CN115851625A - Preparation method of low MDCK cell residue DNA influenza vaccine stock solution - Google Patents

Abstract

The invention discloses a preparation method of a low MDCK cell residue DNA influenza vaccine stock solution, which comprises the steps of after virus is harvested, carrying out glass fiber filtration on virus liquid, and limiting the specific pore diameter of glass fiber to be 0.6-1.0 mu m so as to effectively intercept and filter broken cell fragments; meanwhile, the chemical essence of the glass fiber is silicon dioxide, a large number of hydrogen bonds can be formed between silanol groups on the surface of the silicon dioxide and biomass surface groups generated by cell disruption, and although the hydrogen acting force formed between the silanol groups and the biomass surface groups is weak, the number of the silanol groups and the biomass surface groups is huge, so that biomass impurities such as DNA generated by cell disruption can be firmly adsorbed on the surface of the silicon dioxide in a short time, and the filtration and purification of the harvest liquid are realized.

Description

Preparation method of low MDCK cell residue DNA influenza vaccine stock solution

Technical Field

The invention relates to the field of purification of influenza vaccines, in particular to a preparation method of a monovalent stock solution of a DNA influenza vaccine with low MDCK cell residue.

Background

The influenza virus is spherical in shape, and is a few filamentous in shape, the diameter of the influenza virus is between 80 and 120 nanometers, and the influenza virus structure can be divided into three parts, namely an envelope, a matrix protein and a core from outside to inside.

The traditional influenza vaccine is prepared by breeding virus from chick embryos, although the technology is mature, the risk of exogenous material pollution is very large, and the control means is complicated and has poor operability. Therefore, WHO has recommended the use of passaged cells for the production of influenza vaccines. The MDCK cell line was established by Madin and Darby isolated and cultured in 1958 from kidney tissue of Cocker Spanisl mother Quimby dogs, USA, and the cells are usually grown in an adherent manner to be epithelioid cells and can also be domesticated to be serum-free suspension culture cells. MDCK cell lines have been widely used for the amplification and purification of a variety of viruses. MDCK (Madin-Dady Canine Kidney Cell) is suitable for production of influenza vaccines due to the characteristics of high virus infection efficiency, rapid proliferation, difficult variation and the like. Residual amounts of MDCK cell residual DNA need to be controlled in the MDCK cell influenza vaccine production process to control the potential risk of the final product in terms of tumorigenesis. In the preparation of influenza vaccines by the MDCK cell process, a method for removing residual DNA is very important.

Disclosure of Invention

The invention aims to provide a preparation method of a low MDCK cell residual DNA influenza vaccine stock solution, which aims to solve the problem of removing residual MDCK cell DNA in an MDCK cell influenza vaccine preparation process.

In order to solve the technical problems, the invention provides the following technical scheme:

a preparation method of a monovalent stock solution of a low MDCK cell residual DNA influenza vaccine comprises the following specific preparation steps:

(1) Cell recovery;

(2) Cell passage;

(3) Expanding and passaging WAVE cells;

(4) Culturing in a bioreactor;

(5) Inoculating viruses;

(6) Harvesting viruses;

when the virus is cultured for 48-72 hours, harvesting is started.

(7) Deep filtering and inactivating; and filtering the harvested virus liquid by using a glass fiber filter element to remove cell fragments. The aperture of the glass fiber filter core is 0.6-1.0 μm.

(8) Ultrafiltration concentration and nuclease digestion;

after concentration with an ultrafiltration membrane, washing filtration was performed with a buffer. The buffer solution is phosphate buffer solution. The ultrafiltration membrane is adopted to concentrate the influenza virus, so that the interception function of the ultrafiltration membrane can be effectively utilized to filter the micromolecular DNA and further purify the product. A small portion of the trapped DNA can then be effectively removed by phosphate buffered saline. Preferably, the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 to 10 portions of sodium chloride, 0.2 to 0.4 portion of potassium chloride, 3 to 5 portions of disodium hydrogen phosphate dodecahydrate, 0.2 to 0.4 portion of dipotassium hydrogen phosphate and 800 to 1000 portions of water.

(9)Capto ^TM Performing Core 700 mixed gel molecular sieve chromatography;

Capto ^TM the Core 700 membrane is composed of an activated ligand Core and an inert shell which excludes macromolecules and prevents them from entering the Core through pores in the shell. These macromolecules are collected in the column flow-through, while smaller impurities bind to the ligands within the microspheres. The hydrophobic and positively charged ligands functionalize the core of each microsphere, allowing efficient complex-mode binding of various contaminants small enough to enter the core. The core microsphere technology with the activated ligand core and the inactivated shell layer can effectively capture pollutants and impurities, simultaneously enables target material to flow through, and guarantees the effect of removing impurities while ensuring the recovery of effective substances.

The Capto ^TM The Core 700 film chromatography sample loading linear flow rate is 100cm/h, the retention time is 5min, and the sample loading amount is 1.0-6.0 column volume.

(10)Capto ^TM Core Q ion exchange chromatography;

preferably, said use is Capto ^TM The Core Q ion exchange chromatography is as follows: the sample loading amount is 5-10% of the column volume, the forward linear flow rate is 5mL/min, and the protein peak is collected.

The step can effectively utilize the gel chromatography process to further remove free micromolecular DNA in the system, and effectively ensure the purity of the product.

(11) Splitting virus;

(12) Performing ultrafiltration and dialysis on glass fiber;

(13) And (5) sterilizing and filtering.

