CN116077636A - Production process of adenovirus vector new crown vaccine stock solution - Google Patents
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Abstract
The invention provides a production process of adenovirus vector new crown vaccine stock solution, and relates to the field of biopharmaceutical process. The production process of the adenovirus vector new crown vaccine stock solution comprises the following steps: s1, resuscitating cells; s2, cell expansion; s3, culturing in a reactor; s4, thawing the poison seed; s5 virus infection; s6, virus amplification; s7, harvesting virus liquid; s8, ultrafiltration and enzyme digestion; s9, deep filtration; s10, ion exchange chromatography; s11, molecular sieve chromatography; s12, sterile filtration; s13, preparing and filling. The adenovirus strain is put into a cell reactor, the cells are infected by the virus and are replicated and amplified in the cells, the obtained adenovirus liquid is subjected to steps of filtration, ultrafiltration, enzyme digestion, chromatography and the like to remove redundant impurities, so that adenovirus vector vaccine stock solution is obtained, batch production is realized by arranging a plurality of bioreactors in the reactor culture, and batch production of the new crown vaccine can be realized.
Description
Technical Field
The invention relates to the technical field of biological pharmacy processes, in particular to a production process of adenovirus vector new crown vaccine stock solution.
Background
The novel coronavirus vaccine is a vaccine against the novel coronavirus, and the common virus vaccine development method comprises the following steps: 1) an inactivated viral vaccine, 2) an inactivated viral vaccine, 3) a subunit vaccine, 4) a VLP vaccine, 5) an RNA vaccine, and 6) a recombinant viral vector vaccine.
The new crown vaccine of adenovirus vector uses adenovirus as vector, and replaces some of adenovirus genes with spike glycoprotein genes, also called S protein genes, and the process is recombination. The S protein is a key protein for the invasion of the new coronavirus into human body, and the S protein is recombined on an adenovirus vector, so that the adenovirus also has the characteristics of the new coronavirus. The immune cells produce antibodies specific to the novel coronavirus S protein after inoculation into the body, and retain a portion of the immune cells, i.e., memory immune cells. When the new coronavirus truly invades, the memory immune cells can generate a large amount of antibodies aiming at S protein, the invasion of the new coronavirus into the cells is blocked, the proliferation of the new coronavirus cannot be realized when the new coronavirus cannot enter the human cells, and finally the new coronavirus cannot be destroyed by the immune cells of the human body.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a production process of adenovirus vector new crown vaccine stock solution, which solves the problem that recombinant adenovirus cannot be produced in large scale.
In order to achieve the above purpose, the invention is realized by the following technical scheme: a production process of adenovirus vector new crown vaccine stock solution comprises the following steps:
s1, cell resuscitation
Storing human embryo kidney cells in a liquid ammonia tank, taking out the cells from a seed warehouse when in use, thawing in a water bath at 37 ℃ for resuscitation, rapidly shaking 1mi of the cells, placing the cells in a biosafety cabinet, and inoculating the cells in the biosafety cabinet into a disposable medicine bottle through a disposable pipette;
s2, cell expansion
Transferring the resuscitated cells into CO 2 Culturing in a shaking table, controlling the culture temperature and the carbon dioxide concentration within the condition range, extracting a small amount of initial culture solution from a culture device for counting until the number of cells reaches the process requirement density, butting the culture solution into a wave reactor through a sterile port for amplification culture, and continuously adding the culture solution into the reactor in the amplification process to ensure that the nutrient substances in the culture medium can meet the requirement of cell amplification;
s3, reactor culture
Transferring part of the amplified cells into a stainless steel bioreactor for further culture, supplementing a culture medium to a certain volume in the process of culturing the cells, introducing compressed air, nitrogen, oxygen and carbon dioxide, maintaining a set temperature, pH and dissolved oxygen concentration to ensure that the cells can normally reproduce, periodically replacing a culture solution according to the consumption condition of the culture medium in the bioreactor, and discharging the generated waste culture solution and retaining the cells in the bioreactor;
s4, thawing the toxic seeds
Taking out adenovirus virus seed from the low temperature refrigerator, and thawing in water bath at 37deg.C;
s5, virus infection
The thawed virus seeds are connected into a bioreactor through a sterile interface;
s6, virus amplification
