Purification method of pilin Fim2 / 3 without pertussis component antigen residue

Abstract

The invention discloses a method for purifying pilin Fim < 2 > / 3 without pertussis component antigen residue. Specifically, pertussis thalli are subjected to heat extraction treatment and then subjected to salting-out, anion exchange and cation exchange to obtain a crude product, and the high-purity pertussis pilin Fim2 / 3 without residues such as pertussis toxin PT, pertussis filamentous hemagglutinin FHA and pertussis adhesin PRN is obtained after multi-mode anion composite chromatography. The pertussis mycoprotein Fim < 2 > / 3 prepared by the method has high biological safety due to high purity, for a component pertussis vaccine taking the pertussis mycoprotein Fim < 2 > / 3 as a new component, a detoxification process which must be adopted due to residues of pertussis toxin PT and the like is avoided, the production process of the component pertussis vaccine is reduced, and the production cost is reduced. The utilization rate of the pertussis pilin Fim2 / 3 and the production and preparation efficiency of the component pertussis vaccine are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a method for purifying fimbriae protein Fim2 / 3 without pertussis component antigen residues. Background Art

[0002] Pertussis (whooping cough) is an acute respiratory infectious disease caused by Bordetella pertussis, which mainly occurs in children and adolescents. After infection, it can cause strong symptoms such as spasmodic cough, ventricular asphyxia, cyanosis, vomiting, and severe cases can cause death. Pertussis was once one of the main diseases causing infant deaths worldwide. With the large-scale vaccination of the DTP combined vaccine prepared by combining whole cell pertussis vaccine (wPV) with diphtheria toxoid and tetanus toxoid, the incidence of pertussis disease has been significantly reduced. However, in recent years, a new epidemiological phenomenon of pertussis has been reported internationally - the re-emergence of pertussis. Some areas with high vaccination rates have continued to experience recurrence of pertussis, and the incidence of cough has shown an upward trend (Reference: Nieves DJ, Heininger U. Bordetella pertussis. Microbiol Spectr. 2016 Jun; 4(3). doi: 10.1128 / microbiolspec. EI10-0008-2015. PMID: 27337481.).

[0003] Bordetella is a genus of the genus Bordetella. It is Gram-negative, with darker staining at both ends, a capsule, and no flagella. Bordetella pertussis expresses more than 100 proteins, including a variety of antigens, such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbriae (Fim), etc. Among them, PT is the key virulence factor for Bordetella pertussis to cause disease, and it is also a strong immunogen. In the preparation of the vaccine, a detoxifying agent needs to be added to remove the toxicity of pertussis toxin and retain its immunogenicity. Side effects often occur after whole-cell pertussis vaccination. Sato and other researchers in Japan developed an acellular pertussis component vaccine (aPV) containing PT and FHA (reference: Sato Y, Kimura M, Fukumi H. Development of a pertussis component vaccine in Japan. Lancet. 1984 Jan 21; 1(8369): 122-6. doi: 10.1016 / s0140-6736(84)90061-8. PMID: 6140441.). Studies have shown that aPV can significantly reduce the occurrence of adverse reactions compared with wPV. Currently, aPV vaccines containing multiple antigens are on the market, such as a three-component pertussis vaccine containing PT, FHA, and PRN and a five-component pertussis vaccine containing PT, FHA, PRN, and Fim2 / 3. In clinical trials, the five-component aPV is more effective than the three-component vaccine, and the addition of Fim2 / 3 improves the protective effect of the aPV vaccine.Fim is the fimbriae protein of Bordetella pertussis, mainly including two serotypes, Fim2 and Fim3. It is located on the surface of the bacteria and is difficult to obtain (References: Olin P, Rasmussen F, Gustafsson L, Hallander HO, Heijbel H. Randomised controlled trial of two-component, three-component, and five-component acellular pertussis vaccines compared with whole-cell pertussis vaccine. Ad Hoc Group for the Study of Pertussis Vaccines. Lancet. 1997 Nov 29; 350(9091): 1569-77. doi: 10.1016 / s0140-6736(97)06508-2. Erratum in: Lancet 1998 Feb 7; 351(9100): 454. PMID: 9393335.).

[0004] As early as 1985, Zhang et al. disclosed a method for purifying Fim protein (reference: Zhang JM, Cowell JL, Steven AC, Carter PH, McGrath PP, Manclark CR. Purification and characterization of fimbriae isolated from Bordetella pertussis. Infect Immun. 1985 May; 48(2): 422-7. doi: 10.1128 / iai.48.2.422-427.1985. PMID: 2859248; PMCID: PMC261332.). The method first obtains bacterial cells by centrifugation, resuspends them in a phosphate buffer, performs homogenization and lysis treatment in an ice bath, and performs salt precipitation precipitation on the supernatant collected after centrifugation with 24% ammonium sulfate, and the precipitate is dissolved in a Tris solution with a pH of 9.5 and dialyzed; then, the supernatant is collected by centrifugation, and Fim in the supernatant is precipitated by dialyzing into a phosphate solution with a pH of 6.0, and the precipitate is collected by centrifugation at 27000g for 20 minutes, and the precipitate is dissolved in a Tris solution; 0.1M magnesium chloride is added for precipitation again, and the precipitate is collected and dissolved in a Tris solution with a pH of 9.5 after centrifugation at 27000g for 20 minutes.

[0005] Connaught Laboratory Co., Ltd. has applied for a patent for purifying pili (CN100387618C). The main steps of this method are: homogenize Bordetella pertussis and extract with 10mM potassium phosphate, 150mM sodium chloride and 4M urea; after centrifugation to obtain the supernatant, use a 100-300kDa filter to concentrate and keep it at 75-85℃ for 10-60 minutes; after centrifugation to obtain the supernatant, add 4.5% PEG8000 and continue stirring for 30 minutes; the precipitate collected by centrifugation is dissolved in phosphate buffer and purified by Septhadex 6B or PEI silica gel column. For the acquisition of Fim2 / 3 antigen, the precipitation method is mainly used for purification.

