS-PAGE of FIG. These results show that the impurity protein concentration is reduced through the cooling process.
  • FIG. 30 shows the results of purification through the T-5 method using HPV16 L1 VLP and HPV18 L1 VLP under 60 ° C. heating conditions.
  • Panel A shows the SDS-PAGE results of the first chromatography.
  • LS represents a sample loaded in the column
  • FT represents a sample flowing out without binding to the column.
  • Elution means the fraction eluted after binding to the column resin.
  • Arrows indicate the positions of HPV16 L1 and HPV18 L1.
  • Panel B is the result of SDS-PAGE analysis of the final purified HPV16 L1 and HPV18 L1 by primary and secondary chromatography.
  • Panel C shows the results of analysis of the final purified product by transmission electron microscopy. Electron microscopic analysis confirmed that the purified L1 protein constituted a virus analog.
  • FIG 31 shows the results of the primary chromatography purified HPV18 L1 by T-5 method. Each fraction was analyzed via SDS-PAGE. LS is the loading sample, FT is the flow-through, W is the column washed fraction, E means the fraction eluted by flowing the protein attached to the column with a buffer containing 1 M NaCl. HPV18 L1 shows that it can be purified with high purity during chromatography after heating / cooling process after reducing agent treatment.
  • FIG. 32 shows the results of SDS-PAGE analysis of the HPV18 L1 protein fraction eluted in the first chromatography of FIG. 31 after secondary cation exchange chromatography.
  • LS, FT, and W are as described above. Proteins attached to the column resin were eluted sequentially by flowing a buffer containing 0.6, 0.7, 0.8, 0.9, and 1 M NaCl. HPV18 L1 protein was found to elute in 0.9 M and 1 M NaCl fractions.
  • FIG. 33 shows the results of dynamic light scattering analysis of HPV18 L1 VLP purified by T-5 method. Dynamic light scattering of T-5 HPV18 L1 VLPs was analyzed using the ELS-Z2 system.
  • Panel 34 shows the results of the purification of HPV58 L1 by the T-5 purification method.
  • Panel A is the result of SDS-PAGE analysis of the L1 protein fraction eluted in the first chromatography.
  • Panel B is the result of SDS-PAGE analysis of secondary chromatography. Details of panel B are as described in FIG.
  • FIG 35 shows the SDS-PAGE and Western blot results of the HPV58 L1 protein finally recovered by the first secondary chromatography by T-5 purification method.
  • HPV58 L1 shows that it can be successfully purified by the process of heating and cooling after reducing agent treatment.
  • HPV16 L1 NG2 shows the DNA sequence encoding HPV16 L1 protein (HPV16 L1 NG2). Saccharomyces cerevisiae was transformed into an expression vector with HPV16 L1 NG2 inserted. The transformed cells were used for the expression and purification of HPV16 L1 protein.
  • HPV16 L1 nucleic acid sequence is known as GenBank KC792555.1.
  • HPV18 L1 NG3 shows the DNA sequence encoding HPV18 L1 protein (HPV18 L1 NG3). Saccharomyces cerevisiae was transformed with an expression vector with HPV18 L1 NG3 inserted. The transformed cells were used for the expression and purification of HPV18 L1 protein.
  • the HPV18 L1 nucleic acid sequence is known as GenBank KC792556.1.
  • HPV L1 protein expressing cell lines were plated in "SD-ura", a synthetic complete medium without uracil, and incubated for 4 or 5 days. Single colonies were inoculated in SD-ura liquid medium and incubated for 2 days.
  • SD-ura a synthetic complete medium without uracil
  • Single colonies were inoculated in SD-ura liquid medium and incubated for 2 days.
  • the cultured transformed cells contained 1% yeast extract (Duchefa, Netherlands), 2% peptone (Duchefa), 7% glucose (Duchefa) and 1% galactose (Duchefa). Cultured in YPDG medium.
  • the cultured cells were centrifuged to remove the culture solution, and washed with PBS (phosphate-buffered saline). The washed cells were recovered by centrifugation again and stored at -70 ° C until protein purification.
  • PBS phosphate-buffered saline
  • HPV VLPs using the T-1 method Purification of HPV VLPs using the T-1 method was carried out using heparin chromatography after recovering the protein in cell lysate into pellets by ammonium sulfate precipitation according to a known method [22,23, 24].
