CN106497891B - A kind of production method of influenza virus vaccine - Google Patents

A kind of production method of influenza virus vaccine Download PDF

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CN106497891B
CN106497891B CN201710001455.9A CN201710001455A CN106497891B CN 106497891 B CN106497891 B CN 106497891B CN 201710001455 A CN201710001455 A CN 201710001455A CN 106497891 B CN106497891 B CN 106497891B
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influenza virus
cell
chloride
density
acid
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CN106497891A (en
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刘文友
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Shanghai Qingsai Biotechnology Co ltd
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Abstract

The invention discloses a kind of application of chalcone in vero cell production influenza virus, inventors have surprisingly discovered that the substance is applied in the serum free medium of Vero cell, the addition of expensive growth hormone can be reduced, and it is easy to reach high density, virus titer is high simultaneously, peplos are complete, and product quality is excellent and low production cost.The highest potency of technique influenza virus of the invention reaches 1:1280.

Description

A kind of production method of influenza virus vaccine
Technical field
The present invention relates to a kind of production methods of influenza virus vaccine, belong to biological field, especially field of cell culture.
Background technique
The transformed cells that Vero cell was obtained in 1962 from normal adult African green monkey kidney cell.The cell is adherent The fibroblast of dependence.It can support the diseases such as the proliferation, including encephalitis B, polio, rabies of a variety of viruses Poison has been allowed for producing people's viral vaccine.Vero cell be the World Health Organization approval be most commonly used for viral epidemic disease Seedling production continuous cell line, early in the 80's of 20th century, the Montagnon of Mei Liai research institute (Lyons, France) and It is worked together just is used for Vero cell the production of vaccine for man IPV for the first time.Then it just be used to inactivate rabies vaccine, The production of oral work polio vaccine has more than 30 years history so far.Recent years is using Vero cell as matrix Production of vaccine tending to become strong of active day.
The serum free medium of Vero cell has had more research, but all there is high expensive, needs to add The shortcomings that more valuableness growth hormone, its density is not high after amplification culture.Chinese patent 201210313363.1 is disclosed a kind of suitable For the culture medium and method of Vero cell microcarrier suspension culture, cell culture density is up to 6.5 × 106 Cell Number/milliliter, but the culture medium still has complicated component, disadvantage at high cost, cell culture density also has further improvement Demand.
Neohesperidin dihydrochalcone (abbreviation NHDC) is that the neohesperidin extracted from citrus natural plants passes through hydrogen Flavone derivative made of change is a kind of functional sweetener with debitter and flavor improvement.Products characteristics: 1, sweet tea Degree is high, and heat is small.It is characterized in that sugariness is big (1500-1800 times of sweetness of cane sugar), clean taste, pleasant impression is lasting, and has The effect of splendid shielding bitter taste.Neohesperidin dihydrochalcone appearance is off-white color to yellowish crystalline powder, odorless.Sweet tea Degree is about 1500~1800 times of sucrose, the aobvious sweet tea time be slightly later than saccharin sodium and far faster than glycyrrhizin (5:7:23s), three Stay the sweet tea time be 42:57:133s, also fall between, therefore be relatively close to saccharin sodium.It is dissolved in water, is slightly soluble in ethyl alcohol, it is insoluble In ether and benzene.The structural formula of neohesperidin dihydrochalcone are as follows:
Neohesperidin dihydrochalcone (abbreviation NHDC) is low in cost, inventors have surprisingly discovered that the substance is in Vero cell Serum free medium in produce influenza virus vaccine, it is possible to reduce the addition of expensive growth hormone, specifically, being not required to Recombinant human epidermal growth factor is added, and is easy to reach high density, while virus titer is high, peplos are complete, product Excellent quality and low production cost.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention It is to propose a kind of application of chalcone in vero cell production influenza virus, inventors have surprisingly discovered that the substance is in Vero It is applied in the serum free medium of cell, it is possible to reduce the addition of expensive growth hormone, and be easy to reach high density, while disease Toxic effect valence is high, and peplos are complete, and product quality is excellent and low production cost.
