CN107746797B - A kind of device and method carrying out rabies viruses inactivation, inactivator hydrolysis using disposable sack - Google Patents
A kind of device and method carrying out rabies viruses inactivation, inactivator hydrolysis using disposable sack Download PDFInfo
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Abstract
The present invention provides a kind of device and method that rabies viruses inactivation, inactivator hydrolysis are carried out using disposable sack, wherein inactivate disposable sack, connector there are three opening up, top is inflating port, connect inflation group, bottom is respectively liquid inlet and liquid outlet, is separately connected filling liquid group and drain group, and sack center is equipped with built-in blender；Wherein inflation group is made of bellows filter, silicone tube, pipeline folder, and bellows filter is wrapped with sterile bag；Filling liquid group is sequentially connected by connector B, silicone tube, pipeline folder, threeway and EZD valve respectively, another opening connection silicone tube, pipeline folder and the connector A of threeway；Advantage are as follows: transformation and reasonable employment have been carried out to disposable sack, have been improved inactivation/method for hydrolysis of virus liquid, inactivation of virus risk of failure has been reduced, reduces antigen losses, reduce production cost.
Description
Technical field
The present invention relates to viral vaccine production fields, are gone out more particularly, to a kind of using disposable sack progress rabies viruses
Living, inactivator hydrolysis device and method.
Background technique
The container that current vaccines workshop virus stock solution used uses when inactivating is mainly 20L Nalgene plastic barrel or stainless
Cylinder of steel.Preparation, storage, the transfer of disposable sack required solution mainly for the production of in.It is carried out using 20L barrels or stainless cylinder of steel
Need to carry out high pressure sterilization, cleaning validation, detection detergent/disinfectant remnants etc., disposable sack before rabies viruses inactivation
There is complete producer's verifying to support directly to use, cleaning, sterilization link can be reduced when for large-scale production, reduction is set
Standby occupied space, advantage of lower cost.
Need to carry out the mixing of inactivator and virus stock solution used using magnetic agitation or shaking table using 20L barrels or stainless cylinder of steel,
Mixed process can generate a large amount of foams due to acutely rocking, and be easy to cause insufficient and antigen the loss of inactivation.
Summary of the invention
It is an object of the invention to solve the deficiencies in the prior art, and provides and a kind of carry out mad dog using disposable sack
The device and method that inactivation of virus, inactivator hydrolyze.
The new technical solution of the present invention is: a kind of to carry out rabies viruses inactivation, inactivator hydrolysis side using disposable sack
Method, including the disposable sack of inactivation, the disposable sack of hydrolysis and silicone tube, inactivate disposable sack and offer three connectors,
Top is inflating port A, connects inflation group A, bottom is respectively liquid inlet and liquid outlet, is separately connected filling liquid group and drain
Group, sack center are equipped with built-in blender A；Wherein inflation group A is made of bellows filter A, silicone tube A, pipeline folder A, bellows filter
Device A is wrapped with sterile bag；Filling liquid group is sequentially connected by connector B, silicone tube, pipeline folder, threeway and EZD valve A respectively, and three
Logical another opening connection silicone tube, pipeline folder and connector A；Drain group is successively connected by connector C, silicone tube and EZD valve B
It connects；Disposable sack is hydrolyzed, is opened up there are four connector, top is respectively inflating port B and the connection inactivation one for connecting inflation group B
The inlet of secondary property sack liquid outlet, bottom are respectively liquid outlet and temp probe connector, and sack center is equipped with built-in stir
Mix device B；Wherein inflation group B is made of bellows filter B, silicone tube, pipeline folder, and bellows filter B is wrapped with sterile bag；Inlet
It is connect by feed liquor group with disposable sack liquid outlet is inactivated, feed liquor group is respectively by connector D, silicone tube, pipeline folder, threeway