  • HiTrapTM Heparin HP (GE Healthcare, USA) resin was used for heparin chromatography. Ammonium sulfate and precipitate removed samples were dialyzed with PBST containing 0.325 M NaCl and passed through heparin resin equilibrated with PBST containing 0.325 M NaCl.
  • Heparin resin was washed by pouring 5 times the volume of the buffer (0.325 M NaCl containing PBST) and the protein bound with the resin was eluted by increasing the gradient of NaCl from 0.325 to 2 M. Fractions containing HPV L1 in the eluted fractions were collected and collected by SDS-PAGE analysis. Purified HPV L1 VLPs were dialyzed against PBS + 0.01 Tween 80 (PBST) with 0.33 M NaCl.
  • PBST 0.01 Tween 80
  • Cultured HPV L1 protein expressing cells were mixed in crushing buffer (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2). The cell mixture was again mixed with 0.5 mm glass beads (glass bead, Biospec Product, USA) and vortexed to disrupt the cells. Cell residue was removed by centrifugation at 12000xg for 15 minutes. Cell debris prior to column chromatography was prepared in two different ways. The first method adds ⁇ -ME to the cell lysate so that the final concentration of ⁇ -mercaptoethanol ( ⁇ -mercaptoethanol, ⁇ -ME, Sigma, USA) is 4-6% by weight, and then adjusts the pH to 7.0-7.3. That's how.
  • the cell lysate was dialyzed against the lysis buffer (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4-6 hours, and then the final ⁇ -ME concentration was 4-. It is a method of adding (beta) -ME so that it may become 6%. Subsequently, the cell lysate to which ⁇ -ME was added was placed in a constant temperature water bath at 25 to 65 ° C. for 30 to 50 minutes, and then cooled for 30 minutes to 3 hours or 16 hours at 4 ° C. on ice. The cooled cell lysate was centrifuged at 12000 ⁇ g for 15 minutes to remove the precipitate.
  • the lysis buffer 10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2
  • Heparin resin HiTrapTM Heparin HP, GE Healthcare, USA or POROS ® 50 HE, Applied Biosystems, USA
  • cation exchange resin POROS ® XS, Applied Biosystems, USA
  • Heparin resin or cation exchange resin was treated with cell disruption buffer (10 mM sodium phosphate dibasic, 0.15-0.48 M NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) containing 4-6% of ⁇ -ME prior to passage of the cell lysate. Equilibrated.
  • heparin / cation exchange resin was washed by washing the wash buffer (PBST with 0.35-0.48 M NaCl and 5% ⁇ -ME) at least 5 times the column volume.
  • HPV L1 protein bound to heparin resin was eluted by flowing PBST containing 1 M NaCl, 5% ⁇ -ME, or the final NaCl concentration was 0.6 M, 0.7 M, 0.8 M in PBST containing 5% ⁇ -ME. , 0.9 M, 1 M was prepared by flowing the buffer solution sequentially to elute.
  • the eluate containing L1 protein was concentrated using Amicon Ultra (Millipore, USA) and dialyzed for 20-24 hours in PBST containing 1 M NaCl, 0.2 M ammonium sulfate.
  • the dialysis solution after the first chromatography was further dialyzed against PBST containing 0.3-0.42 M NaCl, followed by secondary chromatography by passing through a heparin resin or a cation exchange resin.
  • Heparin resin and cation exchange resin used for secondary chromatography are the same as those used for primary chromatography.
  • Heparin / Cation exchange resin was equilibrated with PBST containing 0.42 M NaCl before loading the sample. Samples were loaded and washed by pouring PBST containing 0.42 M NaCl at least 5 times the column volume to the resin.
  • HPV L1 protein combined with heparin / cation exchange resin was eluted by increasing NaCl concentration from 0.42 to 2.0 M, or buffer solution prepared with NaCl concentration of 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M was eluted by sequential flow.
  • the elution pattern of the chromatography was measured at a wavelength of 280 nm and collected using an Autochro-2000 program (Young Lin Instrument Co., South Korea). After recovering the fraction eluted with L1 protein was concentrated using Amicon Ultra (Millipore, USA), and dialyzed in PBST containing 0.33 M NaCl.
  • Cells were prepared for disruption in the same manner as described above for untreated purification.
  • the prepared cell lysate was dialyzed in buffer (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4-6 hours.