[0006] Changchun Baike Biotechnology Co., Ltd. has applied for a patent for removing endotoxin from pertussis fimbrial protein 2 / 3 (CN110358802A). The method extracts pertussis bacteria with urea-containing phosphate buffer, precipitates and extracts the supernatant with PEG-8000 and ammonium sulfate (in no particular order), and then purifies it with Q-Sepharose chromatography to obtain endotoxin-free Fim2 / 3.

[0007] Wuhan Institute of Biological Products Co., Ltd. has published a method for purifying pertussis pilus protein (CN112341528A). Pertussis bacteria are treated with a suitable method to obtain a crude pure PRN solution, and the flow-through liquid is collected after chromatography. The flow-through liquid sample is extracted by ammonium sulfate precipitation and Capto Adhere chromatography (in no particular order) to collect the flow-through liquid sample, and finally the pertussis pilus protein is prepared by a hydroxyapatite material chromatography column.

[0008] The above method has many steps, and the final pertussis pilus protein has a low purity, and other impurity proteins such as PT, PRN, FHA, etc. of pertussis cannot be completely removed. In particular, PT protein is a key toxic factor of Bordetella pertussis. Its residue in pilus protein will cause serious safety problems and cannot be directly used in the preparation of multi-component vaccines. Therefore, it is necessary to improve and enhance the production process of large-scale continuous preparation of high-purity pilus protein. Summary of the invention

[0009] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a method for extracting high-purity pertussis pilus protein Fim2 / 3 without residual impurities such as pertussis toxin PT. More specifically, the purpose of the present invention is to provide a stable and efficient method for extracting and purifying pertussis pilus protein Fim2 / 3 without impurities such as pertussis toxin PT, pertussis filamentous hemagglutinin FHA, and pertussis adhesin PRN, which can effectively avoid the detoxification process that has to be taken due to the residual pertussis toxin PT.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] In one aspect, the present invention provides a method for purifying pertussis fimbriae protein Fim2 / 3 by removing pertussis component antigens, comprising:

[0012] (1) suspending Bordetella pertussis using the first solution to obtain a bacterial suspension;

[0013] (2) subjecting the bacterial suspension in step (1) to heat extraction, centrifugation, and collection of the supernatant;

[0014] (3) adding sulfate to the supernatant in step (2) for salting out, and collecting the precipitate;

[0015] (4) using the second solution to redissolve the precipitate in step (3) to obtain a redissolved precipitate, adjusting the conductivity and pH of the redissolved precipitate, purifying the precipitate by anion exchange chromatography, and collecting the chromatography flow-through;

[0016] (5) adjusting the conductivity and pH of the chromatography flow-through in step (4), and then purifying by cation exchange chromatography to obtain crude pertussis fimbriae protein Fim2 / 3;

[0017] (6) adjusting the conductivity and pH of the crude pertussis fimbriae protein Fim2 / 3 in step (5), and then performing multimodal anion complex chromatography to obtain pertussis fimbriae protein Fim2 / 3 from which pertussis component antigens have been removed.

[0018] In some embodiments, the pertussis fimbriae protein Fim2 / 3 is a mixture of natural Bordetella pertussis cell membrane fimbriae proteins Fim2 and Fim3.

[0019] In some embodiments, in step (1), 20 to 40 liters of the first solution are used for suspending every 1 kg of Bordetella pertussis to obtain a bacterial suspension.

[0020] The invention adopts a heat extraction method to release the pertussis fimbriae protein Fim2 / 3 from the cell membrane of Bordetella pertussis.

[0021] The bacterial suspension is subjected to heat extraction at a pH of 8.0 to 9.0, preferably 8.5 to 9.0, and more preferably 8.8, so that the pertussis fimbriae protein Fim2 / 3 separated from the cell membrane can be effectively dissolved in the suspension. If the pH is less than 8.0, the solubility of the pertussis fimbriae protein Fim2 / 3 will gradually decrease, affecting the yield. If the pH is greater than 9.0, the pertussis fimbriae protein Fim2 / 3 may face the risk of protein denaturation.

[0022] During hot extraction, an appropriate amount of sodium chloride can effectively precipitate the highly hydrophobic macromolecules released during the hot extraction process, but too high a sodium chloride concentration may reduce the solubility of the pertussis fimbriae protein Fim2 / 3 and cause precipitation.

[0023] In the present invention, the buffer component of the first solution in step (1) is not limited, and other buffers having a buffering capacity between pH 8.0 and 9.0 can be appropriately selected.

[0024] As a preferred embodiment, the pH of the first solution in step (1) is 8.8.

[0025] The centrifugation method used in the present invention is not limited, and the centrifugation method and centrifugation conditions can be appropriately selected.

[0026] In some embodiments, the first solution is a Tris-NaCl solution (Tris-NaCl solution). As a more preferred embodiment, the concentration of sodium chloride in the first solution is 0.3-0.5 M. As a preferred embodiment, the concentration of sodium chloride in the first solution is 0.3 M, and the concentration of Tris-NaCl is 25 mM.

[0027] In some embodiments, the temperature of the heat extraction is 60° C. to 65° C. As a preferred embodiment, the temperature of the heat extraction is 60° C. When the pertussis fimbriae protein Fim2 / 3 is fully released, a low temperature is selected to help maintain the antigenic epitope of the protein.

