CN108570098B - Separation and purification method of multiple antigen components of pertussis - Google Patents
Separation and purification method of multiple antigen components of pertussis Download PDFInfo
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Abstract
The invention relates to a method for separating and purifying a plurality of antigen components of pertussis, wherein the antigens comprise a Pertussis Toxin (PT) antigen, a Filamentous Hemagglutinin (FHA) antigen and a pertussis adhesin (PRN) antigen, and the method comprises the following steps: (1) performing fermentation culture on the pertussis strains, and respectively harvesting culture solution supernatant and thallus precipitate; (2) PT antigen is separated and purified from culture supernatant, and PRN antigen and FHA antigen are separated and purified from bacterial pellet.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a method for separating and purifying multiple pertussis antigen components.
Background
Pertussis is a virulent infectious disease seriously harming the health of infants, and vaccination is the most ideal prevention and treatment means. The acellular diphtheria-pertussis-tetanus vaccine (DTaP) is a second generation diphtheria-pertussis-tetanus combined vaccine, has been popularized in a large area due to the side effect of the acellular diphtheria-pertussis-tetanus vaccine (DTaP) which is obviously lower than that of a first generation full-thallus diphtheria-pertussis vaccine (DTwP), enters the national planned immunization, and has the annual market demand of more than 6000 million.
The DTaP vaccine can be divided into co-purified vaccine and component vaccine (DTacP) according to the different production methods: the former adopts laggard salting-out and ultracentrifugation processes to extract effective antigens of Pertussis Toxin (PT) and Filamentous Hemagglutinin (FHA), although the yield is high and the production cost is low, the antigen proportion is unstable and the purity is low, and the process is adopted by domestic enterprises at present; the latter is mainly produced by European and American enterprises, adopts a column chromatography process to respectively extract effective components such as PT, FHA and pertussis adhesin (PRN), and has the advantages of high antigen purity, stable proportion, high protective effect and lower side effect. The production process of pertussis effective antigen components of DTacP vaccines which are internationally marketed is mostly established in the last 90 th century, the used separation and purification methods comprise hydroxyapatite-haptoglobin column chromatography, gel filtration, blue gel affinity chromatography and fetuin affinity chromatography, although the process is stable, the used chromatography filler is mostly outdated, for example, affinity ligands used by a certain European company are derived from human globin or bovine fetuin, and the biological safety risk of the ligand sources exists.
Since the 21 st century, with the development of chromatographic technology, more safe and efficient new fillers are used for purifying pertussis antigens, and better effects are achieved. In most of the schemes, a method of tangential flow ultrafiltration concentration or ammonium sulfate precipitation is adopted to concentrate the supernatant of the bacterial liquid, then ultrafiltration is replaced by a low-pH low-salt buffer solution containing 2M urea, and PT and FHA are respectively harvested by changing elution conditions after enrichment through a cation exchange chromatography medium. The flow-through solution obtained by the cation exchange chromatography may be subjected to a multi-binding anion exchange chromatography, or the cell bodies of bordetella pertussis may be dissolved by urea or high-temperature treatment, and subjected to ammonium sulfate precipitation and multi-binding anion exchange chromatography to isolate PRN. Both PT and FHA are very hydrophobic and therefore require 2M urea and high salt solutions to be stable, whereas with cation exchange chromatography a low conductivity sample is required and therefore there is a large loss of PT and FHA during the ultrafiltration step.
Moreover, PT and FHA are positively charged and have certain hydrophobicity in a solution environment with a low pH, so PT in purified FHA is difficult to remove, and FHA needs to be detoxified, which destroys the immunogenicity of FHA.
Disclosure of Invention
The invention relates to a method for separating and purifying a plurality of pertussis effective antigens from a once-cultured Bordetella pertussis culture, wherein the plurality of effective antigens are a Pertussis Toxin (PT) antigen, a Filamentous Hemagglutinin (FHA) antigen and a pertussis adhesin (PRN) antigen;
the method comprises the following steps:
(1) performing fermentation culture on the pertussis strains, and respectively harvesting culture solution supernatant and thallus precipitate;
(2) PT antigen is separated and purified from culture supernatant, and PRN antigen and FHA antigen are separated and purified from bacterial pellet.
The method for separating and purifying the PRN antigen and the FHA antigen from the thallus sediment comprises the following steps:
adding a buffer solution containing 1M sodium chloride and 50mMPB with the pH value of 8.0 and the precipitation amount (wet weight) of 10-15 times of the volume of the bacterial precipitation, uniformly stirring, and leaching at the temperature of 2-8 ℃ for 1-5 hours (preferably 1.5 hours);
and after centrifugation, respectively collecting the leaching supernatant and the leaching precipitate, separating and purifying the FHA antigen from the leaching supernatant, and separating and purifying the PRN antigen from the leaching precipitate.