Infecting cells with viruses, replicating nucleic acid with virus genes in the cells, combining the replicated nucleic acid with carrier protein to form new viruses, thereby realizing the amplification of the viruses, periodically replacing culture solution in the same step S3 in the culture and amplification process to ensure that nutrients in a culture medium can meet the amplification requirement of the viruses in the cells, replacing the culture solution, and transferring a certain amount of amplified cells into a reactor, wherein a retention medium of the reactor can retain the cells but cannot retain the viruses, so that the amplified viruses can be discharged along with the culture solution, analyzing the culture solution discharged every day, collecting the culture solution discharged every time when the recombinant viruses are detected in the discharged culture solution, namely the virus solution, and ending the operation of the reaction kettle after the viruses in the reactor are not amplified;
s7, harvesting virus liquid
The collected virus liquid is filled into a disposable liquid storage bag in a sealing way, all transfer processes after the virus liquid is harvested are conveyed through sealing pipelines, and the pipelines are in a sterile butt joint way, so that waste gas pollutants are avoided;
s8, ultrafiltration and enzyme cutting
Introducing virus liquid into an ultrafilter through a silica gel pipe line for ultrafiltration concentration treatment, retaining substances such as small molecular proteins in the virus liquid and the culture solution in the remaining concentrated solution through an ultrafiltration medium, washing and filtering the concentrated solution by using a washing and filtering buffer solution so that the remaining culture solution in the concentrated solution can be replaced by the washing and filtering buffer solution, removing the washing and filtering buffer solution, adding nuclease into the concentrated solution to degrade large-fragment nucleic acid which is not infected by the virus, adding the washing and filtering buffer solution again after degradation, and removing the degraded large-fragment nucleic acid through the ultrafiltration medium;
s9, deep filtration
Deep filtering the enzyme-digested virus solution by using a multistage filter to remove large particle impurities such as cell residues and the like in the virus solution;
s10, ion exchange chromatography
Introducing the treated concentrated solution into ion exchange equipment, adding ion exchange buffer solution into an ion exchange chromatographic column for eluting by utilizing the difference of virus and impurity charging characteristics, and collecting the eluent in a disposable liquid storage bottle;
s11, molecular sieve chromatography
Introducing the eluent into a molecular sieve chromatographic column, adding a molecular sieve buffer solution into the molecular sieve chromatographic column to perform secondary elution on the ion exchange chromatographic eluent by utilizing the particle size difference of viruses and impurities, and collecting the eluent in a disposable liquid storage bottle;
s12, sterile filtration
Filtering the molecular sieve chromatographic eluent by using a 0.22 mu m filter, collecting the filtered stock solution in a stock solution bottle, and storing in an ultralow temperature refrigerator;
s13, preparing and filling
And (3) preparing the stock solution, and filling the stock solution into a penicillin bottle to obtain the adenovirus vector new crown vaccine.
Preferably, the culture medium in the S2 step of cell expansion comprises Na 2 HPO 4 、KH 2 PO 4 Mixed solution of kcl and nacl.
Preferably, the components of the ultrafiltration and enzyme digestion washing buffer solution in the step S9 are Tri S-base, HC l, naC l and MgC l 2 And sucrose.
Preferably, the ion exchange buffer solution in the S10 step ion exchange chromatography comprises Tri S-base, HC l, naC l and MgC l 2 Mixed solution of sucrose and PS-80.
Preferably, the components of the molecular sieve buffer solution in the S11 step molecular sieve chromatography are Tri S-base, HC l, naC l and MgC l 2 Sucrose, PS-80, ethanol and EDTA.
Preferably, the detailed operation of the S13 formulation and filling is as follows:
a) Taking the semi-finished stock solution out of the stock solution bottle, and performing sterile filtration by using a 0.22 mu m filter;
b) Adding the sterile filtered stock solution into a pre-prepared buffer solution, and diluting the stock solution, wherein the mass ratio of the stock solution to the buffer solution is (1.0-1.5);
c) Cleaning the purchased empty penicillin bottles and rubber plugs by using a bottle cleaning machine and injection water;
d) The penicillin bottle is dried by a dryer after being cleaned, diluted semi-finished products are filled into the penicillin bottle by filling equipment, rubber plugs are pressed on the penicillin bottle and are transferred to a capping machine through rails to be capped and sealed, the outside of the penicillin bottle is cleaned again after capping, and viruses possibly contaminated on the outer bottle are removed;
e) And labeling the finished product on the surface of the penicillin bottle after the finished product is qualified through light inspection, packaging, and then freezing the finally prepared product in a product freezer and transporting the product to leave a factory through a cold chain.