[0028] In some embodiments, the heat-leaching time is 2 to 4 hours. As a preferred embodiment, the heat-leaching time is 2 hours.

[0029] In the present invention, ammonium sulfate is added to the supernatant in step (2) for salting out, which can effectively precipitate the pertussis fimbrial protein Fim2 / 3, while leaving some impurities soluble in the salt solution in the supernatant.

[0030] In some embodiments, the sulfate comprises ammonium sulfate or sodium sulfate, preferably ammonium sulfate.

[0031] In some embodiments, the concentration of ammonium sulfate is ≥15.3% (w / v), preferably, the concentration of ammonium sulfate is 15.3-19.2% (w / v), more preferably, the concentration of ammonium sulfate is 15.3% (w / v). In the present invention, the concentration of ammonium sulfate is the final concentration of ammonium sulfate added to the supernatant.

[0032] When the ammonium sulfate concentration reaches 15.3% (w / v), most of the pertussis fimbrial protein Fim2 / 3 is precipitated. Precipitation may also occur when the concentration is higher than 15.3, but more unnecessary impurities may be introduced.

[0033] The present invention collects and separates the precipitate produced by salting out by centrifugation, and redissolves the precipitate by dissolving it with a second solution. The second solution can be a common buffer used in the art for dissolving proteins, such as 10-100 mM phosphate buffer, Tris buffer, glycine-sodium hydroxide buffer, etc., and its pH can be 8.0-9.0.

[0034] In some embodiments, the second solution is Tris-NaCl buffer (pH 8.8), preferably 0.035M Tris-NaCl (pH 8.8) (0.035M NaCl, 25mM Tris, pH 8.8).

[0035] The present invention performs anion exchange chromatography on the precipitate redissolved in step (3), and collects the chromatography flow-through, thereby purifying the pertussis fimbriae protein Fim2 / 3.

[0036] In some embodiments, the column filler used in the anion exchange chromatography in step (4) may be a DEAE series, but is not limited to the Sepharose type, because the core function of the filler is the DEAE basic functional group rather than the Sepharose filler skeleton.

[0037] In some embodiments, the anion exchange chromatography in step (4) uses a Sepharose DEAE column.

[0038] In some embodiments, the Sepharose DEAE column is 5 to 15 cm high.

[0039] In some embodiments, the chromatographic conditions of the anion exchange chromatography in step (4) are: the pH of the loaded sample is 8.8, the conductivity is ≤6mS / cm, the chromatographic loading volume is ≤30CV, and the chromatographic flow rate is 60cm / h-70cm / h.

[0040] In some embodiments, the pH of the equilibration solution of the anion exchange chromatography in step (4) is 8.8. Preferably, the equilibration solution of the anion exchange chromatography is 0.035M Tris-NaCl buffer (0.035M NaCl, 25mM Tris (pH8.8) solution).

[0041] The present invention performs cation exchange chromatography on the chromatography flow-through obtained in step (4), collects the eluted protein, and thereby further purifies the pertussis fimbriae protein Fim2 / 3.

[0042] In some embodiments, the column filler used in the cation exchange chromatography in step (5) may be an SP series, but is not limited to the Capto type, because the core function of the filler is the SP basic functional group rather than the Capto filler skeleton. In addition, Sepharose-type SP fillers can also achieve similar purification effects.

[0043] In some embodiments, the cation exchange chromatography in step (5) uses a Capto SP ImpRes column.

[0044] In some embodiments, the Capto SP ImpRes column is 5 to 15 cm high.

[0045] In some embodiments, the chromatographic conditions of the cation exchange chromatography in step (5) are: the pH of the sample is 5.0, the conductivity is ≤5mS / cm, the chromatographic loading volume is ≤20cV, and the chromatographic flow rate is 60cm / h-70cm / h.

[0046] In some embodiments, the pH of the equilibrium solution of the cation exchange chromatography in step (5) is 4.5 to 5.0, preferably 5.0.

[0047] In some embodiments, the equilibrium solution for the cation exchange chromatography in step (5) is a 20 mM sodium acetate solution.

[0048] In some embodiments, the eluent of the cation exchange chromatography in step (5) is a sodium chloride sodium acetate buffer. In some preferred embodiments, the sodium chloride concentration in the eluent of the cation exchange chromatography in step (5) is 0.12 to 0.15 M. In some preferred embodiments, the eluent of the cation exchange chromatography in step (5) is a 20 mM sodium acetate-0.13 M sodium chloride solution (pH 5.0).

[0049] In some embodiments, in step (5), after the sample loading operation of the cation exchange chromatography is completed and before the elution step is performed, the chromatography column is washed with a 1% Triton solution (pH 5.0).

[0050] After collecting the cation exchange chromatography eluate, the present invention takes a sample for SDS-PAGE electrophoresis detection to confirm the type and purity of the purified product. The residual component antigen can also be sequenced by mass spectrometry to determine the residual status of other antigens in the eluate.

[0051] The present invention performs multi-mode anion composite chromatography on the crude pertussis fimbriae protein Fim2 / 3 obtained in step (5) to remove other pertussis antigen residues.

[0052] In some embodiments, the multimodal anion complex chromatography uses a Capto Adhere column.

[0053] In some embodiments, the Capto Adhere column is 5 to 15 cm high.

[0054] In some embodiments, the chromatography conditions of the multimodal anion composite chromatography in step (6) are: the pH of the loaded sample is 8.0, the conductivity is 15 mS / cm, the chromatography loading amount is ≤4 cV, and the chromatography flow rate is 80 cm / h-120 cm / h.