  • the precipitate of the dialyzed sample was removed by centrifugation at 12000 ⁇ g for 10 minutes and then passed through a heparin resin equilibrated with the buffer. Thereafter, heparin resin was washed by pouring PBST containing 0.42 M NaCl at 5 times the volume of the resin, and the proteins bound to heparin resin after washing were eluted by flowing PBST containing 1 M NaCl.
  • the ⁇ -me treatment purification method is a purification method in which the heating & chilling process is omitted in the T-5 purification method.
  • HPV L1 expressing cells were prepared as described above for the ⁇ -me treatment purification method and 4-6 hours in buffer (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2). Dialysis. ⁇ -me was added to the final crushed liquor to a final 4-6%.
  • Heparin resin was equilibrated with a buffer containing 4-6% ⁇ -me (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) and then passed through the prepared cell lysate. Heparin resin passed through the cell lysate was washed by pouring PBST containing 4-6% ⁇ -me and 0.42 M NaCl at 5 times the volume of the resin. Proteins attached to heparin resin were eluted by flowing PBST containing 4-6% ⁇ -me and 1 M NaCl.
  • Heating & chilling treatment purification method is a purification method in which the ⁇ -me is omitted in the T-5 purification method.
  • HPV L1 protein purification cells were dialyzed for 4-6 hours in a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) after disruption. The dialyzed crushed liquid was treated at 37-45 ° C. for 30 minutes and cooled on ice for 30 minutes-3 hours.
  • the precipitate of the crushed liquid which had undergone the cooling and cooling process, was removed by centrifugation at 12000xg for 10 minutes, and then passed through a heparin resin equilibrated with a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2). . Thereafter, heparin resin was washed by pouring PBST containing 0.42 M NaCl at 5 times the volume of the resin. After washing, the proteins bound with the resin were eluted by flowing PBST containing 1 M NaCl.
  • the prepared sample was passed through Superose-6 resin (1.5 ⁇ 32 cm, GE Healthcare, USA) equilibrated with PBST containing 0.65 M NaCl (first chromatography) [36].
  • the elution pattern of size exclusion chromatography was measured at a wavelength of 280 nm and collected using an Autochro-2000 program (Young Lin Instrument Co., South Korea).
  • Purification using ammonium sulfate precipitation is a method of replacing the heating & chilling process with ammonium sulfate precipitation after ⁇ -me treatment in the T-5 purification method.
  • Ammonium sulphate precipitation and sediment removal before the chromatography step were carried out according to known methods (22, 24).
  • the cell lysate protein was saturated with 45% ammonium sulfate and precipitated, followed by removal of the precipitate of precipitated contaminants.
  • Samples prepared above prior to chromatography were dialyzed in buffer (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 hours.
  • Heparin resin was equilibrated with the same buffer as the above buffer and then passed through the dialyzed sample (first chromatography). After washing PBST containing 0.42 M NaCl to 5 times the volume of the resin was washed and then flowed PBST containing 1 M NaCl to elute the bound protein. The eluted fractions for secondary chromatography were dialyzed in a buffer containing 0.33 M NaCl. Subsequent processes were performed in the same manner as the secondary chromatography method of the size exclusion chromatography method.
  • Protein concentration was measured using protein serum kit (Bio-Rad Laboratories, USA) using bovine serum albumin (BSA; Pierce, USA) as a standard.
  • SDS-PAGE was performed according to Laemmli's method [35] and western blotting was performed using a known method [26]. Proteins developed on SDS-PAGE gels were visualized by silver staining. HPV using rabbit anti-HPV 16 L1 serum as primary antibody and goat HRP-conjugated anti-rabbit IgG polyclonal antibody (HRP-conjugated goat anti-rabbit IgG, Bethyl, USA) as secondary antibody L1 protein was detected [26]. The intensity of the band was measured by National Institute Health (NIH) open source software Image J and calculated according to known methods [25].
  • NASH National Institute Health
  • Purified HPV 16 L1 protein was adsorbed onto a carbon-coated grid and then stained with phosphotungstic acid or uranyl acetate. Transmission electron micrographs were taken using TEM200CX (JEOL, Japan) at a final magnification of 150,000 ⁇ [25].
  • Dynamic light scattering (DLS) analysis of HPV VLPs was done according to known methods [26]. Purified HPV VLPs were diluted to 0.13 mg / ml using PBST with 0.13 M NaCl and analyzed using DLS-700 system (Otsuka Electronics, Japan) or ELS-Z2 system (Otsuka Electronics, Japan).