And EZD valve C is sequentially connected, another opening connection silicone tube, pipeline folder and the connector E of threeway；Liquid outlet connects out liquid group,
Liquid group is sequentially connected by connector F, silicone tube and EZD valve D out；
It the described method comprises the following steps:
1. then the production cell of passage is placed in microcarrier bioreactor and is expanded by recovery, the passage of cell is produced
Increase culture；
2. treatment fluid is added into microcarrier bioreactor, the perfusion rate of adjustment microcarrier bioreactor is 0, clearly
Production cell surface is washed, hydrophobin fixed virus is inoculated on production cell after cleaning；
3. harvesting virus liquid, it is filtered clarification, is concentrated by ultrafiltration；
4. viral concentration liquid inactivates: being inflated in advance from the top for inactivating disposable sack by inflation group A, viral concentration
Liquid is entered by A mouthfuls of connector, and inactivator is entered by connector B, is carried out top by B mouthfuls of connector with PBS solution and is washed, closes EZD valve
Door A, is placed in 2-8 DEG C of freezer, and lasting stirring inactivation is for 24 hours；
5. the transfer of inactivation of virus liquid: being inflated from the top for hydrolyzing disposable sack by inflation group B, then set in advance
In the water-bath sleeve with TCU temperature-controlling system, by the connector C of the disposable sack of inactivation equipped with inactivation of viruses liquid and hydrolysis
E mouthfuls of connector of disposable sack are connected, and carry out the transfer of virus liquid；
6. hydrolysis removal inactivator: 37 DEG C of heating water bath 2h are so that inactivator complete hydrolysis, by NaOH solution by connector
D enters the disposable sack of hydrolysis, and to adjust inactivation of virus liquid pH to 7.4-7.8, the inactivation of virus liquid after hydrolysis is arranged by connector F
Out；
The production cell is Vero cell, in human diploid cell, hamster kidney cell, chick-embryo cell, duck embryo cells
Any one, the hydrophobin fixed virus are PV plants, Pasteur strain, Pitman-Moore, CVS, Flury LEP, Flury
Any strain in HEP, Kelev, ERA plants；
Inactivator water bath device used when hydrolyzing is TCU temperature control, refrigerant/heating agent double-direction control；
The defecation method of the virus liquid is that rabies viruses harvest liquid is clear by 0.8 μm and 0.65 μm of filter progress
Clearly；
The method for concentration of the virus liquid is that the hydrophobin harvest liquid after clarifying uses the super of 300KD or 750KD
Filter membrane packet or hollow fiber column are concentrated by ultrafiltration, and cycles of concentration is 20-30 times；
Inactivator used is beta-propiolactone, is first diluted beta-propiolactone with 1:40 times of water for injection, then with volume ratio 1:
100 are added in viral concentration liquid；
4. stirring condition is 150-180rpm at 2-8 DEG C to the step, and lasting stirring is for 24 hours.
The beneficial effects of the present invention are: having carried out transformation and reasonable employment to disposable sack, make inactivation/water of virus liquid
Solution method is improved, and is reduced inactivation of virus risk of failure, is reduced antigen losses, reduces production cost.
Material used in disposable sack consolidates very much and highly flexible, and the risk of damage is small, and material is transparent, convenient for going out
The observation of solution state in work/hydrolytic process.With built-in blender, beta-propiolactone can be made to mix well with virus stock solution used.It is dense
Contracting liquid reduces feed liquid and splashes from bottom liquid inlet.Exclusive EZ-Drain valve design can guarantee that inactivation is abundant without safe dead angle.
Inactivation/hydrolytic process need to only pass through the transfer of a virus liquid, and EZ-Drain discharge shifts noresidue, reduce inactivation failure wind
Danger.
Disposable sack is thin compared with the wall of 20L Nalgene plastic barrel or stainless cylinder of steel, and the coefficient of heat transfer is high, matched
TCU temperature-controlling system has the characteristics that heating is quick, temperature control is accurate, can perform well in the hydrolysis of inactivator.
Detailed description of the invention
Fig. 1 is the schematic diagram of the disposable sack of inactivation of virus of the invention.
Fig. 2 is the schematic diagram that extinguishing chemical of the invention hydrolyzes disposable sack.