[0055] In some embodiments, the pH of the equilibration solution of the multimodal anion complex chromatography in step (6) is 7.5 to 8.5, preferably 8.0. As a preferred embodiment, the equilibration solution of the multimodal anion complex chromatography in step (6) is 0.11M sodium chloride, 20mM phosphate buffer (pH 8.0).

[0056] In some embodiments, the eluent of the multimodal anion complex chromatography in step (6) is a phosphate solution of sodium chloride. In some embodiments, the concentration of sodium chloride in the eluent of the multimodal anion complex chromatography in step (6) is 0.25 to 0.4 M. As a preferred embodiment, the concentration of sodium chloride in the eluent of the multimodal anion complex chromatography in step (6) is 0.4 M, the concentration of phosphate is 20 mM, and the pH of the eluent of the multimodal anion complex chromatography is 8.0.

[0057] In a specific embodiment of the present invention, the phosphate in the eluent of the multimodal anion complex chromatography in step (6) is sodium phosphate.

[0058] After collecting the multi-mode anion composite chromatography eluate, the present invention takes a sample for SDS-PAGE electrophoresis detection to confirm the identity and final purity of the purified product. At the same time, mass spectrometry analysis can be used to perform sequence analysis on the residual component antigens to determine the residual status of other antigens in the eluate.

[0059] In the above steps, protein collection and detection can be performed using a detector with a detection wavelength of 280 nm.

[0060] In some embodiments, the pH of the equilibrium solution of the anion exchange chromatography in step (4) is 8.5-9.0, and the conductivity is ≤6 mS / cm, the pH of the equilibrium solution of the cation exchange chromatography in step (5) is 4.5-5.0, and the conductivity is ≤5 mS / cm, and the pH of the equilibrium solution of the multimodal anion composite chromatography in step (6) is 7.5-8.5, and the conductivity is 12-18 mS / cm.

[0061] As a preferred embodiment, the pH of the equilibrium solution of the anion exchange chromatography in step (4) is 8.8 and the conductivity is 5 mS / cm.

[0062] As a preferred embodiment, the pH of the equilibrium solution of the cation exchange chromatography in step (5) is 5.0 and the conductivity is 2.0 mS / cm.

[0063] As a preferred embodiment, the pH of the equilibrium solution of the multimodal anion complex chromatography in step (6) is 8.0 and the conductivity is 15 mS / cm.

[0064] Another aspect of the present invention provides a pertussis fimbriae protein Fim2 / 3, wherein the pertussis fimbriae protein Fim2 / 3 is obtained by the purification method described above.

[0065] In some embodiments, the molecular weight of the pertussis fimbrial protein Fim2 / 3 is 22 kDa to 23 kDa.

[0066] In some embodiments, components such as pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), and pertussis renal adhesin (PRN) in the pertussis pilus protein Fim2 / 3 are detected by mass spectrometry without residue.

[0067] In some embodiments, the electrophoretic purity of the pertussis fimbrial protein Fim2 / 3 is above 96%. As a preferred embodiment, the electrophoretic purity of the pertussis fimbrial protein Fim2 / 3 is above 97%, more preferably above 98%.

[0068] Beneficial effects:

[0069] The present invention heat-extracts pertussis cells, and then obtains a crude product through salting out, anion exchange, and cation exchange, and then obtains a high-purity pertussis pilus protein Fim2 / 3 without pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertussis adhesin PRN, etc. after multi-mode anion composite chromatography. The pertussis pilus protein Fim2 / 3 prepared by the method of the present invention has high biological safety due to its high purity. For the component pertussis vaccine using it as a new component, the detoxification process treatment that must be taken due to the residues of pertussis toxin PT, etc. is avoided, the production process of the component pertussis vaccine is reduced, and the utilization rate of the pertussis pilus protein Fim2 / 3 and the production and preparation efficiency of the component pertussis vaccine are improved.

[0070] The present invention provides a method for purifying high-purity pertussis fimbriae protein. The pertussis fimbriae protein Fim2 / 3 obtained by the method has high purity, no PT, FHA, PRN and other Bordetella pertussis protein residues, has high safety, can be directly used for the preparation of a multi-component vaccine with pertussis fimbriae protein Fim2 / 3 as a newly introduced antigen, and improves the production and preparation efficiency and yield of pertussis fimbriae protein Fim2 / 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The SDS-PAGE electrophoretogram of the heat extraction and salting-out process in Example 1;

[0072] Figure 2 The SDS-PAGE electrophoretograms of the various chromatography processes in Example 1;

[0073] Figure 3 This is the SDS-PAGE electrophoresis diagram of salting out using different concentrations of sodium chloride in Comparative Example 1. DETAILED DESCRIPTION

[0074] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0075] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0076] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in the technical field.

[0077] Example 1 The method of the present invention

[0078] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria (CS pertussis strain provided by Beijing Institute of Biological Products), heat-extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 2050 mL of supernatant.

[0079] 2. Add 313.7 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of the reconstituted product.

[0080] 3. The above precipitate was redissolved, the conductivity was adjusted to 5 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 220 mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution, and then the sample was loaded, and 255 mL of the chromatography flow-through was harvested.

[0081] 4. The conductivity of DEAE chromatography flow-through was adjusted to 4.5 mS / cm with purified water, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 270 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 36 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was obtained.