  • Anti-HPV16 L1 monoclonal antibodies, H16.V5 and H16.E70, were prepared using PBS-T 20 containing 0.3% bovine serum albumin at 0.25 ⁇ g / ml, 0.12 ⁇ g / ml, 0.06 ⁇ g / ml, 0.03 ⁇ g / ml, After diluting to 0.015 ⁇ g / ml, the mixture was reacted with the coated HPV VLPs at 37 ° C. for 90 minutes. After washing three times with PBS-T, HRP-attached anti-mouse IgG antibody (Bethyl, USA) was diluted 1: 5000 in PBS-T 20 containing 0.3% bovine serum albumin and plated at 37 ° C. for 40 minutes. Reacted. The plate was washed five times with PBS-T 20 and then developed a color reaction. Color development was carried out using o -phenylenediamine (Sigma, USA) and absorbance was measured at 492 nm
  • mice Six-week old Balb / c mice were used for immunogenicity evaluation of HPV16 L1 (Orientbio. South Korea). In order to immunize HPV L1 protein with mice, the purity and concentration of L1 protein were confirmed according to the above-described protein quantification method and SDS-PAGE method. Mice were immunized subcutaneously and four times at two week intervals. For single immunization, 1 ng of L1 protein and 200 ⁇ g aluminum hydroxide (Sigma, USA) were administered subcutaneously. Protein amount of 1 ng is based on the quantitative result of T-5 HPV16 L1 VLPs. The HPV16 L1 protein purified by another method was quantified using T-5 HP16 L1 as a standard.
  • the T-5 purification process is shown in FIG.
  • Loading samples for first heparin chromatography were prepared by dividing the dialysis process and the non-dialysis process.
  • the cell lysis buffer was subjected to dialysis, and then a reducing agent ( ⁇ -ME) was added.
  • ⁇ -ME reducing agent
  • both samples were heated to 37-42 ° C. and left for 30-50 minutes, and then placed on ice and cooled to around 0 ° C.
  • Heparin chromatography was performed after removing impurities precipitated by heating / cooling.
  • 2 and 3 are the results of the analysis of the heparin chromatography of the sample after the dialysis process and the sample without the dialysis process by SDS-PAGE. In both cases, it can be seen that L1 protein was recovered with high purity.
  • the L1 protein elution fraction recovered through primary heparin chromatography was further purified via secondary heparin chromatography.
  • 4A shows the results of purification by secondary heparin chromatography.
  • L1 protein bound to heparin resin was eluted by increasing the gradient of NaCl (Fig. 4b).
  • Fig. 4b shows that L1 protein flowing out without binding to heparin resin.
  • FT (flow-through) fraction As a result of SDS-PAGE, L1 protein flowing out without binding to heparin resin was not observed in the FT (flow-through) fraction. It was confirmed that L1 protein bound to heparin resin was eluted by increasing the linear gradient of NaCl (fractions 11-17).
  • 4B is a result of detecting a substance eluted during heparin chromatography at a wavelength of 280 nm.
  • the purity of the L1 protein (T-5 HPV16 L1 VLP) recovered in secondary heparin chromatography was previously known [22. 24] was used to compare the purity of L1 protein (T-1 HPV16 L1 VLP).
  • the T-1 purification method is shown in FIG. 1, and the results of SDS-PAGE analysis of heparin chromatography of the T-1 purification method are shown in FIG. 5.
  • LS refers to a loading sample.
  • the L1 protein was eluted by increasing the NaCl concentration in a gradient gradient from 0.325 M to 2 M, and the L1 protein was eluted between fractions 11 to 15 times.
  • FIG. 6a is a result of comparing the purity of HPV16 L1 VLP purified by T-1 and T-5 method.
  • Two independent purification experiments were performed for purity analysis of VLPs and presented as results of Panel A and Panel B.
  • T-1 HPV16 L1 VLP and T-5 HPV16 L1 VLP were loaded with 500 ng, 250 ng, 125 ng, and 62 ng per well after protein quantification, and visualized by silver staining after SDS-PAGE development.
  • L1 band of 55 kDa was observed in high purity in both VLPs.
  • the intensity of the L1 band of the T-5 HPV 16 VLPs was stronger than that of the T-1 HPV 16 VLPs.
  • FIG. 6B shows the results of two experiments shown in FIG.
  • the L1 amount of the T-1 HPV 16 VLP was adjusted in the same manner as the L1 amount of the T-5 HPV16 VLP.