Wherein: 1 is inflation group A, and 2 be filling liquid group, and 3 be drain group, and 4 press from both sides A for pipeline, and 5 be silicone tube A, and 6 be inflating port A,
7 be bellows filter A, and 8 be EZD valve A, and 9 be built-in blender A, and 10 is inactivate disposable sack, and 11 be liquid inlet, and 12 are
Connector B, 13 be liquid outlet, and 14 be connector A, and 15 be EZD valve B, and 16 be connector F, and 17 be connector C, and 18 be to fill
Gas group B, 19 be inflating port B, and 20 is hydrolyze disposable sack, and 21 be connector E, and 22 be feed liquor group, and 23 be connector D, and 24 are
Inlet, 25 be temp probe connector, and 26 be liquid outlet, and 27 be liquid group out.
Specific embodiment
The present invention will be further described with reference to the accompanying drawing.
It is a kind of to carry out rabies viruses inactivation, inactivator method for hydrolysis, including the disposable sack of inactivation using disposable sack
10, disposable sack 20 and silicone tube are hydrolyzed, disposable sack 10 is inactivated and opens up there are three connector, top is inflating port A6,
Inflation group A1 is connected, bottom is respectively liquid inlet 11 and liquid outlet 13, filling liquid group 2 and drain group 3 is separately connected, in sack
Centre is equipped with built-in blender A9；Wherein inflation group A1 is made of bellows filter A7, silicone tube A5, pipeline folder A4, bellows filter A7
It is wrapped with sterile bag；Filling liquid group 2 is sequentially connected by connector B12, silicone tube, pipeline folder, threeway and EZD valve A8 respectively,
Another opening connection silicone tube, pipeline folder and the connector A14 of threeway；Drain group 3 is by connector C17, silicone tube and EZD valve
B15 is sequentially connected；Disposable sack 20 is hydrolyzed, is opened up there are four connector, top is respectively the inflating port for connecting inflation group B18
B19 and connection inactivate the inlet 24 of disposable 10 liquid outlet 13 of sack, and bottom is respectively that liquid outlet 26 and temp probe connect
Interface 25, sack center are equipped with built-in blender B；Wherein inflation group B18 is made of bellows filter B, silicone tube, pipeline folder, capsule
Formula filter B is wrapped with sterile bag；Inlet 24 is connect by feed liquor group 22 with disposable 10 liquid outlet 13 of sack is inactivated, into
Liquid group 22 is sequentially connected by connector D23, silicone tube, pipeline folder, threeway and EZD valve C respectively, another opening connection of threeway
Silicone tube, pipeline folder and connector E21；Liquid outlet 26 connects out liquid group 27, and liquid group 27 is by connector F16, silicone tube and EZD out
Valve D is sequentially connected；
It the described method comprises the following steps:
1. then the production cell of passage is placed in microcarrier bioreactor and is expanded by recovery, the passage of cell is produced
Increase culture；
2. treatment fluid is added into microcarrier bioreactor, the perfusion rate of adjustment microcarrier bioreactor is 0, clearly
Production cell surface is washed, hydrophobin fixed virus is inoculated on production cell after cleaning；
3. harvesting virus liquid, it is filtered clarification, is concentrated by ultrafiltration；
4. viral concentration liquid inactivates: being inflated in advance from the top for inactivating disposable sack 10 by inflation group A1, virus
Concentrate is entered by A14 mouthfuls of connector, and inactivator is entered by connector B12, is carried out top by B12 mouthfuls of connector with PBS solution and is washed,
EZD valve A8 is closed, is placed in 2-8 DEG C of freezer, lasting stirring inactivation is for 24 hours；
5. the transfer of inactivation of virus liquid: being inflated in advance from the top for hydrolyzing disposable sack 20 by inflation group B18, so
It is placed in the water-bath sleeve with TCU temperature-controlling system, by the connector of the disposable sack 10 of inactivation equipped with inactivation of viruses liquid
C17 is connected with hydrolyze disposable sack 20 E21 mouthfuls of connector, carries out the transfer of virus liquid；
6. hydrolysis removal inactivator: 37 DEG C of heating water bath 2h are so that inactivator complete hydrolysis, by NaOH solution by connector