The invention has the beneficial effects that: after the virus is harvested, glass fiber filtration is carried out on virus liquid, and broken cell fragments are effectively intercepted and filtered by limiting the specific aperture of the glass fiber; meanwhile, the glass fiber is adopted as the filter element material because the chemical nature of the glass fiber is silicon dioxide, a large number of hydrogen bonds can be formed between silanol groups on the surface of the silicon dioxide and biomass surface groups generated by cell disruption, and although the hydrogen acting force formed between the silanol groups and the biomass surface groups is weak, the number of the silanol groups and the biomass surface groups is large, so that biomass impurities such as DNA generated by cell disruption can be firmly adsorbed on the surface of the silicon dioxide in a short time, and the filtration and purification of the harvest liquid are realized.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example 1

A preparation method of a monovalent stock solution of a low MDCK cell residual DNA influenza vaccine comprises the following steps:

s1, cell recovery: taking the seed cells of the working bank from a liquid nitrogen tank, putting the seed cells into warm water at 37 ℃, melting the seed cells, aseptically transferring the seed cells into a square bottle filled with growth liquid, and culturing the seed cells in an incubator at the temperature of 36 ℃;

s2, cell passage: observing under a microscope, after the revived cells grow to be full of a monolayer, digesting with 0.25% trypsin,

adherent cells were shed as follows 1:3, and culturing at the constant temperature of 36 ℃ for 3 days;

s3, expanding and passaging WAVE cells; 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) transfer to WAVE cultureIn the bag cultivation, a culture medium preservation test is required before inoculation in order to control risks in the process; the culture volume is calculated and expanded to 20L with the dilution density of 0.3X 10 ⁶ cells/mL of the desired seed suspension. The cell suspension in 5L flasks was transferred by calculated weight to 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) temperature in Wave of medium 36.5 ℃ at start (20L), rotation speed 18rpm, angle 8 °, air flow rate Day 0-Day 1:0.3L/minDay 2-Day end:0.6L/min, CO ₂ %5% culture for 3 days; if the culture volume after inoculation is less than 20L, the total volume needs to be supplemented to 20L by a feed medium;

s4, bioreactor culture, namely digesting the cells by using 0.25% trypsin, and transferring the cells to a bioreactor for culture. The bioreactor is pre-loaded with polyester chip carrier, 30g per liter of culture volume, and cell inoculation density of 2 × 10 ⁵ cells/mL. Setting culture parameters: temperature 36 ℃, DO30, pH 7.0. Culturing until the cell density reaches more than 1 × 107 cells/mL;

s5, virus inoculation: discarding the culture solution in the bioreactor, adding a replacement solution, adding an influenza virus strain (H1N 1) according to the infection amount of 0.001-0.1 MOI, and setting parameters: the culture was carried out at 33 ℃, DO30, pH 7.3. After 24 hours, the cells were replaced with 0.01M phosphate buffer, and the medium was added after repeating 2 times to continue the culture. Setting parameters: the temperature is 33 ℃, DO is 30, and the pH value is 7.3; the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 part of potassium chloride, 3 parts of disodium hydrogen phosphate dodecahydrate, 0.2 part of dipotassium hydrogen phosphate and 800 parts of water for injection.

S6, harvesting viruses: after the inoculation of influenza virus, virus liquid is obtained at the culture temperature of 33.0 ℃, pH7.25 (Deadband 0.20) and DO40 percent for 48 to 72 hours.

S7, deep filtration and inactivation: immediately filtering the harvested virus liquid by using a glass fiber filter element (with the aperture of 0.6 mu m) to remove cell debris; a deep filter with a deep filtration membrane is adopted, the liquid inlet pressure is less than or equal to 0.2MPa, the liquid is filtered to a stainless steel tank A # with the temperature of 200L 2-8 ℃, and the turbidity of the clear filtrate is less than 20NTU. Cooling the virus clarified liquid to 2-8 ℃ in a 200L inactivation tank A #, and mixing the virus clarified liquid with beta-propiolactone (ml): adding a diluted virus inactivating agent into the inactivated virus liquid according to the weight (g) of 1; after 12 hours of inactivation, the cells were transferred via transfer line to a 1000L inactivation tank B # for 12 hours of continued inactivation. The inactivation temperature is 2-8 ℃, and the stirring revolution is 60-100 rpm;

and inactivating the virus clarified liquid for 24 hours, transferring the virus clarified liquid into a hydrolysis/tank to be detected through a material transferring pipeline, hydrolyzing for 2 hours at 37-38 ℃, sampling, carrying out a virus inactivation verification test, and cooling to 2-8 ℃ to be detected for 10-11 days.

S8, ultrafiltration concentration and nuclease digestion: the virus inactivation solution is ultrafiltered and concentrated to 5-6 kg by a 1000kD PES ultrafiltration membrane package, the ultrafiltration membrane package is washed by PBS (pH7.4) buffer solution to about 10kg, the volume of the buffer solution is 10 times that of the buffer solution, and the buffer solution is dialyzed and replaced by 0.01MPBS buffer solution and transferred to a 20L enzymolysis tank. Adding a proper amount of 1mol/L magnesium chloride solution and non-restriction endonuclease into the concentrated solution to ensure that the final concentration of the magnesium chloride is 0.2mmol/L and the final concentration of the non-restriction endonuclease is 90 +/-10U/ml; stirring uniformly, keeping the temperature at 25 +/-1 ℃ for at least 120 minutes to obtain the enzymatic hydrolysate.

S9、Capto ^TM Performing Core 700 mixed gel molecular sieve chromatography; molecular sieve chromatography filler (Capto) for molecular sieve chromatography ^TM Core 700), the sample loading amount is 1.0-6.0 column volume;

before loading the enzymolysis liquid, carrying out protective filtration on a chromatographic column (PES (glass fiber material, with a pore diameter of 0.8 mu m +0.45 mu m)), wherein the pressure is ensured to be less than 1.5bar in the filtration process, otherwise, a new filter is replaced; 3CV (clarified virus harvest volume) was treated with 0.05M PBS forward linear flow 100cm/h, 1.0M NaOH forward linear flow 100cm/h contact for 15min; 3CV (clarified virus harvest volume) was equilibrated with a forward linear flow rate of 100cm/h using 0.05M PBS; the linear flow rate of sample loading is 100cm/h, the retention time is 5min, and the sample loading amount is 1.0-6.0 column volume; eluting 5CV (clarified virus harvest volume) with 0.01M PBS (forward linear flow rate of H1N1 100cm/H, H3N2 100cm/H, bv 200cm/H, by 200cm/H, respectively), and staying for 5min; elution fraction collection range: collection was initiated when the elution peak (UV 280) reading rose to 140mAU and was terminated when the elution peak (UV 280) reading fell to 140 mAU. Post-treating the collected liquid and uniformly mixing the liquid; 0.05M PBS pH7.4 forward linear flow rate 100cm/h wash 5CV (clarified virus harvest volume) regeneration; 0.1M NaOH 200cm/h 5CV (clarified virus harvest volume) disinfection treatment.