Preferably, the buffer solution comprises Tri s-base, HC l, naC l and MgC l 2 Sucrose, PS-80, ethanol and EDTA.
The invention provides a production process of adenovirus vector new crown vaccine stock solution. The beneficial effects are as follows:
1. the invention comprises the steps of resuscitating, amplifying, culturing and transferring outsourcing human embryo kidney cells into a cell reactor, putting outsourcing adenovirus virus seeds into the cell reactor, infecting the cells by viruses, carrying out replication and amplification in the cells, removing redundant impurities from the obtained adenovirus liquid through steps of filtration, ultrafiltration, enzyme cutting, chromatography and the like, thereby obtaining adenovirus vector vaccine stock solution, preparing the stock solution, and then filling the stock solution into a penicillin bottle to prepare adenovirus vector new crown vaccine, and realizing batch production by arranging a plurality of bioreactors in the reactor culture, so that batch production of the new crown vaccine can be realized, and the production efficiency is improved.
Drawings
FIG. 1 is a schematic illustration of a preparation flow of the present invention;
fig. 2 is a schematic diagram of the filling flow of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples:
as shown in fig. 1-2, the embodiment of the invention provides a production process of adenovirus vector new crown vaccine stock solution, which comprises the following steps:
s1, cell resuscitation
Storing human embryo kidney cells in a liquid ammonia tank, taking out the cells from a seed warehouse when in use, thawing in a water bath at 37 ℃ for resuscitation, rapidly shaking 1mi of the cells, placing the cells in a biosafety cabinet, and inoculating the cells in the biosafety cabinet into a disposable medicine bottle through a disposable pipette;
s2, cell expansion
Transferring the resuscitated cells into CO 2 Culturing in a shaking table, controlling the culture temperature and the carbon dioxide concentration within the condition range, extracting a small amount of initial culture solution from a culture device for counting until the number of cells reaches the process requirement density, butting the culture solution into a wave reactor through a sterile port for amplification culture, and continuously adding the culture solution into the reactor in the amplification process to ensure that the nutrient substances in the culture medium can meet the requirement of cell amplification;
s3, reactor culture
Transferring part of the amplified cells into a stainless steel bioreactor for further culture, supplementing a culture medium to a certain volume in the process of culturing the cells, introducing compressed air, nitrogen, oxygen and carbon dioxide, maintaining a set temperature, pH and dissolved oxygen concentration to ensure that the cells can normally reproduce, periodically replacing a culture solution according to the consumption condition of the culture medium in the bioreactor, and discharging the generated waste culture solution and retaining the cells in the bioreactor; the normal respiration and metabolism of cells in the cell expansion and culture process does not generate malodorous gases such as ammonia and the like, does not contain active substances, and breathes tail gas CO 2 And H 2 O is discharged into a production workshop room through a filter valve with the aperture of 0.22 mu m, is discharged out of the room through an air conditioning coarse, medium and high-efficiency ventilation system, and can isolate microorganisms in the environment from entering a culture medium to influence cell culture. Cell proliferation drives off unused oxygen and CO from respiration 2 The discharge amount is small, and the air enters the environmentThe air quality in the room and the outside is not affected, so that the air is not collected and treated as waste gas;
s4, thawing the toxic seeds
Taking out adenovirus virus seed from the low temperature refrigerator, and thawing in water bath at 37deg.C;
s5, virus infection
The thawed virus seeds are connected into a bioreactor through a sterile interface;
s6, virus amplification
Infecting cells with viruses, replicating nucleic acid with virus genes in the cells, combining the replicated nucleic acid with carrier protein to form new viruses, thereby realizing the amplification of the viruses, periodically replacing culture solution in the same step S3 in the culture and amplification process to ensure that nutrients in a culture medium can meet the amplification requirement of the viruses in the cells, replacing the culture solution, and transferring a certain amount of amplified cells into a reactor, wherein a retention medium of the reactor can retain the cells but cannot retain the viruses, so that the amplified viruses can be discharged along with the culture solution, analyzing the culture solution discharged every day, collecting the culture solution discharged every time when the recombinant viruses are detected in the discharged culture solution, namely the virus solution, and ending the operation of the reaction kettle after the viruses in the reactor are not amplified; during the process, biological aerosol containing virus is produced and discharged into a production workshop room through a filter valve with 0.22 mu m of the reactor, and is discharged out of the room through an air conditioning coarse, medium and high-efficiency ventilation system. The interception medium of the reactor needs to be replaced after the primary culture is finished, waste filter medium is generated, and the reactor is subjected to pure steam inactivation and cleaning after the primary culture is finished;