The method for separating and purifying the PT antigen comprises the following steps:
(1) concentrating and ultrafiltering the supernatant of the bacterial liquid, wherein the concentration multiple is 5-16 times, the ultrafiltration uses 10mM sodium citrate buffer solution containing 2M urea and 0.5M sodium chloride and the pH value is 5.9-6.1, and the washing and filtering multiple is more than or equal to 4;
(2) preparing PT antigen from the bacterial liquid supernatant ultrafiltrate by using a cation exchange chromatography;
the cation exchange chromatography method in the step (2) comprises the following steps:
1) crude pure preparation of PT antigen:
balancing the mixed-mode cation exchange chromatography column by using PBS buffer solution containing 2M urea with pH5.9-6.1, preferably PBS buffer solution containing 20mM PB and 1M sodium chloride, then loading, and balancing the chromatography column by using the same buffer solution;
eluting with 50mM Tris-HCl buffer solution containing 2M urea and 0.5M sodium chloride at the pH of 8.4-8.6, and collecting a crude pure PT antigen solution;
2) pure preparation of PT antigen:
balancing an ion exchange chromatographic column by using a 10mM sodium citrate buffer solution containing 2M urea at the pH value of 5.9-6.1, loading a crude pure PT antigen solution, balancing the chromatographic column by using the same buffer solution after loading until the pH value is 5.9-6.1, and the conductivity is less than or equal to 10 mS/cm;
eluting 8-12 column volumes with 10mM PB buffer solution containing 2M urea and 0.5% Triton X-100 and having pH of 7.3-7.5;
eluting with 10mM PB buffer solution containing 2M urea and 0.05-0.5M sodium chloride and having pH of 7.3-7.5, and collecting pure PT antigen solution.
The method for separating and purifying the FHA antigen comprises the following steps:
(1) salting out: adding 30% (w/v) ammonium sulfate into the leaching supernatant, uniformly stirring, and standing for 18-24 hours at the temperature of 2-8 ℃;
(2) secondary leaching, removing supernatant after salting out is finished, centrifuging the rest precipitate for 40-60 minutes under the condition of centrifugal force 14000-16000 g, collecting the precipitate, adding 10-15 times of the precipitate (wet weight) by volume of 10-15 volume of 10mM sodium citrate buffer solution containing 2M urea with pH value of 6.0 into the precipitate, uniformly stirring, leaching at 2-8 ℃ for 1-2 hours, centrifuging the leaching solution for 40-60 minutes under the condition of centrifugal force 14000-16000 g, collecting supernatant, sterilizing and filtering the supernatant to obtain FHA antigen secondary leaching supernatant;
(3) obtaining purified FHA antigen by chromatography;
the chromatography method in the step (3) comprises the following steps:
1) crude pure preparation of FHA:
using 10mM sodium citrate buffer solution and 2M urea (pH5.9-6.1) containing balanced ion exchange chromatography column, and carrying out secondary extraction on the supernatant of the FHA antigen;
eluting 8-10 column volumes with 10mM PB buffer solution containing 2M urea and 0.5% Triton X-100(pH 7.3-7.5);
eluting with 10mM PB buffer solution containing 2M urea and 0.2-1.0M sodium chloride (pH 7.3-7.5), and collecting crude pure FHA antigen solution;
2) the fine purification preparation of FHA:
equilibrating a Hydroxyapatite (HA) chromatographic column with 10mM PB buffer solution containing 2M urea (pH6.9-7.1); the pH value of the crude pure FHA antigen solution obtained in the step of loading is adjusted to 6.9-7.1, and the conductivity is less than or equal to 20 mS/cm; the optimal conductivity is less than or equal to 15mS/cm
Eluting 8-10 column volumes with 30mM PB buffer solution containing 2M urea and 0.5% Triton X-100(pH 6.9-7.1);
then eluting with 100 plus 200mM PB buffer solution containing 2M urea and 0.2-0.5M sodium chloride (pH6.9-7.1), and collecting the pure FHA antigen solution.