S10、Capto ^TM Core Q ion exchange chromatography; selecting ion exchange chromatography packing (Capto) ^TM Core Q), the loading amount is 5-10% of the column volume; washing 5CV (clarified virus harvest volume) with WFI (water for injection) reverse linear flow rate of 200 cm/h; 1MNaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) sterilization; the sample loading amount is 5-10% of the column volume, and the forward linear flow rate is 5mL/min; an elution step after sample loading, namely pumping a washing solution A (PB) and a washing solution B (5M NaCL) according to a ratio, and performing gradient elution on the NaCL concentration at 0.1M; collecting elution peak components: starting to collect when the reading of an elution peak (namely UV 280) rises to 100mAU, stopping collecting when the reading of the elution peak (namely UV 280) falls to 100mAU, and uniformly mixing the feed liquid after eluting the collection liquid; washing 5CV (clarified virus harvest volume) regeneration by WFI (water for injection) reverse linear flow rate of 200 cm/h; 0.1M NaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) preservation.

S11, virus cracking; the purified solution was added to 1% Triton-100 and magnetically stirred at 4 ℃ for 12 hours to lyse the virus.

S12, performing ultrafiltration dialysis on glass fibers; the lysate was ultrafiltered/dialyzed against a 100kD ultrafiltration membrane. Dialyzing and replacing the mixture by 10 times of volume of 0.01M PBS buffer; washing the membrane module with WFI (water for injection), wherein the washing liquid is not less than 40L/m ² (ii) a 0.5M NaOH test Membrane NWP (Water flux L/M) ² H); washing the membrane package with WFI (water for injection) with a washing liquid of not less than 30L/m ² (ii) a Compressed air is used for testing the integrity of the membrane package; the inlet pressure is less than or equal to 1.8bar; eluting the collected liquid with a mixed mode with the dialysis concentration less than or equal to 10 mg/mL; the inlet pressure is less than or equal to 1.8bar; dialyzing 6-8 times of concentrated feed liquid; controlling the top washing volume according to the concentration of the final collection liquid of 5.56mg/mL before adding the arginine mother liquor, wherein 50mM Acetate (Acetate) has a pH value of 4.6; according to the feed liquid: arginine stock solution was added at a volume ratio of 9; washing the membrane module with WFI (water for injection), wherein the washing liquid is more than or equal to 30L/m ² (ii) a 0.5M NaOH film-coated preservation, and replacing the preservation solution once after online preservation for 2 months; the off-line preservation can be carried out by replacing the preservation solution (0.1M NaOH))。

S13, sterilizing and filtering; filtering the ultrafiltered dialysate with 0.22 μm sterilizing filter to obtain disposable sterile stock solution, and storing at 2-8 deg.C.

Example 2

A preparation method of a monovalent stock solution of a low MDCK cell residual DNA influenza vaccine comprises the following steps:

s1, cell recovery: taking the seed cells of the working bank from a liquid nitrogen tank, putting the seed cells into warm water at 37 ℃, melting the seed cells, aseptically transferring the seed cells into a square bottle filled with growth liquid, and culturing the seed cells in an incubator at the temperature of 36 ℃;

s2, cell passage: observing under a microscope, after the revived cells grow to be full of a monolayer, digesting with 0.25% trypsin,

adherent cells were shed as follows 1:3, and culturing at the constant temperature of 36 ℃ for 3 days;

s3, expanding and passaging WAVE cells; 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) is transferred into a WAVE culture bag, and a culture medium preservation test is required before inoculation in order to control risks in the process; the culture volume is calculated and expanded to 20L with the dilution density of 0.3X 10 ⁶ cells/mL of the desired seed suspension. The cell suspension in 5L flasks was transferred by calculated weight to 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) temperature in Wave of the medium 36.5 ℃ at 18rpm at an angle of 8 DEG, air flow rate Day 0-Day 1:0.3L/min Day 2-Day end:0.6L/min, CO ₂ 5% culture for 3 days; if the culture volume after inoculation is less than 20L, the total volume needs to be supplemented to 20L by a feed medium;

s4, bioreactor culture, namely digesting the cells by using 0.25% trypsin, and transferring the cells to a bioreactor for culture. The bioreactor is pre-loaded with polyester chip carrier, 30g per liter of culture volume, and cell inoculation density of 2 × 10 ⁵ cells/mL. Setting culture parameters: temperature 36 ℃, DO30, pH 7.0. Culturing until the cell density reaches 1 × 10 ⁷ cells/mL or more;

s5, inoculating virus: discarding culture solution in the bioreactor, adding replacement solution, adding influenza virus and virus seeds (H3N 2) according to the infection amount of 0.001-0.1 MOI, and setting parameters: the culture was carried out at 33 ℃, DO30, pH 7.3. After 24 hours, the cells were replaced with 0.01M phosphate buffer, and the medium was added after repeating 2 times to continue the culture. Setting parameters: the temperature is 33 ℃, DO is 30, and the pH value is 7.3; the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 part of potassium chloride, 3 parts of disodium hydrogen phosphate dodecahydrate, 0.2 part of dipotassium hydrogen phosphate and 800 parts of water for injection.