s7, harvesting virus liquid
The collected virus liquid is filled into a disposable liquid storage bag in a sealing way, all transfer processes after the virus liquid is harvested are conveyed through sealing pipelines, and the pipelines are in a sterile butt joint way, so that waste gas pollutants are avoided;
s8, ultrafiltration and enzyme cutting
Introducing virus liquid into an ultrafilter through a silica gel pipe line for ultrafiltration concentration treatment, retaining substances such as small molecular proteins in the virus liquid and the culture solution in the remaining concentrated solution through an ultrafiltration medium, washing and filtering the concentrated solution by using a washing and filtering buffer solution so that the remaining culture solution in the concentrated solution can be replaced by the washing and filtering buffer solution, removing the washing and filtering buffer solution, adding nuclease into the concentrated solution to degrade large-fragment nucleic acid which is not infected by the virus, adding the washing and filtering buffer solution again after degradation, and removing the degraded large-fragment nucleic acid through the ultrafiltration medium;
s9, deep filtration
Deep filtering the enzyme-digested virus solution by using a multistage filter to remove large particle impurities such as cell residues and the like in the virus solution;
s10, ion exchange chromatography
Introducing the treated concentrated solution into ion exchange equipment, adding ion exchange buffer solution into an ion exchange chromatographic column for eluting by utilizing the difference of virus and impurity charging characteristics, and collecting the eluent in a disposable liquid storage bottle;
s11, molecular sieve chromatography
Introducing the eluent into a molecular sieve chromatographic column, adding a molecular sieve buffer solution into the molecular sieve chromatographic column to perform secondary elution on the ion exchange chromatographic eluent by utilizing the particle size difference of viruses and impurities, and collecting the eluent in a disposable liquid storage bottle;
s12, sterile filtration
Filtering the molecular sieve chromatographic eluent by using a 0.22 mu m filter, collecting the filtered stock solution in a stock solution bottle, and storing in an ultralow temperature refrigerator;
s13, preparing and filling
And (3) preparing the stock solution, and filling the stock solution into a penicillin bottle to obtain the adenovirus vector new crown vaccine.
The culture solution in the S2 step of cell expansion comprises Na 2 HPO 4 、KH 2 PO 4 Mixed solution of kcl and nacl.
The components of the ultrafiltration and enzyme digestion washing buffer solution in the S9 step are Tri S-base, HC l, naC l and MgC l 2 And sucrose.
S10 step separationThe ion exchange buffer solution in the sub-exchange chromatography comprises Tris-base, HC l, naC l and MgC l 2 Mixed solution of sucrose and PS-80.
The components of the molecular sieve buffer solution in the S11 step molecular sieve chromatography are Tris-base, HC l, naC l and MgC l 2 Sucrose, PS-80, ethanol and EDTA.
The detailed operation of S13 preparation and filling is as follows:
a) Taking the semi-finished stock solution out of the stock solution bottle, and performing sterile filtration by using a 0.22 mu m filter;
b) Adding the sterile filtered stock solution into a pre-prepared buffer solution, and diluting the stock solution, wherein the mass ratio of the stock solution to the buffer solution is (1.0-1.5);
c) Cleaning the purchased empty penicillin bottles and rubber plugs by using a bottle cleaning machine and injection water;
d) The penicillin bottle is dried by a dryer after being cleaned, diluted semi-finished products are filled into the penicillin bottle by filling equipment, rubber plugs are pressed on the penicillin bottle and are transferred to a capping machine through rails to be capped and sealed, the outside of the penicillin bottle is cleaned again after capping, and viruses possibly contaminated on the outer bottle are removed;
e) And labeling the finished product on the surface of the penicillin bottle after the finished product is qualified through light inspection, packaging, and then freezing the finally prepared product in a product freezer and transporting the product to leave a factory through a cold chain.
