CN111171117B - Purification process of recombinant CA16 virus-like particles, recombinant CA16 virus vaccine and preparation method thereof - Google Patents
Purification process of recombinant CA16 virus-like particles, recombinant CA16 virus vaccine and preparation method thereof Download PDFInfo
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- CN111171117B CN111171117B CN201911390086.2A CN201911390086A CN111171117B CN 111171117 B CN111171117 B CN 111171117B CN 201911390086 A CN201911390086 A CN 201911390086A CN 111171117 B CN111171117 B CN 111171117B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32023—Virus like particles [VLP]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32051—Methods of production or purification of viral material
Abstract
The embodiment of the invention discloses a purification process of recombinant CA16 virus-like particles, a CA16 virus vaccine and a preparation method thereof. The purification process comprises the steps of cell fermentation, crushing, clarification, microfiltration, ultrafiltration and a plurality of chromatography steps, wherein the chromatography steps comprise: ion exchange chromatography; hydrophobic chromatography is carried out by using a hydrophobic chromatography medium Phenyl6 FF; and (5) performing molecular sieve chromatography. The invention can obviously improve the recovery rate of the antigen and the impurity removal rate of the protein.
Description
Technical Field
The invention relates to a preparation technology of a recombinant CA16 virus vaccine, in particular to a purification process of a recombinant CA16 virus-like particle, a recombinant CA16 virus vaccine and a preparation method thereof.
Background
The existing purification process of the recombinant CA16 virus-like particles comprises the following steps: cell disruption solution → clarification, microfiltration, ultrafiltration → ion exchange chromatography captoq (GE) → hydroxyapatite chromatography CHT (berle) → molecular sieve chromatography sephacryls 300HR (GE).
In this process, a hydroxyapatite chromatography medium CHT is used, and the CHT chromatography antigen recovery rate is only 30-45% in the purification process of the recombinant CA16 virus-like particles.
In the process, a strong anion exchange chromatography medium CaptoQ is used, a large amount of load tests are carried out in the purification process of recombinant CA16 virus-like particles, a CA16 ultrafiltration product sample is loaded on a CaptoQ ion exchange chromatography column, the loading capacity of the filler is 1-2mg/mL, and the protein content of the loaded sample is 7.5mg/mL, which indicates that 375-750L of filler is required for 200L of loaded sample, which undoubtedly has great pressure on the type selection of purification equipment and consumes a great amount of cost on the filler and the equipment.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a purification process of recombinant CA16 virus-like particles, which can obviously improve the loading capacity of purified filler, reduce the required volume of the filler, save the filler cost, and improve the antigen recovery rate and the protein impurity removal rate.
The technical problem to be further solved by the invention is to provide a preparation method of the recombinant CA16 virus vaccine, which can obviously improve the loading capacity of the purified filler, reduce the volume required by the filler, save the filler cost, and improve the antigen recovery rate and the protein impurity removal rate.
The technical problem to be further solved by the invention is to provide a recombinant CA16 virus vaccine which has significantly improved antigen recovery rate and protein impurity removal rate.
In order to solve the technical problems, the invention discloses the following technical scheme:
a purification process of recombinant CA16 virus-like particles comprises cell fermentation, crushing, clarification, microfiltration, ultrafiltration and a plurality of chromatography steps, wherein the chromatography steps comprise:
ion exchange chromatography;
hydrophobic chromatography is carried out by using a hydrophobic chromatography medium Phenyl6 FF;
and (5) performing molecular sieve chromatography.
In some possible embodiments, the hydrophobic chromatography specifically comprises:
the high-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5 is used for balancing 2-5 column volumes of a hydrophobic chromatography column (Phenyl6FF), then the sample is loaded, the balance is continued for 1-3 column volumes, the elution is carried out by using the low-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5, and the elution solution UV280nm ultraviolet absorption peak is collected.
In some possible embodiments, the ion exchange chromatography employs the ion exchange chromatography medium NuviaTMHP-Q.
In some possible embodiments, the ion exchange chromatography specifically comprises:
tris salt buffer solution balance ion exchange chromatography column (Nuvia) with pH7.5-8.5TMHP-Q)2-5 column volumes, loading the ultrafiltration protein solution, balancing 2 column volumes, eluting with Tris low salt buffer solution with pH of 7.5-8.5, and collecting eluate UV280nm ultraviolet absorption peak.
In some possible embodiments, molecular sieve chromatography is performed using sephacryl s300 HR.