D23 enters the disposable sack 20 of hydrolysis, and to adjust inactivation of virus liquid pH to 7.4-7.8, the inactivation of virus liquid after hydrolysis is by connecting
Head F16 discharge；
The production cell is Vero cell, in human diploid cell, hamster kidney cell, chick-embryo cell, duck embryo cells
Any one, the hydrophobin fixed virus are PV plants, Pasteur strain, Pitman-Moore, CVS, Flury LEP, Flury
Any strain in HEP, Kelev, ERA plants；
Inactivator water bath device used when hydrolyzing is TCU temperature control, refrigerant/heating agent double-direction control；
The defecation method of the virus liquid is that rabies viruses harvest liquid is clear by 0.8 μm and 0.65 μm of filter progress
Clearly；
The method for concentration of the virus liquid is that the hydrophobin harvest liquid after clarifying uses the super of 300KD or 750KD
Filter membrane packet or hollow fiber column are concentrated by ultrafiltration, and cycles of concentration is 20-30 times；
Inactivator used is beta-propiolactone, is first diluted beta-propiolactone with 1:40 times of water for injection, then with volume ratio 1:
100 are added in viral concentration liquid；
4. stirring condition is 150-180rpm at 2-8 DEG C to the step, and lasting stirring is for 24 hours.
The scale ablation method of 1 rabies vacciness virus liquid of embodiment:
Take the Vero Cell Rabies poison harvest liquid prepared first through 0.8 μm and 0.45 μm of millipore filter filtering, it is clarified
The 300KD ultrafiltration membrane packet ultrafiltration that Millipore is used after filter, before ultrafiltration concentration, first with the flushing liquor (BME for being free of human serum albumin
Liquid, 0.02% glutamine containing mass percent, 24.63% sodium bicarbonate) carry out balance film packet, detect the pH value of phegma, pH value
It is 6.0, is concentrated after meeting the requirements, 25 times of cycles of concentration.From A mouthfuls of transfer virus amalgamation liquids to the disposable sack 10 of inactivation
In.Beta-propiolactone is diluted with 1:40 times of sterilized water for injection first, is added to disease from B mouthfuls of sack according still further to the volume ratio of 1:100
In poison concentration amalgamation liquid, top is carried out by B mouthfuls with PBS solution and is washed.EZD valve A8 is closed, is placed in 2-8 DEG C of freezer, it is lasting to stir
Inactivate 12h, revolving speed 150rpm.One disposable sack 20 of hydrolysis is inflated in advance from E21 mouthfuls of connector, is subsequently placed in
In TCU water-bath sleeve.Inactivation of viruses liquid is transferred in the sack from F16 mouthfuls of connector.37 DEG C of heating water bath 2h are so that inactivation
Agent complete hydrolysis.NaOH solution is entered by G mouthfuls to adjust inactivation of virus liquid pH to 7.4-7.8.H mouthfuls be hydrolysis after virus go out
Liquid outlet living.
2 inactivator of embodiment hydrolyzes situation:
Beta-propiolactone is monitored in 37 DEG C of hydrolysis situations by the analysis method of gas-chromatography.Beta-propiolactone hydrolyzes situation point
Analysis.By Analysis of test results, 299.4ppm of beta-propiolactone when its concentration is not by hydrolyzing when hydrolyzing 150min is down to
34.4ppm, lower than this method quantitative limit hereinafter, illustrating that hydrolysis effect can be fully achieved in 37 DEG C of hydrolysis 150min in beta-propiolactone.
3 cell method of embodiment verifies inactivating efficacy:
By the ablation method of embodiment, take virus stock solution used according to every 3cm2The ratio of cell inoculation 1ml will be mad after inactivation
Dog disease venom is inoculated in MRC-5 cell, and culture is passed on after 7 days, continues culture to 21 days, by cell suspension inoculation Lab-tek chamber
Room glass slide (is purchased from Thermo company), sees whether that there are rabies viruses with immunofluorescence technique to Lab-tek after 3-4 days.
It wherein replaces respectively within 14 days and 21 days cell maintenance medium (MEM liquid contains 4% fetal calf serum), and collects cell conditioned medium and carry out mouse
Encephalocoele function poison, is observed mouse 21 days.Cell negative control and virus positive control are tested while setting up, to determine the effective of test
Property.