[0082] 5. The conductivity of the Capto SP ImpRes chromatography eluate was adjusted to 15 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 8.0 using 1 M NaOH solution to obtain 42 mL of the Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with 0.11 M NaCl and 20 mM sodium phosphate (pH 8.0) solution and then loaded with the sample. 38 mL of the 0.4 M NaCl and 20 mM sodium phosphate (pH 8.0) eluate was harvested, i.e., the pure pertussis fimbriae protein Fim2 / 3, with an electrophoretic purity of 98.2%. The SDS-PAGE electrophoresis results of each stage are shown in Figure 1 and Figure 2. The liquid chromatography-mass spectrometry method developed by Long Zhen, Wei Chen and others was used to detect other antigenic components of pertussis, and no other component antigens remained in the final product (reference: Long Z, Wei C, Zhan Z, Ma X, Li X, Li Y, Yao J, Ji F, Li C, Huang T. Quantitative determination of bioactive proteins in diphtheriatetanus acellular pertussis (DTaP) vaccine by liquid chromatography tandem massspectrometry. J Pharm Biomed Anal. 2019 May 30; 169: 30-40. doi: 10.1016 / j.jpba.2019.02.029. Epub 2019 Feb 21. PMID: 30831450.).

[0083] Example 2

[0084] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 1970 mL of supernatant.

[0085] 2. Add 378.2 g of ammonium sulfate to the supernatant to make it 19.2% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of reconstituted material.

[0086] 3. The above precipitate was re-dissolved, the conductivity was adjusted to 5.5 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 190 mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution, and then the sample was loaded, and 215 mL of the chromatography flow-through was harvested.

[0087] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 4.8 mS / cm with purified water, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 230 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 40 mL of the 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was harvested.

[0088] 5. The conductivity of the Capto SP ImpRes chromatography eluate was adjusted to 15 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 8.0 using 1 M NaOH solution to obtain 47 mL of the Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with 0.11 M NaCl and 20 mM sodium phosphate (pH 8.0) solution and then loaded with the sample. 41 mL of the 0.4 M NaCl and 20 mM sodium phosphate (pH 8.0) eluate was harvested, i.e., the pure product of pertussis fimbriae protein Fim2 / 3. The electrophoretic purity of the final product was 97.3%, and no other component antigen residues were detected.

[0089] Example 3

[0090] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 1950 mL of supernatant.

[0091] 2. Add 298.4 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of reconstituted material.

[0092] 3. The above precipitate was redissolved, the conductivity was adjusted to 4.9 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 210 mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution, and then the sample was loaded, and 240 mL of the chromatography flow-through was harvested.

[0093] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 5.0 mS / cm with purified water, and the pH was adjusted to 4.5 with 30% acetic acid to obtain 260 mL of SP chromatography sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 0.02 M sodium acetate solution (pH 4.5) and then loaded with the sample, and 39 mL of the 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 4.5) eluate was harvested.

[0094] 5. The above Capto SP ImpRes chromatography eluate, 0.2M sodium phosphate buffer (pH8.0) adjusted the conductivity to 15mS / cm, 1M NaOH solution adjusted the pH to 8.0, and obtained 47mL of Capto Adhere sample. Use a Capto Adhere chromatography column with a diameter of 16mm, a height of 10cm, and a volume of 20mL for chromatography, with a flow rate of 80cm / h, and load the sample after balancing the chromatography column with 0.11M NaCl, 20mM sodium phosphate (pH8.0) solution, and harvest 37mL of 0.4M NaCl, 20mM sodium phosphate (pH8.0) eluate, i.e., the pure product of pertussis fimbriae protein Fim2 / 3. The electrophoretic purity of the final product was 96.6%, and no other component antigen residues were detected.

[0095] Example 4

[0096] 1. Use 80L of 0.3M NaCl, 25mM Tris (pH8.8) solution to suspend 4kg of pertussis bacteria, heat extract at 60℃ for 2h, and centrifuge at 4℃ and 10000g for 30min to collect about 78L of supernatant.

[0097] 2. Add 11.9 kg of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 4 L of reconstituted material.

[0098] 3. The above precipitate was re-dissolved and ultrafiltered using a 10KD ultrafiltration membrane and a solution of 0.035M NaCl, 25mM Tris (pH8.8) until the conductivity reached 6mS / cm and the pH reached 8.8, and 4000mL of DEAE chromatography sample was obtained. A DEAE chromatography column with a diameter of 140mm, a height of 10cm, and a volume of 1500mL was used for chromatography at a flow rate of 60cm / h. The column was equilibrated with a solution of 0.035M NaCl, 25mM Tris (pH8.8) and then loaded with 4000mL of sample, and 4293mL of chromatography flow-through was harvested.

[0099] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 5 mS / cm with water for injection, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 6000 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 100 mm, a height of 10 cm, and a volume of 800 mL was used for chromatography at a flow rate of 60 cm / h. The sample was loaded after balancing the chromatography column with a 20 mM sodium acetate (pH 5.0) solution, and then the chromatography column was rinsed with a 1% Triton solution (pH 5.0) for 1 to 2 cV to remove endotoxins, and then eluted with a 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0), and 1200 mL of the eluate was harvested.

[0100] 5. The conductivity of the Capto SP ImpRes chromatography eluate was adjusted to 15 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 8.0 using 1 M NaOH solution to obtain 1300 mL of the Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 100 mm, a height of 10 cm, and a volume of 800 mL was used for chromatography at a flow rate of 80 cm / h. The column was balanced with 0.11 M NaCl, 20 mM sodium phosphate (pH 8.0) solution and then loaded. 350 mL of the 0.4 M NaCl, 20 mM sodium phosphate (pH 8.0) eluate was harvested, i.e., the pure product of pertussis fimbriae protein Fim2 / 3. The electrophoretic purity of the final product was 97.9%, and no other component antigen residues were detected.