In some possible embodiments, the molecular sieve chromatography specifically comprises:
balancing molecular sieve chromatographic column (Sephacryl S300HR) with PBST buffer solution of pH6.0-8.0, balancing 1-3 column volumes, and collecting eluate UV280nm ultraviolet absorption peak to obtain the target protein solution.
In some possible embodiments, the microfiltration step comprises:
microfiltering the collected clear supernatant with 0.2-0.65 μm membrane package, and buffer solution of pH6.8-8.5 to remove macromolecular substances, concentrating by 2-10 times, washing and filtering by 2-5 times, and collecting the filtrate to obtain crude pure protein solution.
In some possible embodiments, the ultrafiltration step comprises:
and (3) carrying out ultrafiltration on the collected crude pure protein liquid by using a 100kd-500kd membrane package and adopting a buffer solution with the pH value of 6.8-8.5 to remove small molecular substances, carrying out 2-10 times of concentration, and carrying out 6-7 times of washing filtration to collect ultrafiltrate to obtain the ultrafiltration protein liquid.
Correspondingly, the invention also discloses a preparation method of the recombinant CA16 virus vaccine, which comprises the purification process.
Correspondingly, the invention also discloses a recombinant CA16 virus vaccine which is prepared by the preparation method.
The invention has the beneficial effects that:
according to the embodiment of the invention, hydrophobic chromatography is carried out by using a hydrophobic chromatography medium Phenyl6FF, so that the antigen expression quantity and the protein impurity removal rate of the target protein solution are obviously improved.
Detailed Description
The following describes in detail an example of a process for purifying recombinant CA16 virus-like particles provided by the present invention; the embodiment mainly comprises the steps of cell fermentation, crushing, clarification, microfiltration, ultrafiltration and a plurality of chromatography steps, wherein the chromatography steps comprise:
ion exchange chromatography;
hydrophobic chromatography is carried out by using a hydrophobic chromatography medium Phenyl6 FF;
and (5) performing molecular sieve chromatography.
In some possible embodiments, the hydrophobic chromatography specifically comprises:
the high-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5 is used for balancing 2-5 column volumes of a hydrophobic chromatography column (Phenyl6FF), then the sample is loaded, the balance is continued for 1-3 column volumes, the elution is carried out by using the low-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5, and the elution solution UV280nm ultraviolet absorption peak is collected.
In some possible embodiments, the ion exchange chromatography employs the ion exchange chromatography medium NuviaTMHP-Q.
In some possible embodiments, the ion exchange chromatography specifically comprises:
tris salt buffer solution balance ion exchange chromatography column (Nuvia) with pH7.5-8.5TMHP-Q)2-5 column volumes, loading the ultrafiltration protein solution, balancing 2 column volumes, and allowingEluting with Tris low salt buffer solution with pH7.5-8.5, and collecting eluate UV280nm ultraviolet absorption peak.
In some possible embodiments, molecular sieve chromatography is performed using sephacryl s300 HR.
In some possible embodiments, the molecular sieve chromatography specifically comprises:
balancing molecular sieve chromatographic column (Sephacryl S300HR) with PBST buffer solution of pH6.0-8.0, balancing 1-3 column volumes, and collecting eluate UV280nm ultraviolet absorption peak to obtain the target protein solution.
In some possible embodiments, the microfiltration step comprises:
microfiltering the collected clear supernatant with 0.2-0.65 μm membrane package, and buffer solution of pH6.8-8.5 to remove macromolecular substances, concentrating by 2-10 times, washing and filtering by 2-5 times, and collecting the filtrate to obtain crude pure protein solution.
In some possible embodiments, the ultrafiltration step comprises:
and (3) carrying out ultrafiltration on the collected crude pure protein liquid by using a 100kd-500kd membrane package and adopting a buffer solution with the pH value of 6.8-8.5 to remove small molecular substances, carrying out 2-10 times of concentration, and carrying out 6-7 times of washing filtration to collect ultrafiltrate to obtain the ultrafiltration protein liquid.
The preparation method of the recombinant CA16 virus vaccine disclosed by the embodiment of the invention comprises the purification process, and the rest is the same as the prior art and is not repeated.
The recombinant CA16 virus vaccine disclosed by the embodiment of the invention is prepared by the preparation method, and is not described in detail.