A kind of production method of influenza virus vaccine, its step are as follows:
(1) culture medium is prepared:
Following content is calculated as with mg/litre:
Neohesperidin dihydrochalcone 0.8-1.5
L-Trp 10
L-cysteine 100
L-Isoleucine 225
L-Leu 80-100
Valine 100
L-arginine hydrochloride 650-800
L-lysine 320-350
L-Methionine 60
Pidolidone 50-82
L-phenylalanine 216
Altheine 800-1000
Glycine 15
L-PROLINE 180
L-Alanine 55
L-Aspartic acid 65-68
Serine 290
L-threonine 50~300
L-Glutamine 650-690
Vitamin C 60-99
Niacinamide 8
Riboflavin 0.6
Thiamine hydrochloride 3.5
Choline chloride 9
D-VB5 calcium 0.3
Folic acid 3.8
Magnesium chloride hexahydrate 50
Anhydrous magnesium sulfate 55
Anhydrous calcium chloride 100
Potassium chloride 450
Sodium chloride 5000
Sodium dihydrogen phosphate-water 200
Disodium hydrogen phosphate 150
Ferrous glycine 0.01
Cupric sulfate pentahydrate 0.0001~0.002；
Yeast extract 8000；
Linoleic acid 0.01~0.1
Linolenic acid 0.08-0.1；
Sodium bicarbonate 3000
D-Glucose 3000~6000
Hypoxanthine sodium salt 6
Lipoic acid 0.1~0.4；
(2) above-mentioned culture medium is used, by Vero cell with 0.3 × 106Cell number/milliliter density is inoculated in 5 liters of stirring-types In bioreactor, cytodex-1 microcarrier concentration is 4 grams per liters, reaches 10.8*10 in cell density6Cell number/milliliter is straight Connecing according to M.O.I is 0.01 inoculation influenza virus, maintains harvest within 70 hours.
The structural formula of neohesperidin dihydrochalcone are as follows:
The invention has the beneficial effects that:
The invention discloses a kind of application of chalcone in vero cell production influenza virus, inventors have surprisingly discovered that The substance is applied in the serum free medium of Vero cell, it is possible to reduce the addition of expensive growth hormone, and be easy to reach High density, while virus titer is high, peplos are complete, and product quality is excellent and low production cost.
Detailed description of the invention
Attached drawing is the cellular morphology Electronic Speculum observation when embodiment of the present invention 3 reaches cell maximal density.
Specific embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1:
Influenza virus A 1 used in embodiment (A/NEW/CALEDONIA/20/99) comes from NIBSC.
(1) following culture mediums are prepared, it is characterised in that it includes following component, and following content is calculated as with mg/litre:
Neohesperidin dihydrochalcone 0.8
L-Trp 10
L-cysteine 100
L-Isoleucine 225
L-Leu 100
Valine 100
L-arginine hydrochloride 650
L-lysine 350
L-Methionine 60
Pidolidone 50
L-phenylalanine 216
Altheine 1000
Glycine 15
L-PROLINE 180
L-Alanine 55
L-Aspartic acid 65
Serine 290
L-threonine 300
L-Glutamine 650
Vitamin C 99
Niacinamide 8
Riboflavin 0.6
Thiamine hydrochloride 3.5
Choline chloride 9
D-VB5 calcium 0.3
Folic acid 3.8
Magnesium chloride hexahydrate 50
Anhydrous magnesium sulfate 55
Anhydrous calcium chloride 100
Potassium chloride 450
Sodium chloride 5000
Sodium dihydrogen phosphate-water 200
Disodium hydrogen phosphate 150
Ferrous glycine 0.01
Cupric sulfate pentahydrate 0.0001；
Yeast extract 8000；
Linoleic acid 0.1
Linolenic acid 0.08；
Sodium bicarbonate 3000
D-Glucose 6000
Hypoxanthine sodium salt 6
Lipoic acid 0.1
(2) above-mentioned culture medium is used, Vero cell is inoculated in 5 liters of stirring-types with 0.3 × 106 cell number/milliliter density In bioreactor, cytodex-1 microcarrier concentration is 4 grams per liters, reaches 5.8*10 in cell density6Cell number/milliliter is direct It is 0.01 inoculation influenza virus according to M.O.I, maintains harvest within 70 hours.
Reactor parameter setting: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, mixing speed 65 Rev/min, fresh medium is replaced according to remaining sugar concentration, with crystal violet method living cell counting density.
Embodiment 2:
(1) following culture mediums are prepared, it is characterised in that it includes following component, and following content is calculated as with mg/litre:
Neohesperidin dihydrochalcone 1.5
L-Trp 10
L-cysteine 100