[0101] The purity of the pure products obtained in Examples 1-4 is shown in Table 1:

[0102] Table 1

[0103] Example Electrophoretic purity Example 1 98.2％ Example 2 97.7％ Example 3 96.6％ Example 4 97.9％

[0104] The mass spectrometry residual test results obtained in Examples 1-4 are shown in Table 2:

[0105] Table 2

[0106]

[0107]

[0108] Example 5

[0109] S1) Weigh 4 kg of pertussis bacteria, add 80 L of 0.3 M Tris-NaCl (pH 8.8) (25 mM Tris, 0.3 M NaCl, pH 8.8) to suspend, heat-extract at 60°C for 2 h, and collect the supernatant by centrifugation;

[0110] S2) slowly adding solid ammonium sulfate to the above supernatant to a final concentration of 15.3% (w / v) to start salting out, stirring the salting out suspension thoroughly and then standing for 4 hours, centrifuging at 16000g for 30 minutes, and dissolving the precipitate after centrifugation with 0.035M Tris-NaCl (pH8.8) (25mM Tris, 0.035M NaCl, pH8.8);

[0111] S3) The above-mentioned precipitate was ultrafiltered using a 10KD ultrafiltration membrane and a 0.035M Tris-NaCl buffer (pH 8.8) until the conductivity reached 5 mS / cm and the pH reached 8.8, and then the precipitate was loaded onto a Sepharose DEAE column for chromatography, with the equilibrium buffer being 0.035M Tris-NaCl (pH 8.8), the flow rate being 60 cm / h, the detection wavelength being 280 nm, and the chromatography flow-through being collected;

[0112] S4) adjusting the conductivity of the above chromatography flow-through to 4.5 mS / cm and the pH to 5.0 with 30% acetic acid and water for injection, loading the sample onto a Capto SP ImpRes column for chromatography at a flow rate of 60 cm / h, balancing the chromatography column with a 20 mM sodium acetate (pH 5.0) solution and then loading the sample, then washing the chromatography column with a 1% Triton solution (pH 5.0) for 1 to 2 cV to remove endotoxins, and eluting with a 20 M sodium acetate-0.13 M sodium chloride (pH 5.0) solution, and collecting the eluate;

[0113] S5) The above chromatography eluate was adjusted to have a conductivity of 15 mS / cm with 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 8.0 with 1 M NaOH solution, and then loaded onto a Capto Adhere column for chromatography at a flow rate of 80 cm / h. The chromatography column was equilibrated with 0.11 M NaCl, 20 mM sodium phosphate (pH 8.0) solution and then loaded, and eluted with 0.4 M NaCl, 20 mM sodium phosphate (pH 8.0) solution, and the eluate was collected.

[0114] The above method was used to purify three different batches of pertussis fimbrial protein Fim2 / 3 and detect them. The electrophoretic purity of the three batches of samples is shown in Table 3, and the residual conditions of PT, FHA, and PRN in the samples detected by mass spectrometry are shown in Table 4.

[0115] Table 3

[0116] batch number Electrophoretic purity Batch 1 100％ Batch 2 99％ Batch 3 100％

[0117] Table 4

[0118]

[0119] Comparative Example 1: Salting out using sodium chloride

[0120] 1. Use 2000 mL of 0.3 M Tris-NaCl (pH 8.8) to suspend 100 g of pertussis bacteria, heat-extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 2200 mL of supernatant.

[0121] 2. Take 1500mL of the supernatant and divide it into 3 equal parts. According to the ratio of solid sodium chloride / supernatant (w / v) of 10%, 15%, and 20%, 50g, 75g, and 100g of solid sodium chloride were added respectively. After fully dissolved, let it stand at 4℃ for 4h, centrifuge at 4℃ and 16000g for 30min, take the supernatant, and dissolve the precipitate with 20mL of 0.035M Tris-NaCl (pH8.8). After electrophoresis detection, Fim protein was still mainly in the supernatant of each gradient, and no enrichment effect was achieved. There were many impurities and the sodium chloride salting-out effect was poor ( Figure 3 ).

[0122] Comparative Example 2 without Capto Adhere chromatography

[0123] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 2050 mL of supernatant.

[0124] 2. Add 313.7 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolving, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with 0.035 M NaCl and 25 mM TrisM (pH 8.8) to obtain 100 mL of reconstituted material.

[0125] 3. The above precipitate was redissolved, the conductivity was adjusted to 5 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 220 mL of DEAE chromatography sample. A volume of 20 mL DEAE chromatography column was chromatographed at a flow rate of 60 cm / h, and the column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution before loading the sample, and 255 mL of chromatography flow-through was harvested.

[0126] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 4.5 mS / cm with purified water, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 270 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample. 36 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was harvested. The final product had an electrophoretic purity of 92.3%. In the mass spectrometry analysis, a large amount of pertussis toxin PT, pertussis adhesin PRN, and pertussis filamentous hemagglutinin FHA residues were present.

[0127] Comparative Example 3: Change of Adhere chromatography loading conditions

[0128] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 2020 mL of supernatant.

[0129] 2. Add 309.1 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolving, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of the reconstituted product.

[0130] 3. The above precipitate was re-dissolved, the conductivity was adjusted to 5mS / cm with purified water, and the pH was adjusted to 8.8 with 1M hydrochloric acid or sodium hydroxide to obtain 200mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16mm, a height of 10cm, and a volume of 20mL was used for chromatography at a flow rate of 60cm / h. The column was equilibrated with 0.035M NaCl, 25mM Tris (pH8.8) solution, and then the sample was loaded, and 245mL of the chromatography flow-through was harvested.

[0131] 4. The conductivity of DEAE chromatography flow-through was adjusted to 5.0 mS / cm with purified water, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 260 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 35 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was obtained.