  • Figure 7 shows the result of SDS-PAGE after adjusting the L1 amount of the two VLPs the same.
  • T-5 HPV16 L1 VLP was found to be 40-65 nm, while the size of T-1 HPV16 L1 VLP was found to be 20-50 nm. Therefore, the type of HPV16 L1 VLP purified by T-5 method was distinguished from that of HPV16 L1 VLP purified by T-1 method. Naturally occurring HPV virions are known to be 50-60 nm in size [29,30]. Thus, these results indicate that the size of the T-5 HPV 16 VLP is closer to that of the original HPV.
  • Dynamic light scattering analysis is widely used to investigate the state of VLP in solution [31].
  • 9 shows representative results of analysis of purified T-1 HPV16 L1 VLPs and T-5 HPV 16 L1 VLPs using the DLS-700 system.
  • 10 shows representative results of each HPV16 L1 VLP analyzed using the ELS-Z2 system.
  • VLP size is expressed as mean ⁇ standard deviation.
  • T-1 HPV16 L1 VLPs were distributed between 29-438 nm
  • T-5 HPV16 L1 VLPs were distributed between 17-233 nm. Therefore, the fluid distribution was different according to the sizes of the two VLPs.
  • 10A shows the Intensity profile of the dynamic light scattering of each VLP.
  • FIG. 10B shows the polydispersity index (P.I.) of each VLP.
  • the T-1 HPV16 L1 VLP was found to exhibit a wider size distribution than the T-5 HPV16 L1 VLP.
  • the polydispersity (P.I.) of the T-5 HPV16 L1 VLPs was found to be lower than the T-1 HPV16 L1 VLPs (FIG. 10B).
  • T-5 HPV16 L1 VLPs were found to be present in a homogeneous form in solution compared to T-1 HPV16 L1 VLPs.
  • the reactivity of anti-HPV 16 L1 monoclonal antibodies against HPV 16 VLPs has been used as an important measure in evaluating the structural excellence and neutralizing antibody inducibility of HPV16 L1 VLPs [26, 32-34].
  • T-1 HPV16 L1 VLP and T-5 HPV16 L1 VLP the most commonly used antibodies H16.V5 and H16.E70 were used [26].
  • Increased responsiveness to both antibodies is closely associated with increased immunogenicity [26, 36].
  • the reactivity with respect to the two antibodies was confirmed that the T-5 HPV16 VLP was significantly superior to the T-1 HPV16 VLP.
  • the L1 protein purity of the T-1 HPV16 L1 VLPs was found to be lower than the T-5 HPV16 L1 VLPs (FIG. 6A, FIG. 6B).
  • the amount of T-1 HPV16 L1 VLPs was adjusted to the amount of T-5 HPV16 L1 VLPs and the amount of L1 protein was confirmed by SDS-PAGE (FIG. 7).
  • 1 ng of HPV16 L1 VLPs was administered with aluminum hydroxide for one time immunization. The amount of 1 ng of protein was based on the protein quantitative value (Bradford protein quantitative value) of T-5 HPV16 L1 VLPs.
  • HPV16 L1 VLPs purified by T-1 and T-5 purification methods were administered subcutaneously four times, two weeks apart.
  • the results of measuring the anti-HPV16 L1 IgG titer in serum after the third and fourth immunizations are shown in FIG. 12.
  • the T-5 HPV16 L1 VLP immune group showed a median of 450 while the T-1 HPV16 L1 VLP immune group had a median of zero.
  • the T-5 HPV16 L1 VLP immune group showed a median of 4050 while the T-1 HPV16 L1 VLP immune group had a median of 300. Therefore, T-5 HPV16 L1 VLPs were found to induce anti-HPV16 L1 IgG antibody titers 10 times higher than T-1 HPV16 L1 VLPs.
  • the first heparin chromatography was able to obtain a high purity L1 protein.
  • Two kinds of experiments were conducted to specifically investigate the effect of heating / cooling process on the purity of L1 protein after reducing agent treatment.
  • the first experiment no process is also that conditions (untreated purification process, non-treatment) to, the reducing agent, only processing conditions ( ⁇ -ME treatment purification method, ⁇ -ME treatment method), and a reducing agent treatment after heating / cooling process
  • the purity of L1 protein after heparin chromatography was compared between rough conditions (T-5 purification method. ⁇ -ME + heating & chilling).
  • Table 1 and Table 2 show the protein amount and removal rate of impurity proteins in the loading sample of each condition for heparin chromatography in the first and second experiments.