One embodiment of the present invention can be achieved by the following process steps:
firstly, inoculating a recombinant hansenula polymorpha working seed batch strain which is constructed by a DNA recombination technology and expresses CA16 virus-like particles to a fermentation tank for methanol-induced expression of recombinant antigens through primary seed culture, secondary seed culture and tertiary seed culture, centrifugally washing after the fermentation is finished, and harvesting thalli, and freezing and storing in a refrigerator at the temperature of-60 ℃;
secondly, the frozen thalli is resuspended by adopting a 4 ℃ crushing buffer solution, and cells are crushed for 2 to 4 times under the condition of the pressure of 1100 and 1500 bar; preferably, the engineering bacteria are crushed for 2 times under the pressure of 1350 bar;
thirdly, microfiltering the collected clear supernatant by using a 0.2-0.65 mu m membrane package and adopting a buffer solution with the pH value of 6.8-8.5 to remove macromolecular substances, carrying out 2-10 times of concentration, and carrying out 2-5 times of washing filtration to collect permeate to obtain crude pure protein liquid;
performing ultrafiltration on the collected crude pure protein liquid by a 100-500 kd membrane package by using a buffer solution with the pH value of 6.8-8.5 to remove small molecular substances, performing 2-10 times of concentration, performing 6-7 times of washing filtration, and collecting ultrafiltrate to obtain an ultrafiltration protein liquid;
tris salt buffer solution balance ion exchange chromatographic column (Nuvia) with pH7.5-8.5TMHP-Q)2-5 column volumes, loading the ultrafiltration protein solution, balancing 2 column volumes, eluting with Tris low salt buffer solution with pH of 7.5-8.5, and collecting eluate UV280nm ultraviolet absorption peak;
sixthly, after 2 to 5 column volumes of the high-salt ammonium sulfate buffer solution with the pH value of 7.5 to 8.5 balance hydrophobic chromatography column (Phenyl6FF), loading, continuously balancing 1 to 3 column volumes, eluting by using a low-salt ammonium sulfate buffer solution with the pH value of 7.5 to 8.5, and collecting an eluent UV280nm ultraviolet absorption peak;
seventhly, balancing the molecular sieve chromatographic column (Sephacryl S300HR) with the PBST buffer solution with the pH value of 6.0-8.0, continuously balancing 1-3 column volumes, and collecting an eluent UV280nm ultraviolet absorption peak, namely the target protein solution.
Compared with the prior art, the embodiment of the invention has the following advantages:
in the step of ion exchange chromatography, compared with the prior art, the method has the advantages of high loading capacity, reduced filler cost, equipment investment and maintenance cost saving, reduced equipment occupation space and more benefit for process amplification;
the hydrophobic chromatography step replaces the CHT chromatography in the prior art, so that the nearly twice antigen recovery rate of the step is improved;
the antigen recovery rate of the VLP antigen purified by the purification scheme route is higher than that of the original process, all detection items conform to pharmacopeia standards, and the expected target is achieved;
the novel vaccine belongs to a genetic engineering recombinant vaccine with extremely high safety, and does not cause inverse toxicity and potential RNA carcinogenic risk;
the hand-foot-and-mouth disease caused by the CA16 virus is on the rise, but at present, no vaccine for preventing the hand-foot-and-mouth disease from appearing on the market is available at home and abroad, so the product can successfully appear on the market and has great social value and economic value.
In order to further explain the technical means and effects of the present embodiment for achieving the predetermined invention, the specific steps of the purification process of the recombinant CA16 virus-like particles of the present embodiment are described in detail below by way of example data.
Example 1:
the recombinant CA16 yeast expression strain is subjected to fermentation culture in a 30L fermentation tank
The method comprises the steps of using strains constructed by a DNA recombination technology, obtaining high-efficiency expression strains (original strains) through stable culture and screening, establishing original seeds, main seed batches and working seed batches through passage of the original strains, and inoculating the working seed batches to a fermentation tank through primary seed culture, secondary seed culture and tertiary seed culture to perform methanol-induced expression of recombinant antigens. The method comprises the steps of performing engineering bacteria fermentation by adopting a high-density formula culture medium, performing fermentation growth stage, intermittently supplementing glycerol to enable bacteria to grow rapidly, performing fermentation induction stage, continuously supplementing methanol to enable the bacteria to express viroid-like granular protein efficiently, performing fermentation culture for 88-91 hours, putting the bacteria into a tank, and performing washing and filtration twice in equal volume by adopting a centrifugal mode (high-speed centrifugation or butterfly centrifugation) or a 0.65-micron membrane-covered microfiltration mode. The wet weight of the bacteria at the end of fermentation can reach 240-350g/L, and the antigen expression amount can reach 50000-100000U/ml.