The buffer solution comprises Tr is-base, HC l, naC l and MgC l 2 Sucrose, PS-80, ethanol and EDTA.
The method comprises the steps of resuscitating, amplifying, culturing and transferring outsourcing human embryo kidney cells into a cell reactor, putting outsourcing adenovirus virus seeds into the cell reactor, carrying out replication and amplification on virus infected cells in the cells, removing redundant impurities from the obtained adenovirus liquid through steps of filtering, ultrafiltration, enzyme cutting, chromatography and the like, thereby obtaining adenovirus vector vaccine stock solution, preparing the stock solution, then filling the stock solution into a penicillin bottle, obtaining adenovirus vector new crown vaccine, realizing batch production in the reactor culture by arranging a plurality of bioreactors, realizing batch production of the new crown vaccine, and improving the production efficiency.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A production process of adenovirus vector new crown vaccine stock solution is characterized in that: the method comprises the following steps:
s1, cell resuscitation
Storing human embryo kidney cells in a liquid ammonia tank, taking out the cells from a seed warehouse when in use, thawing in a water bath at 37 ℃ for resuscitation, rapidly shaking for 1min, placing the cells in a biosafety cabinet, and inoculating the cells into a disposable medicine bottle in the biosafety cabinet through a disposable pipette;
s2, cell expansion
Transferring the resuscitated cells into CO 2 Culturing in a shaking table, controlling the culture temperature and the carbon dioxide concentration within the condition range, extracting a small amount of initial culture solution from a culture device for counting until the number of cells reaches the process requirement density, butting the culture solution into a wave reactor through a sterile port for amplification culture, and continuously adding the culture solution into the reactor in the amplification process to ensure that the nutrient substances in the culture medium can meet the requirement of cell amplification;
s3, reactor culture
Transferring part of the amplified cells into a stainless steel bioreactor for further culture, supplementing a culture medium to a certain volume in the process of culturing the cells, introducing compressed air, nitrogen, oxygen and carbon dioxide, maintaining a set temperature, pH and dissolved oxygen concentration to ensure that the cells can normally reproduce, periodically replacing a culture solution according to the consumption condition of the culture medium in the bioreactor, and discharging the generated waste culture solution and retaining the cells in the bioreactor;
s4, thawing the toxic seeds
Taking out adenovirus virus seed from the low temperature refrigerator, and thawing in water bath at 37deg.C;
s5, virus infection
The thawed virus seeds are connected into a bioreactor through a sterile interface;
s6, virus amplification
Infecting cells with viruses, replicating nucleic acid with virus genes in the cells, combining the replicated nucleic acid with carrier protein to form new viruses, thereby realizing the amplification of the viruses, periodically replacing culture solution in the same step S3 in the culture and amplification process to ensure that nutrients in a culture medium can meet the amplification requirement of the viruses in the cells, replacing the culture solution, and transferring a certain amount of amplified cells into a reactor, wherein a retention medium of the reactor can retain the cells but cannot retain the viruses, so that the amplified viruses can be discharged along with the culture solution, analyzing the culture solution discharged every day, collecting the culture solution discharged every time when the recombinant viruses are detected in the discharged culture solution, namely the virus solution, and ending the operation of the reaction kettle after the viruses in the reactor are not amplified;
s7, harvesting virus liquid
The collected virus liquid is filled into a disposable liquid storage bag in a sealing way, all transfer processes after the virus liquid is harvested are conveyed through sealing pipelines, and the pipelines are in a sterile butt joint way, so that waste gas pollutants are avoided;
s8, ultrafiltration and enzyme cutting