  • Table 1 the conditions without any treatment and only the reducing agent treated 24-25% of the protein was removed before heparin chromatography, while the heating / cooling after the reducing agent treated 66% of the protein. Removed.
  • Table 2 34-40% of protein was removed before heparin chromatography under untreated conditions and only under heating / cooling, while 70% of protein was removed under heating / cooling after reducing agent treatment. Confirmed.
  • FIG. 14 shows the fractions recovered after the first heparin chromatography in the same volume (5 ⁇ L, 2.5 ⁇ L, 1.2 ⁇ L) and confirmed by SDS-PAGE and Western blotting. Without any treatment, the recovered amount of L1 protein was found to drop significantly. In addition, it contains a large amount of impurity protein in the recovery solution was confirmed that the purity is also very low. Under the condition that only the reducing agent ⁇ -ME was treated, the amount of recovered L1 protein was increased, but it was confirmed that the purity was not good because it contained many impurity proteins in the recovery solution. On the other hand, in the case of heating / cooling after reducing agent ⁇ -ME treatment, L1 protein was identified as a major band in SDS-PAGE and Western blotting, and it was confirmed that L1 protein had excellent purity as well as recovery rate.
  • the protein in the recovery solution was quantified and the same amount of protein (1 ⁇ g, 0.5 ⁇ g, 0.25 ⁇ g) was loaded and confirmed by SDS-PAGE and Western blotting (FIG. 15). ). As can be seen from the results, the purity of the L1 protein was confirmed to be the highest under the heating / cooling process after the addition of ⁇ -ME.
  • Figure 16 shows the conditions of no treatment (non-treatment), heat-cooling only (heating & chilling), heat-cooled conditions after reducing agent treatment (T-5 purification method.
  • ⁇ -ME + heating & chilling The elution fraction of the primary heparin chromatography was analyzed by SDS-PAGE. The fraction recovered after the first heparin chromatography was loaded into the same volume (5 ⁇ L, 2.5 ⁇ L, 1.2 ⁇ L) and confirmed by SDS-PAGE and Western blotting. The recovered amount of L1 protein in was found to drop significantly. In addition, it contains a large amount of impurity protein in the recovery solution was confirmed that the purity is also very low. On the other hand, in case of heating / cooling process after reducing agent ⁇ -ME, L1 protein was identified as a major band in SDS-PAGE and Western blotting.
  • the purification process by ammonium sulfate precipitation is shown in FIG. 17.
  • the purification method by ammonium sulfate precipitation replaces the heating & chilling process with ammonium sulfate precipitation after ⁇ -ME treatment in the T-5 purification method, which is distinguished from the T-1 purification method of FIG. 1.
  • the method by ⁇ -ME treatment ( ⁇ -ME method) is the same as that shown in the above embodiment.
  • the eluted fractions of the protein bound to the column were loaded in equal volumes (10, 5, 2.5 ⁇ L) and analyzed by SDS-PAGE and Western blotting (FIG. 18, FIG. 19).
  • L1 protein showed excellent purity, while the method of ammonia sulfate precipitation and ⁇ -ME only showed low L1 protein purity (FIG. 18).
  • the primary chromatographic results of the ammonium sulphate precipitation method and the heat-cooled method after ⁇ -ME treatment showed that L1 protein in the loading sample (LS) was bound to the column in the heat-cooled condition after ⁇ -ME treatment.
  • the method using ammonium sulphate precipitation confirmed that some of the L1 protein of the loading sample (LS) did not bind to the column and flowed out flow-through (FT) (FIG. 19).
  • FIG. 17 Purification by size exclusion chromatography (SEC) is shown in FIG. 17.
  • the method of size exclusion chromatography was based on previously known methods [20]. The method by ammonium sulfate precipitation is the same as described above. Representative results by size exclusion chromatography are shown in FIG. 20.
  • HPV16 L1 protein was eluted with high purity in fractions 3-9 and this fraction was used to compare L1 protein purity by primary chromatography (FIG. 21).
  • FIG. 22 is a result of SDS-PAGE, Western blot analysis after purification of the first chromatography elution fraction of FIG. Second chromatographic elution fractions of each condition were loaded in equal volumes (2, 1, 0.5 ⁇ L). As a result of the second chromatographic elution fraction analysis, it was confirmed that L1 protein recovery was the highest under the condition of heat-cooling after ⁇ -ME treatment.