4 antigenic content of embodiment analysis verifying inactivating efficacy:
By the method for embodiment, in single concentrate and stoste sample detection rabies virus antigen content, using dual anti-folder
Heart method measures antigenic content.In measurement, sample (measuring antibody or antigen therein) and enzyme-labelled antigen or antibody by difference
The step of and the antigen or antibody of surface of solid phase carriers react.Make the antigen-antibody formed on solid phase carrier with the method for washing
Compound is separated with other substances, finally combines the amount of tested substance in enzyme amount and the sample on solid phase carrier at certain ratio
Example.After the substrate of enzyme reaction is added, substrate becomes color products by enzymatic, and the amount of tested substance is straight in the amount and sample of product
Correlation is connect, therefore can be according to the depth qualitative or quantitative analysis of color reaction.
Claims (1)
1. a kind of carry out rabies viruses inactivation, inactivator method for hydrolysis, including the disposable sack of inactivation using disposable sack
(10), disposable sack (20) and silicone tube are hydrolyzed, it is characterised in that: inactivate disposable sack (10) and open up that there are three connections
Mouthful, top is inflating port A(6), connect inflation group A(1), bottom is respectively liquid inlet (11) and liquid outlet (13), difference
Filling liquid group (2) and drain group (3) are connected, sack center is equipped with built-in blender A(9)；Wherein inflation group A(1) by bellows filter A
(7), silicone tube A(5), pipeline press from both sides A(4) composition, bellows filter A(7) be wrapped with sterile bag；Filling liquid group (2) is respectively by connecting
Head B(12), silicone tube, pipeline folder, threeway and EZD valve A(8) be sequentially connected, another opening of threeway connects silicone tube, pipeline
Folder and connector A(14)；Drain group (3) is by connector C(17), silicone tube and EZD valve B(15) it is sequentially connected；Hydrolysis is primary
Property sack (20), open up there are four connector, top is respectively to connect inflation group B(18) inflating port B(19) and connection inactivate
The inlet (24) of disposable sack (10) liquid outlet (13), bottom is respectively liquid outlet (26) and temp probe connector
(25), sack center is equipped with built-in blender B；Wherein inflation group B(18) it is made of bellows filter B, silicone tube, pipeline folder, capsule
Formula filter B is wrapped with sterile bag；Inlet (24) is by feed liquor group (22) and inactivates disposable sack (10) liquid outlet
(13) it connecting, feed liquor group (22) is sequentially connected by connector D(23), silicone tube, pipeline folder, threeway and EZD valve C respectively, and three
Logical another opening connection silicone tube, pipeline folder and connector E(21)；Liquid outlet (26) connects out liquid group (27), out liquid group
(27) it is sequentially connected by connector F(16), silicone tube and EZD valve D；
It the described method comprises the following steps:
1. by recovery, the passage of cell is produced then the production cell of passage being placed in expand in microcarrier bioreactor and be trained
It supports；
2. treatment fluid is added into microcarrier bioreactor, the perfusion rate of adjustment microcarrier bioreactor is 0, cleaning life
Cell surface is produced, is inoculated with hydrophobin fixed virus on production cell after cleaning；
3. harvesting virus liquid, it is filtered clarification, is concentrated by ultrafiltration；
4. viral concentration liquid inactivates: passing through inflation group A(1 from the top for inactivating disposable sack (10)) it is inflated in advance, virus
Concentrate by connector A(14) mouth enter, inactivator by connector B(12) enter, with PBS solution by connector B(12) mouth into
Row top is washed, and EZD valve A(8 is closed), it is placed in 2-8 DEG C of freezer, lasting stirring inactivation is for 24 hours；
5. the transfer of inactivation of virus liquid: passing through inflation group B(18 from the top for hydrolyzing disposable sack (20)) it is inflated in advance, so