The PRN antigen separation and purification method comprises the following steps:
(1) extraction: adding 5 times volume (v/w) of injection water into the leaching precipitation for redissolving, adding 8M urea (w/v), stirring for 2 to 3 hours at the stirring speed of 20 to 30rpm, and then adding the injection water to dilute the solution until 2M urea is stirred for 2 to 3 hours for extraction;
(2) salting out, centrifuging after extraction, collecting supernatant, adding solid ammonium sulfate into the supernatant according to 30% of the volume of the supernatant, stirring at the speed of 20-30 rpm, and standing for 18-24 hours at the temperature of 2-8 ℃;
(3) secondary leaching: removing supernatant after salting out, centrifuging the precipitate, and collecting the precipitate; adding 10mM PB buffer (pH 6.9-7.1) with the volume 10-15 times of the precipitation (wet weight) into the precipitate, stirring at 20-30 rpm, and leaching for 1 hour; after the extraction is finished, centrifugally collecting the supernatant of the secondary extraction, and sterilizing and filtering to obtain the supernatant of the secondary extraction of the PRN antigen;
(4) chromatography to obtain purified PRN antigen;
the chromatography method in the step (4) comprises the following steps:
1) crude pure preparation of PRN antigen:
using 10mM PB buffer solution containing 1M sodium chloride (pH 6.9-7.1), equilibrating hydrophobic chromatography column (6-8 CV),
the supernatant is extracted twice with PRN antigen, and then eluted with 10mM sodium acetate buffer solution containing 0.05-0.2M (preferably 0.2M) sodium chloride (pH 4.4-4.6), and the solution of crude PRN antigen is collected.
2) Fine purification preparation of PRN antigen:
balancing an ion exchange chromatography column (5-8 CV) with 10mM sodium acetate buffer solution (pH4.9-5.1), adjusting the pH value of the sampled crude pure PRN antigen solution to 4.9-5.1, and adjusting the conductivity to be less than or equal to 5 mS/cm;
eluting with 10mM sodium acetate buffer solution containing 0.02-1M (preferably 0.1M) sodium chloride (pH 4.9-5.1), and collecting the purified PRN antigen solution.
The invention has the advantages that: (1) the whole process from the pretreatment of large-tank culture to the coarse purification is a high-salt buffer solution, which is beneficial to ensuring the stability of the PT antigen in the purification process and improving the recovery rate. (2) In the late stage of the culture of the pertussis bacteria, part of the bacteria are crushed to release protease, so that FHA contained in the supernatant of the bacteria liquid is easily hydrolyzed by the protease, and only FHA on the surface of the bacteria is extracted by the process, so that the integrity of FHA protein is better. (3) PT and other hybrid proteins in the thallus leaching supernatant are few, so that the purity of the obtained FHA is higher, the risk of PT toxicity in the final product is reduced, and the obtained FHA antigen does not need to be detoxified, so that the immunogenicity is better. (4) PRN protein is easy to be hydrolyzed into 65KDa protein, PRN antigen obtained from thallus has high purity and good integrity, and does not need to be detoxified, so that the immunogenicity is better.
Drawings
FIG. 1, crude pure electrophoresis of PT antigen.
FIG. 2, crude pure chromatogram of PT antigen.
FIG. 3, fine electrophoresis of PT antigen.
FIG. 4, PT antigen purification chromatogram.
FIG. 5, rough pure electrophoresis picture of FHA antigen.
FIG. 6, crude and pure chromatogram of FHA antigen.
FIG. 7 shows the electrophoresis pattern of FHA antigen.
FIG. 8, FHA antigen purification chromatogram.
FIG. 9 shows a crude pure electrophoretogram of PRN antigen.
FIG. 10, PRN antigen crude pure chromatogram.
FIG. 11 shows a PRN antigen-purified electrophoretogram.
FIG. 12, PRN antigen purification chromatogram.
Detailed Description
Example 1 fermentation culture of Bordetella pertussis
Test materials:
strain: pertussis phase I CS strain, CMCC 58003. Is from the institute of food and drug testing in China. Pertussis phase I CS strain, CMCC 58003. Is from the institute of food and drug testing in China.
Culture medium: acellular pertussis SSM full-synthetic culture medium
The main equipment is as follows: bioho full-automatic fermentation system (50L,500L,5000L) (Bioengineering AG)
The test method comprises the following steps:
opening batch strains of working seeds of pertussis bacteria, inoculating to a modified bag-Jiangshi culture medium containing 15% of sheep blood, culturing at 35 ℃ for 72 hours, transferring to an active carbon semi-comprehensive culture medium, and culturing at 37 ℃ for 48 hours; after 3 passages (charcoal semi-complex medium) and further incubation at 37 ℃ for 48 hours, the cells were scraped in 250ml of phosphate buffer (PBS buffer, 137mM NaCl, 2.7mM KCl, 10mM Na)2HPO4、2mM KH2PO4(ii) a pH7.2) to obtain a bacterial suspension. Then 150ml of the bacterial suspension was inoculated into a 50L fermentor, and the total cell-free pertussis SSM total integrated medium of the 50L fermentor was 35L (121 ℃, 30 min autoclaving), and cultured with aeration and agitation at 35-36 ℃.