[0132] 5. The conductivity of the Capto SP ImpRes chromatography eluent was adjusted to 10 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 7.2 with 1 M NaOH solution to obtain about 40 mL of Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with 0.11 M NaCl and 20 mM sodium phosphate (pH 8.0) solution before loading the sample. The Fim sample was eluted with 0.4 M NaCl and 20 mM sodium phosphate (pH 8.0) solution, i.e., the pure pertussis fimbriae protein Fim2 / 3. The final product had an electrophoretic purity of 95.4%, and pertussis toxin PT and pertussis adhesin PRN residues were detected by mass spectrometry. Comparative Example 4: Changes in Capto Adhere chromatography loading conditions

[0133] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 2020 mL of supernatant.

[0134] 2. Add 309.1 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolving, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of the reconstituted product.

[0135] 3. The above precipitate was re-dissolved, the conductivity was adjusted to 5mS / cm with purified water, and the pH was adjusted to 8.8 with 1M hydrochloric acid or sodium hydroxide to obtain 200mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16mm, a height of 10cm, and a volume of 20mL was used for chromatography at a flow rate of 60cm / h. The column was equilibrated with 0.035M NaCl, 25mM Tris (pH8.8) solution, and then the sample was loaded, and 245mL of the chromatography flow-through was harvested.

[0136] 4. The conductivity of DEAE chromatography flow-through was adjusted to 5.0 mS / cm with purified water, and the pH was adjusted to 5.0 with 30% acetic acid to obtain 260 mL of SP sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 35 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was obtained.

[0137] 5. The conductivity of the SP chromatography eluent was adjusted to 25 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 7.2 using 1 M NaOH solution to obtain about 40 mL of the sample on Capto Adhere. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with 0.11 M NaCl and 20 mM sodium phosphate (pH 8.0) solution and then loaded with the sample. The Fim sample was eluted with 0.4 M NaCl and 20 mM sodium phosphate (pH 8.0) solution, i.e., the pure product of pertussis fimbriae protein Fim2 / 3. The electrophoretic purity of the final product was 96.2%, and pertussis toxin PT residues were detected by mass spectrometry.

[0138] Comparative Example 5 Capto Adhere chromatography elution conditions changed

[0139] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 1950 mL of supernatant.

[0140] 2. Add 298.4 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of reconstituted material.

[0141] 3. The above precipitate was redissolved, the conductivity was adjusted to 5 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 210 mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution, and then the sample was loaded, and 240 mL of the chromatography flow-through was harvested.

[0142] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 5.0 mS / cm with purified water, and the pH was adjusted to 4.5 with 30% acetic acid to obtain 260 mL of SP chromatography sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 39 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was harvested.

[0143] 5. The above Capto SP ImpRes chromatography eluent was adjusted to 15 mS / cm in conductivity using 0.2 M sodium phosphate buffer (pH 8.0), and pH was adjusted to 8.0 using 1 M NaOH solution to obtain 47 mL of Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with 0.11 M NaCl and 20 mM sodium phosphate (pH 8.0) solution and then loaded with the sample. The Fim sample was eluted with 0.5 M NaCl and 20 mM sodium phosphate (pH 8.0) solution, i.e., the pure pertussis fimbriae protein Fim2 / 3. The final product had an electrophoretic purity of 95.1%, and pertussis toxin PT and pertussis adhesin PRN residues were detected in mass spectrometry analysis.

[0144] Comparative Example 6 Capto Adhere chromatography elution conditions changed

[0145] 1. Use 2000 mL of 0.3 M NaCl, 25 mM Tris (pH 8.8) solution to suspend 100 g of pertussis bacteria, heat extract at 60°C for 2 h, and centrifuge at 4°C and 10,000 g for 30 min to collect about 1950 mL of supernatant.

[0146] 2. Add 298.4 g of ammonium sulfate to the supernatant to make it 15.3% (w / v) for salting out. After fully dissolved, let it stand at 4°C for 4 h. Centrifuge at 4°C and 16000 g for 30 min. Discard the supernatant and collect the precipitate. Dissolve the precipitate with a solution of 0.035 M NaCl and 25 mM Tris (pH 8.8) to obtain 100 mL of reconstituted material.

[0147] 3. The above precipitate was redissolved, the conductivity was adjusted to 5 mS / cm with purified water, and the pH was adjusted to 8.8 with 1 M hydrochloric acid or sodium hydroxide to obtain 210 mL of DEAE chromatography sample. A DEAE chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with 0.035 M NaCl, 25 mM Tris (pH 8.8) solution, and then the sample was loaded, and 240 mL of the chromatography flow-through was harvested.

[0148] 4. The conductivity of the DEAE chromatography flow-through was adjusted to 5.0 mS / cm with purified water, and the pH was adjusted to 4.5 with 30% acetic acid to obtain 260 mL of SP chromatography sample. A Capto SP ImpRes chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 60 cm / h. The column was equilibrated with a 20 mM sodium acetate (pH 5.0) solution and then loaded with the sample, and 39 mL of 0.02 M sodium acetate-0.13 M sodium chloride solution (pH 5.0) eluate was harvested.

[0149] 5. The Capto SP ImpRes chromatography eluent was adjusted to a conductivity of 15 mS / cm using 0.2 M sodium phosphate buffer (pH 8.0), and the pH was adjusted to 8.0 using 1 M NaOH solution to obtain 47 mL of Capto Adhere sample. A Capto Adhere chromatography column with a diameter of 16 mm, a height of 10 cm, and a volume of 20 mL was used for chromatography at a flow rate of 80 cm / h. The column was equilibrated with a 0.11 M NaCl, 20 mM sodium phosphate (pH 8.0) solution and then loaded with the sample. The Fim sample was eluted with a 0.2 M NaCl, 20 mM sodium phosphate (pH 8.0) solution. Almost no sample was eluted.