S6, harvesting viruses: after the inoculation of influenza virus, virus liquid is obtained at the culture temperature of 33.0 ℃, pH7.25 (Deadband 0.20) and DO40 percent for 48 to 72 hours.

S7, deep filtration and inactivation: immediately filtering the harvested virus liquid by using a glass fiber filter element (with the aperture of 0.6 mu m) to remove cell debris; a deep filter with a deep filtration membrane is adopted, the liquid inlet pressure is less than or equal to 0.2MPa, the liquid is filtered to a stainless steel tank A # with the temperature of 200L 2-8 ℃, and the turbidity of the clear filtrate is less than 20NTU. Cooling the virus clarified liquid to 2-8 ℃ in a 200L inactivation tank A #, and mixing the virus clarified liquid with beta-propiolactone (ml): adding a diluted virus inactivating agent into the inactivated virus liquid according to the weight (g) of 1; after 12 hours of inactivation, the cells were transferred via transfer line to a 1000L inactivation tank B # for 12 hours of continued inactivation. The inactivation temperature is 2-8 ℃, and the stirring revolution is 60-100 rpm;

and inactivating the virus clarified liquid for 24 hours, transferring the virus clarified liquid into a hydrolysis/tank to be detected through a material transferring pipeline, hydrolyzing for 2 hours at 37-38 ℃, sampling, carrying out a virus inactivation verification test, and cooling to 2-8 ℃ to be detected for 10-11 days.

S8, ultrafiltration concentration and nuclease digestion: the virus inactivation solution is ultrafiltered and concentrated to 5-6 kg by a 1000kD PES ultrafiltration membrane, the ultrafiltration membrane is washed by PBS (pH7.4) buffer solution to about 10kg by top washing until the volume is 10 times that of the ultrafiltration membrane, and the ultrafiltration membrane is dialyzed and replaced by 0.01MPBS buffer solution, and then the ultrafiltration membrane is transferred to a 20L enzymolysis tank. Adding a proper amount of 1mol/L magnesium chloride solution and non-restriction endonuclease into the concentrated solution to ensure that the final concentration of the magnesium chloride is 0.2mmol/L and the final concentration of the non-restriction endonuclease is 90 +/-10U/ml; stirring uniformly, keeping the temperature at 25 +/-1 ℃ for at least 120 minutes to obtain the enzymatic hydrolysate.

S9、Capto ^TM Performing Core 700 mixed gel molecular sieve chromatography; molecular sieve chromatographic separationChromatography of the packing Material with molecular sieves (Capto) ^TM Core 700), the sample loading amount is 1.0-6.0 column volume;

before loading the enzymolysis liquid, carrying out protective filtration on a chromatographic column (PES (glass fiber material, with a pore diameter of 0.8 mu m +0.45 mu m)), wherein the pressure is ensured to be less than 1.5bar in the filtration process, otherwise, a new filter is replaced; 3CV (clarified virus harvest volume) was treated with 0.05M PBS forward linear flow 100cm/h, 1.0M NaOH forward linear flow 100cm/h contact for 15min; 3CV (clarified virus harvest volume) was equilibrated with a forward linear flow rate of 100cm/h using 0.05M PBS; the linear flow velocity of the sample loading is 100cm/h, the retention time is 5min, and the sample loading amount is 1.0-6.0 column volume; eluting 5CV (clarified virus harvest volume) with 0.01M PBS (forward linear flow rate of H1N1 100cm/H, H3N2 100cm/H, bv 200cm/H, by 200cm/H, respectively), and staying for 5min; elution component collection range: collection was initiated when the elution peak (UV 280) reading rose to 140mAU and was terminated when the elution peak (UV 280) reading fell to 140 mAU. Post-treating the collected liquid and uniformly mixing the liquid; 0.05M PBS pH7.4 forward linear flow rate 100cm/h washing 5CV (clarified virus harvest volume) regeneration; 0.1M NaOH 200cm/h 5CV (clarified virus harvest volume) disinfection treatment.

S10、Capto ^TM Core Q ion exchange chromatography; selecting ion exchange chromatography filler (Capto) ^TM Core Q), the sample loading amount is 5-10% of the column volume; 5CV (clarified virus harvest volume) was flushed with WFI (water for injection) reverse linear flow rate 200 cm/h; 1MNaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) sterilization; the sample loading amount is 5-10% of the column volume, and the forward linear flow rate is 5mL/min; an elution step after sample loading, namely pumping a washing solution A (PB) and a washing solution B (5M NaCL) according to a ratio, and performing gradient elution on the NaCL concentration at 0.1M; collecting elution peak components: starting to collect when the reading of an elution peak (namely UV 280) rises to 100mAU, stopping collecting when the reading of the elution peak (namely UV 280) falls to 100mAU, and uniformly mixing the feed liquid after eluting the collected liquid; 5CV (clarified virus harvest volume) regeneration by washing with WFI (water for injection) reverse linear flow rate 200 cm/h; 0.1M NaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) preservation.

S11, virus cracking; the purified solution was added to 1% Triton-100 and magnetically stirred at 4 ℃ for 12 hours to lyse the virus.

S12, performing ultrafiltration and dialysis on glass fibers; the lysate was ultrafiltered/dialyzed against a 100kD ultrafiltration membrane. Dialyzing and replacing the mixture by 10 times of volume of 0.01M PBS buffer; washing the membrane module with WFI (water for injection), wherein the washing liquid is not less than 40L/m ² (ii) a 0.5M NaOH test Membrane pack NWP (Water flux L/M) ² H); washing the membrane with WFI (water for injection) with a washing liquid of 30L/m or more ² (ii) a Compressed air is used for testing the integrity of the membrane package; the inlet pressure is less than or equal to 1.8bar; eluting the collected liquid with a mixed mode with the dialysis concentration less than or equal to 10 mg/mL; the inlet pressure is less than or equal to 1.8bar; dialyzing 6-8 times of concentrated feed liquid; controlling the volume of the top washing according to the concentration of the final collection liquid of 5.56mg/mL before adding the arginine mother liquor, wherein 50mM Acetate (Acetate) is pH4.6; according to the feed liquid: arginine stock solution was added at a volume ratio of 9; washing the membrane module with WFI (water for injection), wherein the washing liquid is more than or equal to 30L/m ² (ii) a 0.5M NaOH film is coated for storage, and the storage solution is replaced once after online storage for 2 months; the off-line storage can be changed by 6 months.