Culturing to bacterial concentration of 1.0 × 1010～1.6×1010And/ml, and harvesting when the pH value is 7.6-8.0, and using the strain as an amplification tank culture strain. The seed solution is transferred to a 500L fermentation tank containing 350L acellular pertussis SSM complete comprehensive culture medium, and is subjected to aeration stirring culture at 35-36 ℃. Culturing to bacterial concentration of 1.0 × 1010～1.6×1010And/ml, and harvesting when the pH value is 7.6-8.0, and using the strain as a large-tank culture strain. Transferring the seed solution to a 5000L fermentation tank containing 3500L acellular pertussis SSM total comprehensive culture medium, and culturing at 35-36 deg.C under ventilation and stirring. After inoculation of the strain, the initial culture concentration in the large tank is 0.02X 1010～0.1×1010And/ml. Culturing in large tank until the bacterial concentration reaches 1.6 × 1010～2.4×1010And/ml, the hemagglutination titer is not lower than 1:128, the culture is stopped when the pH value is 7.8-8.2, the tank is stopped for sampling, pure bacteria inspection is carried out, and the person with the mixed bacteria grows is discarded.
And (3) test results: the concentration of the bacteria gradually increased with the increase of the culture time, and at the end of the culture (at 38 hours), the concentration of the bacteria was 2.2X 1010Per ml, hemagglutination titer 1: 512.
example 2 centrifugation of fermentation broth in Large tank
Experimental materials and samples: bai Japanese cough liquid 3500L
The main equipment is as follows: disk centrifuge, CLARA 250 of Afalaval
The test method comprises the following steps:
centrifuging 500 liters of pertussis fermentation harvest bacterial liquid by using a disc centrifuge, wherein the centrifugal feeding flow is 1000-1500L/h, the slag discharge time is 5-10min, reducing the feeding flow and shortening the slag discharge time to ensure that the turbidity of the supernatant of the centrifuged bacterial liquid is less than or equal to 30NTU, and collecting bacterial precipitates.
And (3) test results:
475 liters of supernatant of the bacterial liquid are harvested, and the turbidity value is 11.4 NTU.
Example 3 isolation and purification of PT antigen from fermentation broth supernatant
1. Ultrafiltration treatment of fermentation broth
Test materials and samples:
and (3) ultrafiltration membrane packing: pellicon2 Biomax 30, Milipore
Tangential flow treatment system: CUF200SA Membrane-pack Ultrafiltration System, Milipore
Sample preparation: pertussis fermentation is carried out to obtain bacterial liquid, and the supernatant liquid after the thalli are centrifugally separated has the sample amount of 475 liters.
The test method comprises the following steps:
and (3) performing ultrafiltration concentration on the supernatant obtained after the thalli separation by using a polyether sulfone membrane with molecular weight cut-off of 10KD/30KD, and keeping the pressure of the feeding end in the concentration process at 15psi-25 psi. And when the volume is concentrated to about 50 liters, washing and filtering by using 3-5 times of volume of displacement liquid (10mM sodium citrate +2M urea +0.5M NaCl, pH 5.9-6.1) until the conductivity of the harvest liquid is more than or equal to 30mS/cm, rinsing the system by using about 10 liters of displacement liquid, and finally harvesting 60L of feed liquid.
2. Chromatographic purification of Pertussis Toxin (PT) antigen
Test materials and samples
A chromatographic column: BPG-140/500 GE
A purification system: AKTA pilot
Hydrophobic filler:
HCX Millipore
ion exchange packing: capto SP GE
Sample preparation: example 3 60L of the harvest after the previous step of concentration by ultrafiltration
2.1, crude pure preparation of PT antigen:
(1) mixing 3L
HCX medium was loaded into a BPG-140/500 chromatography column and equilibrated to 4-5 column volumes with pH6.0, 2M urea +20mMPB +1M sodium chloride.
(2) 60L of the supernatant obtained from the ultrafiltration was taken, and the liquid was passed through a column at a flow rate of 120-.
(3) After the sample loading is finished, the chromatographic column is balanced by using a buffer solution of 2M urea, 20mMPB and 1M sodium chloride with the pH value of 6.0 until the absorption value at A280 is stable to be less than or equal to 10mAU on AKTA.
(4) Eluting with 2M urea of pH 8.5, 50mM Tris-HCl and 0.5M sodium chloride at a flow rate of 120-.
(5) Adding a buffer solution of 2M urea and 10mM sodium citrate with pH of 6.0 into the collected crude pure PT antigen solution for dilution until the conductivity is less than or equal to 10mS/cm and the pH is between 5.9 and 6.1 for later use.
The experimental results are as follows: FIG. 1 is a PT crude pure electrophoretogram, and FIG. 2 is a PT crude pure chromatogram
2.2, preparing PT antigen in a pure way:
(6) 3L of Capto SP medium was loaded onto a BPG-140/500 column and equilibrated to 4-5 column volumes with pH 6.02M urea plus 10mM sodium citrate buffer. The diluted sample was passed through the column at a flow rate of 120-.