L-Isoleucine 225
L-Leu 80
Valine 100
L-arginine hydrochloride 800
L-lysine 320
L-Methionine 60
Pidolidone 82
L-phenylalanine 216
Altheine 800
Glycine 15
L-PROLINE 180
L-Alanine 55
L-Aspartic acid 68
Serine 290
L-threonine 50
L-Glutamine 690
Vitamin C 60
Niacinamide 8
Riboflavin 0.6
Thiamine hydrochloride 3.5
Choline chloride 9
D-VB5 calcium 0.3
Folic acid 3.8
Magnesium chloride hexahydrate 50
Anhydrous magnesium sulfate 55
Anhydrous calcium chloride 100
Potassium chloride 450
Sodium chloride 5000
Sodium dihydrogen phosphate-water 200
Disodium hydrogen phosphate 150
Ferrous glycine 0.01
Cupric sulfate pentahydrate 0.002；
Yeast extract 8000；
Linoleic acid 0.01
Linolenic acid 0.1；
Sodium bicarbonate 3000
D-Glucose 3000
Hypoxanthine sodium salt 6
Lipoic acid 0.4.
(2) above-mentioned culture medium is used, by Vero cell with 0.3 × 106Cell number/milliliter density is inoculated in 5 liters of stirring-types In bioreactor, cytodex-1 microcarrier concentration is 4 grams per liters, and it is direct to reach 5.8*106 cell number/milliliter in cell density It is 0.01 inoculation influenza virus according to M.O.I, maintains harvest within 70 hours.
Reactor parameter setting: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, mixing speed 65 Rev/min, fresh medium is replaced according to remaining sugar concentration, with crystal violet method living cell counting density.
Embodiment 3:
(1) following culture mediums are prepared, it is characterised in that it includes following component, and following content is calculated as with mg/litre:
Neohesperidin dihydrochalcone 1.0
L-Trp 10
L-cysteine 100
L-Isoleucine 225
L-Leu 90
Valine 100
L-arginine hydrochloride 750
L-lysine 340
L-Methionine 60
Pidolidone 70
L-phenylalanine 216
Altheine 900
Glycine 15
L-PROLINE 180
L-Alanine 55
L-Aspartic acid 67
Serine 290
L-threonine 145
L-Glutamine 670
Vitamin C 83
Niacinamide 8
Riboflavin 0.6
Thiamine hydrochloride 3.5
Choline chloride 9
D-VB5 calcium 0.3
Folic acid 3.8
Magnesium chloride hexahydrate 50
Anhydrous magnesium sulfate 55
Anhydrous calcium chloride 100
Potassium chloride 450
Sodium chloride 5000
Sodium dihydrogen phosphate-water 200
Disodium hydrogen phosphate 150
Ferrous glycine 0.01
Cupric sulfate pentahydrate 0.001；
Yeast extract 8000；
Linoleic acid 0.02
Linolenic acid 0.09；
Sodium bicarbonate 3000
D-Glucose 4000
Hypoxanthine sodium salt 6
Lipoic acid 0.3.
(2) above-mentioned culture medium is used, by Vero cell with 0.3 × 106Cell number/milliliter density is inoculated in 5 liters of stirring-types In bioreactor, cytodex-1 microcarrier concentration is 4 grams per liters, reaches 5.8*10 in cell density6Cell number/milliliter is direct It is 0.01 inoculation influenza virus according to M.O.I, maintains harvest within 78 hours.
Reactor parameter setting: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, mixing speed 65 Rev/min, fresh medium is replaced according to remaining sugar concentration, with crystal violet method living cell counting density.
Embodiment 4:
The measurement of Influenza virus titer is by viral sample to be checked in the every hole of A~G row adjacent 5 of 96 hole tissue culturing plates 90 μ L DMEM, 10% FBS culture solution is added in column.10 μ L viral samples to be checked are added in the every hole of A row, and Multi-channel liquid transfer device is mixed It closes 3~5 times, then out of the transfer of every hole 10 μ L to B row corresponding aperture, mixes again, successively dilute sample to G row, so Every hole is added 2. 5 × 10 afterwards5 MDCK cell is placed in 33 DEG C of humidifying, 5 %CO2Incubator absorbs every hole culture after overnight Liquid adds 200 μ L DMEM/0. 000 2% (m/V) trypsase, is placed in 33 DEG C of humidifying, 5% CO23 d of incubator culture. Add in 50 μ L, 1% chicken red blood cells suspension to all holes, record every hole sample agglutination pattern and calculate the metering of 50% cell infection ( TCID50)
Experimental result is as follows:
Viral hemoagglutination titre HAU
Embodiment 1 1:640
Embodiment 2 1:640
Embodiment 3 1:1280
As it can be seen that the Influenza virus titers of culture of the invention are high, it is at low cost.
Embodiment 5:
Electronic Speculum observation being carried out to virion after harvest, as a result as shown in the picture, it is seen that influenza virus envelopes structure understands, Show complete, typical influenza virus particles state.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms are not Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this The range of invention is defined by the claims and their equivalents.