It is placed in the water-bath sleeve with TCU temperature-controlling system, by the connection of the disposable sack of inactivation (10) equipped with inactivation of viruses liquid
Head C(17) it is connected with the connector E(21 of the disposable sack (20) of hydrolysis) mouth, carry out the transfer of virus liquid；
6. hydrolysis removal inactivator: 37 DEG C of heating water bath 2h are so that inactivator complete hydrolysis, by NaOH solution by connector D
(23) enter and hydrolyze disposable sack (20), to adjust inactivation of virus liquid pH to 7.4-7.8, the inactivation of virus liquid after hydrolysis is by even
Connector F(16) discharge；
The cell that produces is any in Vero cell, human diploid cell, hamster kidney cell, chick-embryo cell, duck embryo cells
, the hydrophobin fixed virus is PV plants, Pasteur strain, Pitman-Moore, CVS, Flury LEP, Flury
Any strain in HEP, Kelev, ERA plants；
Inactivator water bath device used when hydrolyzing is TCU temperature control, refrigerant/heating agent double-direction control；
The defecation method of the virus liquid is to clarify rabies viruses harvest liquid by 0.8 μm and 0.65 μm of filter；
The method for concentration of the virus liquid is ultrafiltration membrane of the hydrophobin harvest liquid after clarifying using 300KD or 750KD
Packet or hollow fiber column are concentrated by ultrafiltration, and cycles of concentration is 20-30 times；
Inactivator used is beta-propiolactone, is first diluted beta-propiolactone with 1:40 times of water for injection, then added with volume ratio 1:100
It is added in viral concentration liquid；
4. stirring condition is 150-180rpm at 2-8 DEG C to the step, and lasting stirring is for 24 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711051901.3A CN107746797B (en) | 2017-10-31 | 2017-10-31 | A kind of device and method carrying out rabies viruses inactivation, inactivator hydrolysis using disposable sack |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711051901.3A CN107746797B (en) | 2017-10-31 | 2017-10-31 | A kind of device and method carrying out rabies viruses inactivation, inactivator hydrolysis using disposable sack |
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| Publication Number | Publication Date |
|---|---|
| CN107746797A CN107746797A (en) | 2018-03-02 |
| CN107746797B true CN107746797B (en) | 2019-03-05 |
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| CN201711051901.3A Active CN107746797B (en) | 2017-10-31 | 2017-10-31 | A kind of device and method carrying out rabies viruses inactivation, inactivator hydrolysis using disposable sack |
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| CN1584022A (en) * | 2003-08-19 | 2005-02-23 | 三菱重工业株式会社 | Virus inactivating agent, virus inactivating method, filter with the virus inactivating and air conditioner therewith |
| CN103285390A (en) * | 2012-12-27 | 2013-09-11 | 瑞普（保定）生物药业有限公司 | Method for preparing rabies vaccine |
| CN203280811U (en) * | 2013-03-26 | 2013-11-13 | 晋峰 | Improved disposable plasma virus inactivation filter |
| CN103392691A (en) * | 2013-08-14 | 2013-11-20 | 北京瑞健高科生物科技有限公司 | Container and method for processing, sterilizing and saving biological tissues |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584022A (en) * | 2003-08-19 | 2005-02-23 | 三菱重工业株式会社 | Virus inactivating agent, virus inactivating method, filter with the virus inactivating and air conditioner therewith |
| CN103285390A (en) * | 2012-12-27 | 2013-09-11 | 瑞普（保定）生物药业有限公司 | Method for preparing rabies vaccine |
| CN203280811U (en) * | 2013-03-26 | 2013-11-13 | 晋峰 | Improved disposable plasma virus inactivation filter |
| CN103392691A (en) * | 2013-08-14 | 2013-11-20 | 北京瑞健高科生物科技有限公司 | Container and method for processing, sterilizing and saving biological tissues |
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Denomination of invention: A device and method for inactivating rabies virus and hydrolyzing inactivating agents using disposable bags Effective date of registration: 20230614 Granted publication date: 20190305 Pledgee: Weihai commercial bank Limited by Share Ltd. Dongying branch Pledgor: SHANDONG YIDU BIOTECHNOLOGY CO.,LTD. Registration number: Y2023980043805 |
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