(7) After the loading is finished, the pH value is 6.0, and the buffer solution of 2M urea and 10mM sodium citrate is used for rebalancing until the ultraviolet monitoring protein A280 is stable to be less than or equal to 10 mAU.
(8) 8-10 column volumes were eluted with 2M Urea +10mM PB + 0.5% triton X-100 buffer pH 7.4.
(9) The column was equilibrated with 2M urea +10M MPB buffer pH7.4 until AKTA showed a plateau at A280 to ≤ 10 mAU.
Using the following buffers 1) to 3):
buffer 1)10mM PB +2M Urea +0.05M sodium chloride pH 7.4;
buffer 2)10mM PB +2M Urea +0.1M sodium chloride pH 7.4;
buffer 3)10mM PB +2M Urea +0.4M sodium chloride pH7.4,
the three buffers were eluted sequentially at a flow rate of 120-. Wherein the elution peak collected from buffer solution (buffer solution 2)) of 10mM PB +2M urea +0.1M sodium chloride of pH7.4 is pure PT antigen solution, and is stored at 2-8 deg.C
The experimental results are as follows: FIG. 3 is a PT fine purification electrophoretogram, and FIG. 4 is a PT fine purification chromatogram
Example 4 extraction of fermented cell pellets, collection of supernatant and extraction of pellets
Test materials and samples:
a desk centrifuge: j-26XP, Beckman
Sample preparation: the pertussis-harvested bacterial liquid obtained in example 2 was centrifuged to separate bacterial cells, and the sample amount was 6.0 kg.
The test method comprises the following steps:
adding 6kg of pertussis bacterial sediment into 50mM PB buffer solution containing 1M sodium chloride and having a volume 10-15 times of the sediment (wet weight) and a pH of 8.0, leaching for 1-2 hours, and centrifuging (15182g, 40-60 minutes)
Collecting the leaching supernatant for separation and purification of FHA, and collecting the leaching precipitate for separation and purification of PRN.
Example 5 isolation and purification of pertussis Filamentous Hemagglutinin (FHA) antigen
1. Salting-out treatment of the leach supernatant
(1) Ammonium sulfate was added to the extract supernatant obtained in the examples so that the ammonium sulfate concentration became 30% by weight/volume, and the mixture was allowed to stand for 18 to 24 hours, centrifuged (15182g, 40 to 60 minutes), and the precipitate was collected.
(2) Adding 10-15 times (wet weight) of the precipitate volume of 10mM sodium citrate buffer solution containing 2M urea and having pH of 6.0 into the precipitate, uniformly stirring, leaching at 2-8 ℃ for 1-2 hours, centrifuging the leaching solution for 40-60 minutes under the condition of a centrifugal force of 15182g, collecting the supernatant, sterilizing and filtering the supernatant to obtain the supernatant of the secondary extraction of the FHA antigen.
2. Chromatographic purification of pertussis Filamentous Hemagglutinin (FHA) antigen
Test materials and samples
A chromatographic column: BPG-140/500 GE XK-50/30 GE
A purification system: AKTA pilot
Ion exchange packing: capto SP GE
Ion exchange packing: CHT Type 1 Bio-Rad
Sample preparation: 30L of supernatant fluid of pertussis filamentous hemagglutinin FHA twice leaching
2.1 Rough purification preparation of FHA
(1) 3L of Capto SP medium was loaded onto a BPG-140/500 column and equilibrated to 4-5 column volumes with pH 6.02M urea plus 10mM sodium citrate buffer.
(2) The pertussis filamentous hemagglutinin secondary leaching supernatant passes through a chromatographic column at the flow rate of 120-200ml/min, and the absorption value of the protein A280 is monitored by ultraviolet.
(3) After the sample loading is finished, the buffer solution of pH6.02M urea and 10mM sodium citrate is used for rebalancing until the ultraviolet monitoring protein A280 is stable to be less than or equal to 10 mAU.
(4) 6-8 column volumes were eluted with pH 7.42M Urea +10mM PB + 0.5% triton X-100 buffer.
(5) The column was equilibrated with pH 7.42M urea +10m MPB buffer until AKTA showed a plateau at A280 to < 10 mAU.
(6) Respectively using:
pH7.410mMPB +2M urea +0.3M sodium chloride,
pH7.410mMPB +2M urea +0.4M sodium chloride,
pH7.410mMPB +2M Urea +1M sodium chloride
The three buffers were eluted sequentially at a flow rate of 120-. Wherein: the elution peak collected from the buffer solution of 10mM PB +2M urea +0.4M NaCl at pH7.4 was the crude FHA antigen solution.
(7) The crude FHA antigen solution is stored at 2-8 ℃.
The experimental results are as follows: FIG. 5 is a FHA crude pure electrophoretogram, and FIG. 6 is a FHA crude pure chromatogram
2.2 Fine purification preparation of FHA
(8) And adding a buffer solution of pH7.010mM PB +2M urea into the collected crude pure FHA antigen solution for dilution until the conductivity is less than or equal to 15mS/cm and the pH value is 6.9-7.1 for later use.