[0150] The purity of the pure products obtained in Comparative Examples 2-6 is shown in Table 5 below:

[0151] Table 5

[0152] Comparative Example Electrophoretic purity Comparative Example 2 92.3％ Comparative Example 3 95.4％ Comparative Example 4 96.2％ Comparative Example 5 95.1％ Comparative Example 6 /

[0153] The residual impurity proteins in the pertussis pilus protein detected by mass spectrometry in Comparative Examples 2-6 are shown in Table 6:

[0154] Table 6

[0155]

[0156] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for purifying pertussis fimbriae protein Fim2 / 3 by removing pertussis component antigens, comprising: (1) suspending Bordetella pertussis using the first solution to obtain a bacterial suspension; (2) subjecting the bacterial suspension in step (1) to heat extraction, centrifugation, and collection of the supernatant; (3) adding sulfate to the supernatant in step (2) for salting out, and collecting the precipitate; (4) using the second solution to redissolve the precipitate in step (3) to obtain a redissolved precipitate, adjusting the conductivity and pH of the redissolved precipitate, purifying the precipitate by anion exchange chromatography, and collecting the chromatography flow-through; (5) adjusting the conductivity and pH of the chromatography flow-through in step (4), and then purifying by cation exchange chromatography to obtain crude pertussis fimbriae protein Fim2 / 3; (6) adjusting the conductivity and pH of the crude pertussis fimbriae protein Fim2 / 3 in step (5), and then performing multimodal anion complex chromatography to obtain pertussis fimbriae protein Fim2 / 3 from which pertussis component antigens have been removed.

2. The purification method according to claim 1, wherein The pH of the first solution in step (1) is 8.5 to 9.0, preferably pH 8.8; Preferably, the first solution is a tris(hydroxymethyl)aminomethane-sodium chloride solution, more preferably, the concentration of sodium chloride in the first solution is 0.3-0.5M, preferably, the concentration of sodium chloride in the first solution is 0.3M, and the concentration of tris(hydroxymethyl)aminomethane is 25mM.

3. The purification method according to claim 1, wherein The temperature of the hot extraction in step (2) is 60°C to 65°C, preferably 60°C; Preferably, the hot-leaching time is 2 to 4 hours, more preferably, the hot-leaching time is 2 hours.

4. The purification method according to claim 1, wherein The sulfate described in step (3) includes ammonium sulfate and sodium sulfate. Preferably, the sulfate is ammonium sulfate. Preferably, the concentration of ammonium sulfate is ≥15.3% (w / v). Preferably, the concentration of ammonium sulfate is 15.3-19.2% (w / v). More preferably, the concentration of ammonium sulfate is 15.3% (w / v).

5. The purification method according to claim 1, wherein The anion exchange chromatography described in step (4) uses a Sepharose DEAE column, Preferably, the Sepharose DEAE column is 5 to 15 cm high. Preferably, the chromatographic conditions of the anion exchange chromatography are: the pH of the loaded sample is 8.8 and the conductivity is ≤6 mS / cm.

6. The purification method according to claim 1, wherein The cation exchange chromatography described in step (5) uses a CaptoSPImpRes column. Preferably, the Capto SPImpRes column is 5 to 15 cm high. Preferably, the chromatographic conditions of the cation exchange chromatography are: the pH of the loaded sample is 5.0, and the conductivity is ≤5 mS / cm.

7. The purification method according to claim 1, wherein The multimodal anion complex chromatography in step (6) uses a Capto Adhere column. Preferably, the Capto Adhere column is 5 to 15 cm high. Preferably, the chromatography conditions of the multi-mode anion composite chromatography are: the pH of the loaded sample is 8.0 and the conductivity is 15 mS / cm.

8. The purification method according to any one of claims 1 to 7, wherein: The pH of the equilibrium solution of the anion exchange chromatography in step (4) is 8.5 to 9.0, the pH of the equilibrium solution of the cation exchange chromatography in step (5) is 4.5 to 5.0, and the pH of the equilibrium solution of the multimodal anion composite chromatography in step (6) is 7.5 to 8.5; Preferably, the pH of the equilibrium solution of the anion exchange chromatography in step (4) is 8.8; Preferably, the pH of the equilibrium solution of the cation exchange chromatography in step (5) is 5.0; Preferably, the pH of the equilibrium solution of the multimodal anion complex chromatography in step (6) is 8.0; Preferably, the eluent of the cation exchange chromatography in step (5) is a sodium chloride-sodium acetate buffer. More preferably, the concentration of sodium chloride in the eluent of the cation exchange chromatography in step (5) is 0.12 to 0.15 M. Preferably, the concentration of sodium acetate in the eluent of the cation exchange chromatography in step (5) is 20 mM, the concentration of sodium chloride is 0.13 M, and the pH of the eluent of the cation exchange chromatography is 5.

0. Preferably, the eluent of the multimodal anion complex chromatography in step (6) is a phosphate solution of sodium chloride, more preferably, the concentration of sodium chloride in the eluent of the multimodal anion complex chromatography in step (6) is 0.25-0.4 M, preferably, the eluent of the multimodal anion complex chromatography in step (6) is a sodium phosphate solution of sodium chloride, more preferably, the concentration of sodium chloride in the eluent of the multimodal anion complex chromatography in step (6) is 0.4 M, the concentration of sodium phosphate is 20 m M, and the pH of the eluent of the multimodal anion complex chromatography is 8.

0.

9. A pertussis fimbriae protein Fim2 / 3, wherein The pertussis fimbriae protein Fim2 / 3 is obtained by the purification method described in any one of claims 1-8.

10. The pertussis fimbriae protein Fim2 / 3 according to claim 9, wherein The molecular weight of the pertussis fimbrial protein Fim2 / 3 is 22 kDa to 23 kDa.

Images