Claims (1)

1. a kind of method with vero cell production influenza virus, its step are as follows:
(1) following culture mediums are prepared, it is characterised in that it includes following component, and following content is calculated as with mg/litre:
Neohesperidin dihydrochalcone 1.0
L-Trp 10
L-cysteine 100
L-Isoleucine 225
L-Leu 90
Valine 100
L-arginine hydrochloride 750
L-lysine 340
L-Methionine 60
Pidolidone 70
L-phenylalanine 216
Altheine 900
Glycine 15
L-PROLINE 180
L-Alanine 55
L-Aspartic acid 67
Serine 290
L-threonine 145
L-Glutamine 670
Vitamin C 83
Niacinamide 8
Riboflavin 0.6
Thiamine hydrochloride 3.5
Choline chloride 9
D-VB5 calcium 0.3
Folic acid 3.8
Magnesium chloride hexahydrate 50
Anhydrous magnesium sulfate 55
Anhydrous calcium chloride 100
Potassium chloride 450
Sodium chloride 5000
Sodium dihydrogen phosphate-water 200
Disodium hydrogen phosphate 150
Ferrous glycine 0.01
Cupric sulfate pentahydrate 0.001；
Yeast extract 8000；
Linoleic acid 0.02
Linolenic acid 0.09；
Sodium bicarbonate 3000
D-Glucose 4000
Hypoxanthine sodium salt 6
Lipoic acid 0.3；
(2) above-mentioned culture medium is used, by Vero cell with 0.3 × 106It is anti-that cell number/milliliter density is inoculated in 5 liters of stirring-type biologies It answers in device, cytodex-1 microcarrier concentration is 4 grams per liters, reaches 5.8*10 in cell density6Cell number/milliliter directly according to M.O.I is 0.01 inoculation influenza virus, maintains harvest within 78 hours；
Reactor parameter setting: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, mixing speed is 65 revs/min Clock replaces fresh medium according to remaining sugar concentration, with crystal violet method living cell counting density；
The influenza virus is influenza virus A 1, is A/NEW/CALEDONIA/20/99, comes from NIBSC.
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CN105779376A (en) * 2016-03-24 2016-07-20 友康恒业生物科技（北京）有限公司 Serum-free medium for separating influenza viruses and purpose and preparation method thereof
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