  • HPV16 L1 VLP purified under each condition, the reactivity with H16.E70, an HPV16 neutralizing monoclonal antibody, was compared by ELISA (FIG. 23B).
  • H16.E70 an HPV16 neutralizing monoclonal antibody
  • ELISA ELISA
  • concentration of HPV16 L1 VLP purified by pre-exclusion chromatography-based purification method (SEC) and ammonium sulfate precipitation method was purified by ⁇ -ME treatment and heat-cooling process. The concentration was adjusted to the same concentration of HPV16 L1 VLP (T-5 HPV16 L1 VLP).
  • virus-like particles (VLPs) recovered by ⁇ -ME treatment and heat-cooling were purified and recovered by conventionally known methods such as size exclusion chromatography and ammonium sulfate precipitation. The purity was higher than that of the particles, and the antigenic properties of the recovered VLPs were also excellent.
  • FIG. 23 Three final HPV16 L1 VLPs purified in FIG. 23 were immunized subcutaneously by 1 ng. Preimmune As shown in FIG. 23A, the concentration of each VLP was equally adjusted. 1 ng of VLP was administered in combination with 200 ⁇ g of aluminum hydroxide upon mouse immunization. Immunization progressed four times every two weeks. Neutralizing activity in serum of 4 immunized mice was determined to be 16% neutralizing activity in the group immunized with HPV16 L1 VLP purified by size exclusion chromatography-based method (SEC), and by ammonium sulfate precipitation. In the group immunized with purified HPV16 L1 VLP, neutralizing activity was 28%.
  • SEC size exclusion chromatography-based method
  • HPV16 L1 VLP purified by the method of heating and cooling after the reducing agent treatment showed 50% neutralizing activity.
  • HPV16 L1 VLP purified by T-5 method showed better immunogenicity than HPV16 L1 VLP purified by known method.
  • 25 and 26 show the effect of removing impurity proteins with heating temperature after reducing agent treatment in HPV16 L1 VLP and HPV18 L1 VLP purification.
  • the mixture was reacted for 15 minutes under conditions of starting temperature, 25, 30, 35, 40, 45, 50, 55, 60, and 65 ° C. These samples were then left on ice for 1 hour and then centrifuged at 12000 ⁇ g to remove precipitates. The results of measuring the protein concentration of each sample from which the precipitate was removed are shown in Panel A. Each sample was loaded at the same volume and analyzed on SDS-PAGE is shown in Panel B.
  • Impurity protein removal rate with heating was found to increase significantly above 35 °C ( Figure 25A, Figure 25B and Figure 26A, Figure 26B).
  • the HPV16 L1 protein remained in the cell disruption solution even at 65 ° C. and 60 ° C. heating conditions, and the HPV18 L1 protein remained in the cell disruption fluid even at 60 ° C. heating conditions.
  • the purity of HPV16 L1 protein was found to be excellent at 35-50 ° C (FIG. 25D) and the protein purity of HPV18 L1 was the best at 45-60 ° C (Fig. 25D). 26D).
  • the impurity protein was removed while the cell lysate was heated at 35-60 ° C. while the L1 protein remained stable.
  • the thermal stability of these L1 proteins under reducing agent treatment conditions may facilitate the separation of L1 proteins from impurity proteins through heating.
  • FIG. 27 shows a result of comparing heating (heating) only after reducing agent treatment and heating and chilling (heating & chilling) after heating.
  • panel A shows the results of measuring the protein concentration under the conditions of heating only, cooling conditions after heating.
  • Panels B and C show western blot results of heating, heating and chilling under 45 ° C and 50 ° C heating conditions. Samples of each condition were loaded in equal volumes (5, 2.5, 1.25 ⁇ L) for Western blot analysis. As can be seen in panel A, the total protein content was found to be reduced after heating and cooling rather than heating alone. On the other hand, L1 protein was found to show no reduction (Fig. 27B, Fig. 27C). These results show that the cooling process increases the purity of the L1 protein.
  • FIG. 28 shows the results of analysis of SDS-PAGE and Western blot for the condition of heating after reducing agent treatment only (H) and the condition of heating and cooling (HC).
  • H reducing agent treatment only
  • HC heating and cooling
  • FIG. 30 shows the results of SDS-PAGE analysis of the first chromatography.
  • LS and FT represent loading samples and flow-through. Elution means the fraction in which the L1 protein is eluted.