(9) 0.4L of CHT Type 1 medium was loaded into an XK-50/30 chromatography column and 4-5 column volumes were equilibrated with buffer pH 7.010mM PB +2M urea.
(10) The diluted sample is passed through a chromatographic column at a flow rate of 40-60ml/min and the absorbance at protein A280 is monitored by UV. After the loading is finished, the buffer solution of pH7.010mM PB +2M urea is used for rebalancing until the ultraviolet monitoring protein A280 is stable to be less than or equal to 10 mAU.
(11) 6-8 column volumes were eluted with pH 7.02M urea +30mM PB + 0.5% triton X-100 buffer.
(12) The column was equilibrated with 2M urea +30M MPB buffer pH7.0 until AKTA showed a plateau at A280 to < 10 mAU.
(13) Respectively using:
pH7.0100 mM PB +2M urea +0.3M sodium chloride,
pH7.0200mM PB +2M Urea +0.4M sodium chloride
The buffer solution of (2L per eluent) was continuously eluted at a flow rate of 40-60ml/min, and the eluates were collected separately. Wherein the elution peak collected by the buffer solution of 200M MPB, 2M urea and 0.4M sodium chloride with the pH value of 7.0 is the pure FHA antigen solution.
(14) The pure FHA antigen solution is stored at the temperature of 2-8 ℃.
The experimental results are as follows: FIG. 7 is a FHA crude electrophoretogram, and FIG. 8 is a FHA crude chromatogram
Example 6 isolation and purification of Bordetella pertussis adhesin (PRN) antigen
1. Extraction salting-out of leaching precipitates
(1) The leached precipitate harvested in example 4 was dissolved in water for injection containing 8M urea in a ratio of 1:5(W/V) and the mixture was stirred at 2-8 ℃ for at least 2 hours or left to stand overnight.
(2) After dilution to 4 volumes, the mixture was stirred for at least 2 hours or left overnight, centrifuged (12000g, 1h) to collect the supernatant, and the precipitate was discarded.
(3) Ammonium sulfate was added to the supernatant to give a concentration of 20% w/v to 30% w/v, and the mixture was allowed to stand for 1 to 24 hours, centrifuged (12000g, 1 hour), and the precipitate was collected.
(4) The precipitate was dissolved in 10mM sodium phosphate buffer solution (pH 6.9-7.1), and the supernatant was collected by centrifugation (12000g, 1 hour) and clarified with a 0.22 μm filter to obtain a crude pertussis-adhesin extract.
2. Chromatographic purification of pertussis adhesin (PRN) antigen
Test materials and samples
A chromatographic column: BPG-140/500 GE XK-50/30 GE
A purification system: AKTA pilot
Ion exchange packing: capto SP GE
Ion exchange packing: capto adhere GE
Sample preparation: 30L of pertussis adhesin crude extract
2.1 crude purification preparation of PRN antigen
(1) The 3L Capto adhere medium was loaded into BPG-140/500 chromatography column, and 6-8 column volumes were equilibrated with pH7.010mM PB +1M sodium chloride buffer.
(2) The pertussis adhesin crude extract passes through a chromatographic column at the flow rate of 120-.
(3) After the loading is finished, the buffer solution of pH7.010mM PB +1M sodium chloride is used for rebalancing until the value of the ultraviolet monitoring protein A280 is less than or equal to 25 mAU.
(4) With pH4.510mM CH3And (4) leaching the COONa +1M sodium chloride buffer solution until the ultraviolet monitoring protein A280 is stable to be less than or equal to 10 mAU.
(5) Are used separately
pH4.5、10mMCH3COONa +0.2M sodium chloride,
pH4.5、10mMCH3COONa +0.1M sodium chloride,
pH4.5、10mM CH3COONa
The buffer solution of (2) was continuously eluted at a flow rate of 120-. Wherein the pH is 4.5 and the concentration is 10mM CH3The elution peak collected in COONa +0.2M sodium chloride buffer was the crude purified PRN antigen solution.
(6) The crude purified PRN antigen solution was stored at 2-8 ℃.
The experimental results are as follows: FIG. 9 shows a PRN crude pure electrophoretogram, and FIG. 10 shows a PRN crude pure chromatogram
2.2 Fine purification preparation of PRN antigen
(7) To the collected solution of crude purified PRN antigen, pH 5.010mM CH was added3And (4) diluting the COONa buffer solution until the conductivity is less than or equal to 5mS/cm and the pH value is 4.9-5.1 for later use.
(8) 0.4L of Capto adhere media was loaded onto an XK-50/30 column using pH5.010mM CH3COONa buffer equilibrates for 3-4 column volumes.