S13, sterilizing and filtering; filtering the ultrafiltered dialysate with 0.22 μm sterilizing filter to obtain disposable sterile stock solution, and storing at 2-8 deg.C.

Example 3

A preparation method of a monovalent stock solution of a low MDCK cell residual DNA influenza vaccine comprises the following steps:

s1, cell recovery: taking the seed cells of the working bank from a liquid nitrogen tank, putting the seed cells into warm water at 37 ℃, melting the seed cells, aseptically transferring the seed cells into a square bottle filled with growth liquid, and culturing the seed cells in an incubator at the temperature of 36 ℃;

s2, cell passage: observing under a microscope, after the revived cells grow to be full of a monolayer, digesting with 0.25% trypsin,

adherent cells were shed as follows 1:3, and culturing at the constant temperature of 36 ℃ for 3 days;

s3, expanding and passaging WAVE cells; 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) transferred into WAVE culture bagsControlling risks in the process, and performing a culture medium preservation test before inoculation; the culture volume is calculated and expanded to 20L with the dilution density of 0.3X 10 ⁶ cells/mL of the desired seed suspension. The cell suspension in 5L flasks was transferred by calculated weight to 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) temperature in Wave of the medium 36.5 ℃ at 18rpm at an angle of 8 DEG, air flow rate Day 0-Day 1:0.3L/min Day 2-Day end:0.6L/min, CO ₂ %5% culture for 3 days; if the culture volume after inoculation is less than 20L, the total volume needs to be supplemented to 20L by a feed medium;

s4, bioreactor culture, namely digesting the cells by using 0.25% trypsin, and transferring the cells to a bioreactor for culture. The bioreactor was pre-loaded with polyester chip carriers, 30g per liter of culture volume and cell seeding density of 2X 105cells/mL. Setting culture parameters: temperature 36 ℃, DO30, pH 7.0. Culturing until the cell density reaches more than 1 × 107 cells/mL;

s5, virus inoculation: discarding culture solution in a bioreactor, adding replacement solution, adding influenza virus and virus seeds (BV) according to the infection amount of 0.001-0.1 MOI, and setting parameters as follows: the temperature was 33 ℃, DO30, pH 7.3. After 24 hours, the cells were replaced with 0.01M phosphate buffer, and the medium was added after repeating 2 times to continue the culture. Setting parameters: the temperature is 33 ℃, DO is 30, and the pH value is 7.3; the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 part of potassium chloride, 3 parts of disodium hydrogen phosphate dodecahydrate, 0.2 part of dipotassium hydrogen phosphate and 800 parts of water for injection.

S6, harvesting viruses: after inoculation of influenza virus, virus solution was harvested at 33.0 ℃ at pH7.25 (DeadBand 0.20) and DO40% for 48-72 hours.

S7, deep filtration and inactivation: immediately filtering the harvested virus liquid by using a glass fiber filter element (with the aperture of 0.6 mu m) to remove cell debris; a deep filter with a deep filtration membrane is adopted, the liquid inlet pressure is less than or equal to 0.2MPa, the liquid is filtered to a stainless steel tank A # with the temperature of 200L 2-8 ℃, and the turbidity of the clear filtrate is less than 20NTU. Cooling the virus clarified liquid to 2-8 ℃ in a 200L inactivation tank A #, and mixing the virus clarified liquid with beta-propiolactone (ml): adding a diluted virus inactivating agent into the inactivated virus liquid in a weight (g) of 1; after 12 hours of inactivation, the cells were transferred via transfer line to a 1000L inactivation tank B # for 12 hours of continued inactivation. The inactivation temperature is 2-8 ℃, and the stirring revolution is 60-100 rpm;

and inactivating the virus clarified liquid for 24 hours, transferring the virus clarified liquid into a hydrolysis/tank to be detected through a material transferring pipeline, hydrolyzing for 2 hours at 37-38 ℃, sampling, carrying out a virus inactivation verification test, and cooling to 2-8 ℃ to be detected for 10-11 days.

S8, ultrafiltration concentration and nuclease digestion: the virus inactivation solution is ultrafiltered and concentrated to 5-6 kg by a 1000kD PES ultrafiltration membrane package, the ultrafiltration membrane package is washed by PBS (pH7.4) buffer solution to about 10kg, the volume of the buffer solution is 10 times that of the buffer solution, and the buffer solution is dialyzed and replaced by 0.01MPBS buffer solution and transferred to a 20L enzymolysis tank. Adding a proper amount of 1mol/L magnesium chloride solution and non-restriction endonuclease into the concentrated solution to ensure that the final concentration of the magnesium chloride is 0.2mmol/L and the final concentration of the non-restriction endonuclease is 90 +/-10U/ml; stirring uniformly, keeping the temperature at 25 +/-1 ℃ for at least 120 minutes to obtain the enzymatic hydrolysate.