Separation and purification of CA16 virus-like particles
The engineering bacteria are resuspended by adopting a crushing buffer solution, the cells are crushed for 2 times under the condition of the pressure of 1100-1500bar, and the cell crushing rate reaches 70-90 percent.
Microfiltering by 0.65 μm, ultrafiltering by 300kd, and loading on NuviaTMHP-Q chromatographic column, collecting eluate UV280nm ultraviolet absorption peak,
after treatment, the mixture is loaded on a Phenyl6FF chromatographic column, an eluent UV280nm ultraviolet absorption peak is collected,
and then loading the protein solution to a Sephacryl S300HR chromatographic column, and collecting an ultraviolet absorption peak of eluent UV280nm to obtain a purified target protein solution.
The experimental results are as follows:
TABLE I comparative data for the respective filler loadings
TABLE II, Linear flow Rate comparison data for each Filler
| Name of Filler | Linear flow rate of packing |
| CaptoQ | 60cm/hr |
| CHT | 300cm/hr |
| 6FF | 48cm/h |
| HP-Q | 150cm/hr |
Table three, detection results in the prior art:
TABLE IV and test results of example 1
The test result shows that the sample of the embodiment of the invention is loaded on NuviaTMThe HP-Q high-loading strong anion exchange chromatographic column has the filler loading capacity of 10-16mg/mL which is more than 10 times of that of the prior art with the same volume, and can achieve more than 70% of antigen recovery rate and more than 90% of protein impurity removal rate. The loading capacity of the filler is obviously improved, the required volume of the filler is reduced, the filler cost is saved, the equipment investment and the maintenance cost are saved, the occupied space of the equipment is reduced, and the process linear amplification is facilitated.
The sample of the embodiment of the invention is loaded on hydrophobic chromatography Phenyl6FF, the antigen recovery rate is more than 90%, the protein impurity removal rate is higher, and the cost performance is high.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all should be considered as belonging to the protection scope of the invention.
Claims (5)
1. A purification process of recombinant CA16 virus-like particles sequentially comprises the steps of cell fermentation, crushing, clarification, microfiltration, ultrafiltration and a plurality of chromatography, and is characterized in that the chromatography steps sequentially comprise:
adopts ion exchange chromatography medium NuviaTMHP-Q was subjected to ion exchange chromatography comprising:
tris salt buffer solution balance ion exchange chromatography column Nuvia with pH7.5-8.5TMLoading the ultrafiltration protein liquid after 2-5 column volumes of HP-Q, balancing 2 column volumes, eluting with Tris low salt buffer solution with pH of 7.5-8.5, and collecting eluate UV280nm ultraviolet absorption peak;
hydrophobic chromatography was performed using the hydrophobic chromatography medium Phenyl6FF, comprising:
the high-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5 is used for balancing hydrophobic chromatography column Phenyl6FF 2 for 2-5 column volumes, then the sample is loaded, the balance for 1-3 column volumes is continued, then the low-salt ammonium sulfate buffer solution with the pH value of 7.5-8.5 is used for elution, and the eluent UV280nm ultraviolet absorption peak is collected;
molecular sieve chromatography using sephacryl s300HR, comprising:
and (3) balancing the molecular sieve chromatographic column Sephacryl S300HR with the PBST buffer solution with the pH value of 6.0-8.0, continuously balancing 1-3 column volumes, and collecting an ultraviolet absorption peak of eluent UV280nm to obtain the target protein solution.
2. The process of claim 1, wherein the microfiltration step comprises:
microfiltering the collected clear supernatant with 0.2-0.65 μm membrane package, and buffer solution of pH6.8-8.5 to remove macromolecular substances, concentrating by 2-10 times, washing and filtering by 2-5 times, and collecting the filtrate to obtain crude pure protein solution.
3. The process for purifying recombinant CA16 virus-like particles according to claim 2, wherein the ultrafiltration step comprises:
and (3) carrying out ultrafiltration on the collected crude pure protein liquid by using a 100kd-500kd membrane package and adopting a buffer solution with the pH value of 6.8-8.5 to remove small molecular substances, carrying out 2-10 times of concentration, and carrying out 6-7 times of washing filtration to collect ultrafiltrate to obtain the ultrafiltration protein liquid.
4. A method for preparing a recombinant CA16 virus vaccine, which is characterized by comprising the purification process of any one of 1-3.
5. A recombinant CA16 virus vaccine prepared by the method of claim 4.
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