(9) The diluted sample is passed through a chromatographic column at a flow rate of 40-60ml/min and the absorbance at protein A280 is monitored by UV.
(10) After the loading is finished, pH5.010mM CH is used3And (4) the buffer solution of COONa is re-balanced until the ultraviolet monitoring protein A280 is stable to be less than or equal to 10 mAU.
(11) Respectively using:
pH5.010mM CH3COONa +0.05M sodium chloride,
pH5.010mM CH3COONa +0.1M sodium chloride,
pH5.010mM CH3COONa +0.5M sodium chloride
The buffer solution of (2L per eluent) was continuously eluted at a flow rate of 40-60ml/min, and the eluates were collected separately. Wherein the elution peak collected from the buffer of pH5.010mM CH3COONa +0.1M sodium chloride is the purified PRN antigen solution.
(12) The purified PRN antigen solution is stored at 2-8 ℃.
The experimental results are as follows: FIG. 11 shows a PRN purified electrophoretogram, and FIG. 10 shows a PRN purified chromatogram
Example 7 antigen recovery assay and quality Studies for multiple purifications
The results of the recovery rates of the three antigens prepared in six batches according to the methods of examples 1 to 6 were examined as shown in Table 1 below.
TABLE 1 recovery of purified antigen
The results of the purified three pertussis toxin antigen solutions are shown in Table 2, the results of the purified three pertussis toxin antigen solutions are shown in Table 3, and the results of the purified three pertussis adhesin antigen solutions are shown in Table 4
TABLE 2 test results of six pertussis toxin antigen solutions
TABLE 3 assay results for six batches of pertussis filamentous hemagglutinin antigen solutions
TABLE 4 quality control results of six pertussis adhesin antigen solutions
The results show that three batches of pilot scale stock solutions produced according to the determined process all meet the requirements of the manufacturing and verification regulations (draft) of the product, and the stock solution preparation process is stable, controllable in quality, good in repeatability and capable of realizing batch production.
Finally, it should be noted that the above examples only help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.
Claims (3)
1. A method for separating and purifying a plurality of pertussis effective antigens, namely pertussis toxin PT antigen, filamentous hemagglutinin FHA antigen and pertussis adhesin PRN antigen, from a single culture of bordetella pertussis, comprising the steps of:
(1) performing fermentation culture on the pertussis strains, and respectively harvesting culture solution supernatant and thallus precipitate;
(2) separating and purifying PT antigen from the supernatant of the culture solution, and separating and purifying PRN antigen and FHA antigen from the thallus precipitate;
the method for separating and purifying the PT antigen comprises the following steps:
1) concentrating and ultrafiltering the supernatant of the bacterial liquid, wherein the concentration multiple is 5-16 times, the ultrafiltration uses 10mM sodium citrate buffer solution containing 2M urea and 0.5M sodium chloride and the pH value is 5.9-6.1, and the washing and filtering multiple is more than or equal to 4;
2) preparing PT antigen from the bacterial liquid supernatant ultrafiltrate by using a cation exchange chromatography; the method of cation exchange chromatography comprises the following steps:
a) crude pure preparation of PT antigen:
balancing the mixed-mode cation exchange chromatography column by using PBS buffer solution containing 2M urea with pH5.9-6.1, then loading, and balancing the chromatography column by using the same buffer solution;
eluting with 50mM Tris-HCl buffer solution containing 2M urea and 0.5M sodium chloride at the pH of 8.4-8.6, and collecting a crude pure PT antigen solution;
b) pure preparation of PT antigen:
balancing an ion exchange chromatographic column by using a 10mM sodium citrate buffer solution containing 2M urea at the pH value of 5.9-6.1, loading a crude pure PT antigen solution, balancing the chromatographic column by using the same buffer solution after loading until the pH value is 5.9-6.1, and the conductivity is less than or equal to 10 mS/cm;
eluting 8-10 column volumes with 10mM PB buffer solution containing 2M urea and 0.5% Triton X-100 and having pH of 7.4-7.6;
eluting with 10mM PB buffer solution containing 2M urea and 0.05-0.5M sodium chloride and having pH of 7.4-7.6, and collecting pure PT antigen solution;
the method for separating and purifying the PRN antigen and the FHA antigen from the thallus sediment comprises the following steps:
adding 1M sodium chloride with the pH =8.0 and the buffer solution of 50mM PB into the thallus precipitate, uniformly stirring, and leaching for 1-5 hours at the temperature of 2-8 ℃, wherein the volume of the buffer solution is 10-15 times of the wet weight of the precipitate;
centrifuging, collecting a leaching supernatant and a leaching precipitate respectively, separating and purifying FHA antigen from the leaching supernatant, and separating and purifying PRN antigen from the leaching precipitate;