S9、Capto ^TM Performing Core 700 mixed gel molecular sieve chromatography; molecular sieve chromatography filler (Capto) for molecular sieve chromatography ^TM Core 700), the sample loading amount is 1.0-6.0 column volume;

before loading the enzymolysis liquid, carrying out protective filtration on a chromatographic column (PES (glass fiber material, with a pore diameter of 0.8 mu m +0.45 mu m)), wherein the pressure is ensured to be less than 1.5bar in the filtration process, otherwise, a new filter is replaced; 3CV (clarified virus harvest volume) was treated with 0.05M PBS forward linear flow 100cm/h, 1.0M NaOH forward linear flow 100cm/h contact for 15min; 3CV (clarified virus harvest volume) was equilibrated with a forward linear flow rate of 100cm/h using 0.05M PBS; the linear flow velocity of the sample loading is 100cm/h, the retention time is 5min, and the sample loading amount is 1.0-6.0 column volume; eluting 5CV (clarified virus harvest volume) with 0.01M PBS (forward linear flow rate of H1N1 100cm/H, H3N2 100cm/H, bv 200cm/H, by 200cm/H, respectively), and staying for 5min; elution fraction collection range: collection was initiated when the elution peak (UV 280) reading rose to 140mAU and was terminated when the elution peak (UV 280) reading fell to 140 mAU. Post-treating the collected liquid and uniformly mixing the liquid; 0.05M PBS pH7.4 forward linear flow rate 100cm/h wash 5CV (clarified virus harvest volume) regeneration; 0.1M NaOH 200cm/h 5CV (clarified virus harvest volume) disinfection treatment.

S10、Capto ^TM Core Q ion exchange chromatography; selecting ion exchange chromatography packing (Capto) ^TM Core Q), the sample loading amount is 5-10% of the column volume; 5CV (clarified virus harvest volume) was flushed with WFI (water for injection) reverse linear flow rate 200 cm/h; 1MNaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) sterilization; the sample loading amount is 5-10% of the column volume, and the forward linear flow rate is 5mL/min; an elution step after sample loading, namely pumping a washing solution A (PB) and a washing solution B (5M NaCL) according to a ratio, and performing gradient elution on the NaCL concentration at 0.1M; collecting elution peak components: starting to collect when the reading of an elution peak (namely UV 280) rises to 100mAU, stopping collecting when the reading of the elution peak (namely UV 280) falls to 100mAU, and uniformly mixing the feed liquid after eluting the collected liquid; washing 5CV (clarified virus harvest volume) regeneration by WFI (water for injection) reverse linear flow rate of 200 cm/h; 0.1M NaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) preservation.

S11, virus cracking; the purified solution was added to 1% Triton-100 and magnetically stirred at 4 ℃ for 12 hours to lyse the virus.

S12, performing ultrafiltration dialysis on glass fibers; the lysate was ultrafiltered/dialyzed against a 100kD ultrafiltration membrane. Dialyzing and replacing the mixture by 10 times of volume of 0.01M PBS buffer; washing the membrane module with WFI (water for injection), wherein the washing liquid is not less than 40L/m ² (ii) a 0.5M NaOH test Membrane pack NWP (Water flux L/M) ² H); washing the membrane with WFI (water for injection) with a washing liquid of 30L/m or more ² (ii) a Compressed air is used for testing the integrity of the membrane package; the inlet pressure is less than or equal to 1.8bar; eluting the collected liquid with a mixed mode with the dialysis concentration less than or equal to 10 mg/mL; the inlet pressure is less than or equal to 1.8bar; dialyzing 6-8 times of concentrated feed liquid; controlling the volume of the top washing according to the concentration of the final collection liquid of 5.56mg/mL before adding the arginine mother liquor, wherein 50mM Acetate (Acetate) is pH4.6; according to the feed liquid: arginine stock solution was added at a volume ratio of 9; washing the membrane module with WFI (water for injection), wherein the washing liquid is more than or equal to 30L/m ² (ii) a 0.5M NaOH film is coated for storage, and the storage solution is replaced once after online storage for 2 months; the off-line storage can be changed by 6 months.

S13, sterilizing and filtering; filtering the ultrafiltered dialysate with 0.22 μm sterilizing filter to obtain disposable sterile stock solution, and storing at 2-8 deg.C.

Example 4

A preparation method of a monovalent stock solution of a low MDCK cell residual DNA influenza vaccine comprises the following steps:

s1, cell recovery: taking the seed cells of the working bank from a liquid nitrogen tank, putting the seed cells into warm water at 37 ℃, melting the seed cells, aseptically transferring the seed cells into a square bottle filled with growth liquid, and culturing the seed cells in an incubator at the temperature of 36 ℃;

s2, cell passage: observing under a microscope, after the revived cells grow to be full of a monolayer, digesting with 0.25% trypsin,

adherent cells were exfoliated as per 1:3, and culturing at the constant temperature of 36 ℃ for 3 days;

s3, expanding and passaging WAVE cells; 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) is transferred into a WAVE culture bag, and a culture medium preservation test is required before inoculation in order to control risks in the process; the culture volume is calculated and expanded to 20L with the dilution density of 0.3X 10 ⁶ cells/mL of the desired seed suspension. The cell suspension in 5L flasks was transferred by calculated weight to 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2 g/L) temperature in Wave of the medium 36.5 ℃ at 18rpm at an angle of 8 DEG, air flow rate Day 0-Day 1:0.3L/min Day 2-Day end:0.6L/min, CO ₂ %5% culture for 3 days; if the culture volume after inoculation is less than 20L, the total volume needs to be supplemented to 20L by a feed medium;

s4, bioreactor culture, namely digesting the cells by using 0.25% trypsin, and transferring the cells to a bioreactor for culture. The bioreactor was pre-loaded with polyester chip carriers, 30g per liter of culture volume and cell seeding density of 2X 105cells/mL. Setting culture parameters: temperature 36 ℃, DO30, pH 7.0. Culturing until the cell density reaches more than 1 × 107 cells/mL;

s5, virus inoculation: removing culture solution in the bioreactor, adding replacement solution, adding influenza virus and virus seeds (BY) according to the infection amount of 0.001-0.1 MOI, and setting parameters as follows: the culture was carried out at 33 ℃, DO30, pH 7.3. After 24 hours, the cells were replaced with 0.01M phosphate buffer, and the medium was added after repeating 2 times to continue the culture. Setting parameters: the temperature is 33 ℃, DO is 30, and the pH value is 7.3; the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 parts of sodium chloride, 0.2 part of potassium chloride, 3 parts of disodium hydrogen phosphate dodecahydrate, 0.2 part of dipotassium hydrogen phosphate and 800 parts of water for injection.