  • Panel B is the result of analysis by SDS-PAGE of the final product after secondary chromatography. Each L1 protein was purified to high purity.
  • Panel C shows the results of the final purified product analyzed by transmission electron microscopy. Purification of the L1 protein after heating to 60 ° C. resulted in the formation of virus like particles.
  • Figure 31 shows the SDS-PAGE results of the first chromatography on the purification of HPV18 L1 VLPs by T-5 method.
  • the reducing agent before primary heparin chromatography was added to the cell debris and then heated to 45 ° C. and cooled on ice.
  • 31 shows that impurity proteins were effectively removed by first heparin chromatography.
  • 32 shows the SDS-PAGE results of secondary cation exchange chromatography. In secondary chromatography, impurities were eluted in fractions containing 0.6 M and 0.7 M NaCl and L1 protein was eluted in fractions containing 0.9 M and 1 M NaCl. As a result, it was confirmed that the HPV18 L1 protein could be purified with high purity through the T-5 method.
  • 33 shows the results of dynamic light scattering analysis of the final purified T-5 HPV18 L1 VLPs.
  • 34A shows the result of SDS-PAGE analysis of the HPV58 L1 fraction separated by primary chromatography when HPV58 L1 VLPs were purified by T-5 method.
  • HPV58 L1 protein was identified as the major band.
  • 34B is the result of SDS-PAGE analysis of the separation of HPV58 L1 VLPs through secondary chromatography.
  • HPV58 L1 protein was eluted throughout the fractions containing 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M NaCl.
  • Figure 35 shows the results of analysis of the final purified HPV58 L1 VLPs by T-5 method by SDS-PAGE and Western blot. The T-5 method confirmed that the HPV58 L1 VLP was purified to high purity.
  • Kim HJ, Lim SJ, Kim JY, Kim SY, Kim H-J (2009) A method for removing contaminating protein during purification of human papillomavirus type 18 L1 protein from Saccharomyces cerevisiae. Arch Pharm Res 32: 1759-1766.
  • the present invention relates to a method for purifying Human Papillomavirus (HPV) L1 protein with high purity and high efficiency. According to the purification method of the present invention, not only can greatly improve the purification purity of the HPV L1 protein, but also the VLP of the purified HPV L1 protein has an excellent immunogenicity by forming a structure more similar to the original HPV virion.
  • HPV Human Papillomavirus
PCT/KR2013/006823 2012-07-30 2013-07-30 인유두종바이러스 바이러스 유사입자의 고효율 정제방법 WO2014021604A1 (ko)

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CN201380040425.9A CN104507956B (zh) 2012-07-30 2013-07-30 人乳头瘤病毒病毒样颗粒的高效提纯方法
EP13824788.7A EP2881401B1 (en) 2012-07-30 2013-07-30 High efficiency method for purifying human papillomavirus virus-like particles
CA2880420A CA2880420C (en) 2012-07-30 2013-07-30 High efficiency method for purifying human papillomavirus virus-like particles
AU2013297306A AU2013297306B2 (en) 2012-07-30 2013-07-30 High efficiency method for purifying human papillomavirus virus-like particles
MX2015001413A MX361186B (es) 2012-07-30 2013-07-30 Metodo de alta eficiencia para purificar particulas similares a virus del virus del papiloma humano.
US14/418,004 US9994618B2 (en) 2012-07-30 2013-07-30 High efficiency method for purifying human papillomavirus virus-like particles
JP2015525348A JP6014763B2 (ja) 2012-07-30 2013-07-30 ヒトパピローマウイルスのウイルス様粒子の高効率精製方法
BR112015002126-3A BR112015002126B1 (pt) 2012-07-30 2013-07-30 Método de purificação das proteínas l1 do papiloma vírus humano

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CN109750050A (zh) * 2017-11-07 2019-05-14 上海泽润生物科技有限公司 重组人乳头瘤病毒45亚型蛋白表达

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JP2017528137A (ja) * 2014-09-11 2017-09-28 カディラ ヘルスケア リミティド 卓越した免疫学的特性を有する卓越したヒトパピローマウイルス抗原及びそれを含むワクチン
CN109750050A (zh) * 2017-11-07 2019-05-14 上海泽润生物科技有限公司 重组人乳头瘤病毒45亚型蛋白表达
CN109750050B (zh) * 2017-11-07 2023-08-18 上海泽润生物科技有限公司 重组人乳头瘤病毒45亚型蛋白表达

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