Introducing virus liquid into an ultrafilter through a silica gel pipe line for ultrafiltration concentration treatment, retaining substances such as small molecular proteins in the virus liquid and the culture solution in the residual concentrated liquid through an ultrafiltration medium, washing and filtering the concentrated buffer solution by using a washing and filtering buffer solution so that the residual culture solution in the concentrated liquid can be replaced by the washing and filtering buffer solution, removing the residual culture solution by using the ultrafiltration medium, adding nuclease into the concentrated solution to degrade large-fragment nucleic acid which is not infected by the virus, adding the washing and filtering buffer solution again after degradation, and removing the degraded large-fragment nucleic acid by using the ultrafiltration medium;
s9, deep filtration
Deep filtering the enzyme-digested virus solution by using a multistage filter to remove large particle impurities such as cell residues and the like in the virus solution;
s10, ion exchange chromatography
Introducing the treated concentrated solution into ion exchange equipment, adding ion exchange buffer solution into an ion exchange chromatographic column for eluting by utilizing the difference of virus and impurity charging characteristics, and collecting the eluent in a disposable liquid storage bottle;
s11, molecular sieve chromatography
Introducing the eluent into a molecular sieve chromatographic column, adding a molecular sieve buffer solution into the molecular sieve chromatographic column to perform secondary elution on the ion exchange chromatographic eluent by utilizing the particle size difference of viruses and impurities, and collecting the eluent in a disposable liquid storage bottle;
s12, sterile filtration
Filtering the molecular sieve chromatographic eluent by using a 0.22 mu m filter, collecting the filtered stock solution in a stock solution bottle, and storing in an ultralow temperature refrigerator;
s13, preparing and filling
And (3) preparing the stock solution, and filling the stock solution into a penicillin bottle to obtain the adenovirus vector new crown vaccine.
2. The process for producing adenovirus vector new corona vaccine stock solution according to claim 1, wherein the process comprises the following steps: the culture solution in the S2 step of cell expansion comprises Na 2 HPO 4 、KH 2 PO 4 Mixed solution of KCl and NaCl.
3. The process for producing adenovirus vector new corona vaccine stock solution according to claim 1, wherein the process comprises the following steps: the components of the ultrafiltration and enzyme digestion medium washing and filtering buffer solution in the step S9 are Tris-base, HCl, naCl, mgCl 2 And sucrose.
4. The process for producing adenovirus vector new corona vaccine stock solution according to claim 1, wherein the process comprises the following steps: the saidThe component of the ion exchange buffer solution in the S10 step ion exchange chromatography is Tris-base, HCl, naCl, mgCl 2 Mixed solution of sucrose and PS-80.
5. The process for producing adenovirus vector new corona vaccine stock solution according to claim 1, wherein the process comprises the following steps: the components of the molecular sieve buffer solution in the S11 step molecular sieve chromatography are Tris-base, HCl, naCl, mgCl 2 Sucrose, PS-80, ethanol and EDTA.
6. The process for producing adenovirus vector new corona vaccine stock solution according to claim 1, wherein the process comprises the following steps: the detailed operation of the S13 preparation filling is as follows:
a) Taking the semi-finished stock solution out of the stock solution bottle, and performing sterile filtration by using a 0.22 mu m filter;
b) Adding the sterile filtered stock solution into a pre-prepared buffer solution, and diluting the stock solution, wherein the mass ratio of the stock solution to the buffer solution is (1.0-1.5);
c) Cleaning the purchased empty penicillin bottles and rubber plugs by using a bottle cleaning machine and injection water;
d) The penicillin bottle is dried by a dryer after being cleaned, diluted semi-finished products are filled into the penicillin bottle by filling equipment, rubber plugs are pressed on the penicillin bottle and are transferred to a capping machine through rails to be capped and sealed, the outside of the penicillin bottle is cleaned again after capping, and viruses possibly contaminated on the outer bottle are removed;
e) And labeling the finished product on the surface of the penicillin bottle after the finished product is qualified through light inspection, packaging, and then freezing the finally prepared product in a product freezer and transporting the product to leave a factory through a cold chain.
7. The process for producing adenovirus vector new corona vaccine stock solution according to claim 6, wherein the process comprises the following steps: the buffer solution comprises Tris-base, HCl, naCl, mgCl 2 Sucrose, PS-80, ethanol and EDTA.
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