S6, harvesting viruses: after the inoculation of influenza virus, virus liquid is obtained at the culture temperature of 33.0 ℃, pH7.25 (Deadband 0.20) and DO40 percent for 48 to 72 hours.

S7, deep filtration and inactivation: immediately filtering the harvested virus liquid by using a glass fiber filter element (with the aperture of 0.6 mu m) to remove cell debris; a deep filter with a deep filtration membrane is adopted, the liquid inlet pressure is less than or equal to 0.2MPa, the liquid is filtered to a stainless steel tank A # with the temperature of 200L 2-8 ℃, and the turbidity of the clear filtrate is less than 20NTU. Cooling the virus clarified liquid to 2-8 ℃ in a 200L inactivation tank A #, and mixing the virus clarified liquid with the beta-propiolactone according to the volume (ml): adding a diluted virus inactivating agent into the inactivated virus liquid in a weight (g) of 1; after 12 hours of inactivation, the cells were transferred via transfer line to a 1000L inactivation tank B # for 12 hours of continuous inactivation. The inactivation temperature is 2-8 ℃, and the stirring revolution is 60-100 rpm;

and inactivating the virus clarified liquid for 24 hours, transferring the virus clarified liquid into a hydrolysis/tank to be detected through a material transferring pipeline, hydrolyzing for 2 hours at 37-38 ℃, sampling, carrying out a virus inactivation verification test, and cooling to 2-8 ℃ to be detected for 10-11 days.

S8, ultrafiltration concentration and nuclease digestion: the virus inactivation solution is ultrafiltered and concentrated to 5-6 kg by a 1000kD PES ultrafiltration membrane package, the ultrafiltration membrane package is washed by PBS (pH7.4) buffer solution to about 10kg, the volume of the buffer solution is 10 times that of the buffer solution, and the buffer solution is dialyzed and replaced by 0.01MPBS buffer solution and transferred to a 20L enzymolysis tank. Adding a proper amount of 1mol/L magnesium chloride solution and non-restriction endonuclease into the concentrated solution to ensure that the final concentration of the magnesium chloride is 0.2mmol/L and the final concentration of the non-restriction endonuclease is 90 +/-10U/ml; stirring uniformly, keeping the temperature at 25 +/-1 ℃ for at least 120 minutes to obtain the enzymatic hydrolysate.

S9、Capto ^TM Performing Core 700 mixed gel molecular sieve chromatography; molecular sieve chromatography filler (Capto) for molecular sieve chromatography ^TM Core 700), the sample loading amount is 1.0-6.0 column volume;

before loading the enzymolysis liquid, carrying out protective filtration on a chromatographic column (PES (glass fiber material, with a pore diameter of 0.8 mu m +0.45 mu m)), wherein the pressure is ensured to be less than 1.5bar in the filtration process, otherwise, a new filter is replaced; 3CV (clarified virus harvest volume) was treated with 0.05M PBS forward linear flow 100cm/h, 1.0M NaOH forward linear flow 100cm/h contact for 15min; 3CV (clarified virus harvest volume) was equilibrated using a forward linear flow rate of 100cm/h in 0.05M PBS; the linear flow velocity of the sample loading is 100cm/h, the retention time is 5min, and the sample loading amount is 1.0-6.0 column volume; eluting 5CV (clarified virus harvest volume) with 0.01M PBS (forward linear flow rate of H1N1 100cm/H, H3N2 100cm/H, bv 200cm/H, by 200cm/H, respectively), and staying for 5min; elution fraction collection range: collection was initiated when the elution peak (UV 280) reading rose to 140mAU and was terminated when the elution peak (UV 280) reading fell to 140 mAU. Post-treating the collected liquid and uniformly mixing the liquid; 0.05M PBS pH7.4 forward linear flow rate 100cm/h washing 5CV (clarified virus harvest volume) regeneration; 0.1M NaOH 200cm/h 5CV (clarified virus harvest volume) disinfection treatment.

S10、Capto ^TM Core Q ion exchange chromatography; selecting ion exchange chromatography packing (Capto) ^TM Core Q), the sample loading amount is 5-10% of the column volume; washing 5CV (clarified virus harvest volume) with WFI (water for injection) reverse linear flow rate of 200 cm/h; 1MNaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) sterilization; the sample loading amount is 5-10% of the column volume, and the forward linear flow rate is 5mL/min; an elution step after sample loading, namely pumping a washing solution A (PB) and a washing solution B (5M NaCL) according to a ratio, and performing gradient elution on the NaCL concentration at 0.1M; collecting elution peak components: starting to collect when the reading of an elution peak (namely UV 280) rises to 100mAU, stopping collecting when the reading of the elution peak (namely UV 280) falls to 100mAU, and uniformly mixing the feed liquid after eluting the collected liquid; washing 5CV (clarified virus harvest volume) regeneration by WFI (water for injection) reverse linear flow rate of 200 cm/h; 0.1M NaOH reverse linear flow rate 100cm/h3CV (clarified virus harvest volume) preservation.

S11, virus cracking; the purified solution was added to 1% Triton-100 and magnetically stirred at 4 ℃ for 12 hours to lyse the virus.