the method for separating and purifying the FHA antigen comprises the following steps:
1) salting out: adding 30% (w/v) ammonium sulfate into the leaching supernatant, uniformly stirring, and standing for 18-24 hours at the temperature of 2-8 ℃;
2) secondary leaching, removing supernatant after salting out is finished, centrifuging the rest precipitate for 40-60 minutes under the condition of centrifugal force 14000-16000 g, collecting the precipitate, adding 10mM sodium citrate buffer solution containing 2M urea, with the volume being 10-15 times of the wet weight of the precipitate and the pH =5.9-6.1, into the precipitate, uniformly stirring, leaching at 2-8 ℃ for 1-2 hours, centrifuging the leaching solution for 40-60 minutes under the condition of centrifugal force 14000-16000 g, collecting supernatant, sterilizing and filtering the supernatant to obtain FHA antigen secondary leaching supernatant;
3) obtaining purified FHA antigen by chromatography, wherein the chromatography method comprises the following steps:
a) crude pure preparation of FHA:
using 10mM sodium citrate buffer solution containing 2M urea with the pH value of 5.9-6.1 to balance an ion exchange chromatographic column, and carrying out secondary extraction on the supernatant of the FHA antigen;
eluting 6-9 column volumes with 10mM PB buffer solution containing 2M urea and 0.5% Triton X-100 at pH 7.4-7.6;
eluting with 10mM PB buffer solution containing 2M urea and 0.2-1.2M sodium chloride at pH 7.4-7.6, and collecting crude pure FHA antigen solution;
b) the fine purification preparation of FHA:
balancing a hydroxyapatite chromatography column by using 10mM PB buffer solution containing 2M urea and having the pH value of 6.9-7.1; loading the crude pure FHA antigen solution, and balancing the crude pure FHA antigen solution to pH6.9-7.1 with the same buffer solution, wherein the conductivity is less than or equal to 20 mS/cm;
eluting 6-9 column volumes with 30mM PB buffer solution containing 2M urea and 0.5% Triton X-100 at pH 6.9-7.1;
eluting with 200mM PB buffer solution containing 2M urea and 0.2-0.5M sodium chloride at pH6.9-7.1, and collecting pure FHA antigen solution;
the PRN antigen separation and purification method comprises the following steps:
1) extraction: adding 5 times volume (v/w) of injection water into the leaching precipitation for redissolving, adding 8M urea (w/v), stirring for 2 to 3 hours at the stirring speed of 20 to 30rpm, and then adding the injection water to dilute the solution until 2M urea is stirred for 2 to 3 hours for extraction;
2) salting out, centrifuging after extraction, collecting supernatant, adding solid ammonium sulfate into the supernatant according to 30% of the volume of the supernatant, stirring at the speed of 20-30 rpm, and standing for 18-24 hours at the temperature of 2-8 ℃;
3) secondary leaching: removing supernatant after salting out, centrifuging the precipitate, and collecting the precipitate; adding 10mM PB buffer solution with the pH value of 6.9-7.1, the volume of which is 10-15 times of the wet weight of the precipitate, stirring at 20-30 rpm, and leaching for 1 hour; after the extraction is finished, centrifugally collecting the supernatant of the secondary extraction, and sterilizing and filtering to obtain the supernatant of the secondary extraction of the PRN antigen;
4) obtaining purified PRN antigen by chromatography, said chromatography comprising:
a) crude pure preparation of PRN antigen:
balancing the hydrophobic chromatographic column by using 10mM PB buffer solution containing 1M sodium chloride and having pH of 6.9-7.1, and loading the PRN antigen to extract supernatant for the second time;
eluting with a 10mM sodium acetate buffer solution containing 0-0.2M sodium chloride and having a pH of 4.4-4.6, and collecting a crude pure PRN antigen solution;
b) fine purification preparation of PRN antigen:
balancing an ion exchange chromatography column by using a 10mM sodium acetate buffer solution with the pH value of 4.9-5.1, adjusting the pH value of a crude pure PRN antigen solution to be 4.9-5.1, and adjusting the conductivity to be less than or equal to 5 mS/cm;
eluting with 10mM sodium acetate buffer solution with pH of 4.9-5.1 and containing 0.02-1M sodium chloride, and collecting the pure PRN antigen solution.
2. The method of claim 1,
the method for separating and purifying the PRN antigen and the FHA antigen from the bacterial pellet comprises the following steps: adding 1M sodium chloride and 50mM PB buffer solution with the volume of 10-15 times of the wet weight of the precipitate and the pH =8.0 into the bacterial precipitate, uniformly stirring, and leaching for 1.5 hours at the temperature of 2-8 ℃.
3. The method according to claim 1 or 2, characterized in that in the crude pure preparation of the PT antigen: the PBS buffer was a PBS buffer containing 20mM PB and 1M sodium chloride.
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