S12, performing ultrafiltration dialysis on glass fibers; the lysate was ultrafiltered/dialyzed against a 100kD ultrafiltration membrane. Dialyzing and replacing the mixture by 10 times of volume of 0.01M PBS buffer; washing the membrane module with WFI (water for injection), wherein the washing liquid is not less than 40L/m ² (ii) a 0.5M NaOH test Membrane pack NWP (Water flux L/M) ² H); washing the membrane with WFI (water for injection) with a washing liquid of 30L/m or more ² (ii) a Compressed air is used for testing the integrity of the membrane package; the inlet pressure is less than or equal to 1.8bar; eluting the collected liquid with a mixed mode with the dialysis concentration less than or equal to 10 mg/mL; the inlet pressure is less than or equal to 1.8bar; dialyzing 6-8 times of concentrated feed liquid; controlling the volume of the top washing according to the concentration of the final collection liquid of 5.56mg/mL before adding the arginine mother liquor, wherein 50mM Acetate (Acetate) is pH4.6; according to the feed liquid: arginine stock solution was added at a volume ratio of 9; washing the membrane module with WFI (water for injection), wherein the washing liquid is more than or equal to 30L/m ² (ii) a 0.5M NaOH film-coated preservation, and replacing the preservation solution once after online preservation for 2 months; the off-line storage can be changed by 6 months.

S13, sterilizing and filtering; filtering the ultrafiltered dialysate with 0.22 μm sterilizing filter to obtain disposable sterile stock solution, and storing at 2-8 deg.C.

Example 5

This example differs from example 1 in that: in the virus harvesting step, a glass fiber filter core is not adopted for filtering, and other conditions are kept unchanged.

Example 6

The present example differs from example 1 in that: not using Capto ^TM The Core 700 film was chromatographed, the remaining conditions remaining unchanged.

Example 7

Compared with embodiment 2, the present embodiment is different in that: in the virus harvesting step, a glass fiber filter core is not adopted for filtering, and other conditions are kept unchanged.

Example 8

This example differs from example 2 in that: not using Capto ^TM Core 700 film chromatography, remainderThe conditions remained unchanged.

Example 9

This example differs from example 3 in that: in the virus harvesting step, a glass fiber filter core is not adopted for filtering, and other conditions are kept unchanged.

Example 10

This example differs from example 3 in that: not using Capto ^TM The Core 700 film was chromatographed, the remaining conditions remaining unchanged.

Example 11

This example differs from example 4 in that: in the virus harvesting step, a glass fiber filter core is not adopted for filtration, and other conditions are kept unchanged.

Example 12

This example differs from example 3 in that: not using Capto ^TM The Core 700 film was chromatographed, the remaining conditions remaining unchanged.

The antigen and DNA levels during the procedures of examples 1-12 were tested and the specific test results are shown in Table 1:

table 1: examples 1 to 12 test data

The test results in the table show that the technical scheme adopted by the application can effectively solve the problem of removal of residual MDCK cell DNA in the MDCK cell influenza vaccine preparation process, and the final DNA content of the product is obviously increased and the product purity is obviously adversely affected after the key technical process described by the application is lost.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A preparation method of a low MDCK cell residual DNA influenza vaccine stock solution is characterized by comprising the following steps:

s1, cell recovery;

s2, cell passage;

s3, culturing in a Wave reactor;

s4, culturing in a bioreactor;

s5, inoculating viruses;

s6, harvesting viruses;

s7, deep filtration and inactivation, wherein after the virus is harvested, a glass fiber filter core is adopted for filtration; the aperture of the glass fiber filter element is 0.6-1.0 μm;

s8, performing ultrafiltration concentration and nuclease digestion;

S9、Capto ^TM performing chromatography by using a Core 700 mixed gel molecular sieve;

S10、Capto ^TM core Q ion exchange chromatography;

s11, virus cracking;

s12, performing ultrafiltration dialysis on glass fibers;

and S13, sterilizing and filtering.

2. The method of claim 1, wherein the Capto in S9 is a Capto ^TM The flow rate of the Core 700 mixed gel molecular sieve during chromatography is 100-200 cm/h.

3. The method for preparing a low MDCK cell residual DNA influenza vaccine stock solution of claim 1, wherein Capto is used in S10 ^TM Core Q ion exchange chromatography, the loading amount is 5-10% of the column volume, and the linear flow rate is 5mL/min.

4. The method for preparing a stock solution of a vaccine against DNA influenza with low MDCK cell residual according to claim 1, wherein the step S8 is performed by washing with a buffer solution after dialysis with an ultrafiltration membrane.

5. The method for preparing a stock solution of a low MDCK cell residual DNA influenza vaccine of claim 4, wherein in S8, the buffer is a phosphate buffer; the phosphate buffer solution is prepared from the following raw materials in parts by weight: 8 to 10 portions of sodium chloride, 0.2 to 0.4 portion of potassium chloride, 3 to 5 portions of disodium hydrogen phosphate dodecahydrate, 0.2 to 0.4 portion of dipotassium hydrogen phosphate and 800 to 1000 portions of water.

6. The method for preparing a low MDCK cell residual DNA influenza vaccine stock solution of claim 1, wherein the medium in S3 consists of: 18L Xeno-S001S (27.21 g/L) +5M NaOH (1.25 mL/L) + NaHCO ₃ (2g/L)。

7. The method for preparing a stock solution of a low MDCK cell residual DNA influenza vaccine of claim 1, wherein the virus is lysed by adding 1% Triton-100 to the purified solution in S11 under magnetic stirring at 4 ℃ for 12 hours.

8. The method for preparing a stock solution of a low MDCK cell residual DNA influenza vaccine according to claim 1, wherein the ultrafiltration dialysate is filtered through a 0.22 μm sterilizing filter into a disposable sterile stock solution of influenza vaccine monovalent stock solution at 2-8 ℃ in S13.

9. The method for preparing a stock solution of a vaccine against low MDCK cell residual DNA influenza according to claim 1, wherein the virus solution is harvested at a culture temperature of 33.0 ℃, pH7.25 (Deadband 0.20), DO40%, and 48 to 72 hours after the inoculation of the influenza virus in S6.

10. The method for preparing a stock solution of a DNA influenza vaccine with low MDCK cell residue according to claim 1, wherein in S12, the lysate is ultrafiltered/dialyzed using a 100kD ultrafiltration membrane module, and is dialyzed and replaced with 10 volumes of 0.01M PBS buffer; the membrane-covered component is washed by injection water, and the washing liquid is more than or equal to 40L/m ² 。