CN103768589B - Hollow-fibre membrane is utilized to remove the method for residual DNA in encephalitis B used for human being vaccine product - Google Patents
Hollow-fibre membrane is utilized to remove the method for residual DNA in encephalitis B used for human being vaccine product Download PDFInfo
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- CN103768589B CN103768589B CN201210398655.XA CN201210398655A CN103768589B CN 103768589 B CN103768589 B CN 103768589B CN 201210398655 A CN201210398655 A CN 201210398655A CN 103768589 B CN103768589 B CN 103768589B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The present invention relates to a kind of utilize hollow-fibre membrane to remove the method for residual DNA in encephalitis B used for human being vaccine product, it is characterized in that: with encephalitis B used for human being vaccine harvest liquid as raw material, use hollow-fibre membrane micro-filtration method, remove the foreign protein in encephalitis B used for human being vaccine harvest liquid and DNA, collect the liquid after filtering and make encephalitis B used for human being vaccine concentrated solution through ultrafiltration concentration system, after inactivation, use sieve chromatography purification system that encephalitis B used for human being vaccine inactivation liquid is purified.The present invention, on the premise of not affecting vaccine product biological activity, carries out microfiltration process by hollow-fibre membrane to virus harvest liquid, effectively removes and remains in a certain proportion of DNA therein, so that finished product Vaccines DNA residual quantity reaches criterion of acceptability.
Description
Technical field
The present invention relates to a kind of encephalitis B used for human being vaccine product is removed DNA and the method for foreign protein, particularly relate to
A kind of hollow-fibre membrane is utilized to remove the method for residual DNA in encephalitis B used for human being vaccine product.The hollow that the present invention mentions is fine
Dimension film is hollow fiber microfiltration membrane, and its material main component is PP or PVDF etc..
Background technology
Encephalitis B used for human being vaccine is to be inoculated in by encephalitis b virus on applicable zooblast, releases through breeding restrovirus
Being put in culture fluid, the host cell of some remnants or its fragment are as well as being mixed in culture fluid around here, and clarified, concentration is gone out
Live, after purification, extract and obtain in vaccine for virus of encephalitis B stock solution, free host DNA can be produced and be combined with antigen protein
Host DNA, possibly together with substantial amounts of host protein in stock solution, concentrating and purifying technique can remove a certain proportion of free DNA,
But still have residual DNA and exist, the DNA being particularly combined with virus or antigen protein can remain in purification stock solution, as removed
Not thorough, the DNA of this part residual can be injected into into the human body together along with finished product vaccine, may cause bad in human body
Reaction, or there is the possibility causing cancer to occur.
Summary of the invention
The purpose of the present invention is that solution deficiencies of the prior art, provides one and utilizes hollow-fibre membrane to go
Except the method for residual DNA in encephalitis B used for human being vaccine product.The present invention on the premise of not affecting vaccine product biological activity,
By hollow-fibre membrane, virus harvest liquid is carried out microfiltration process, effectively remove and remain in a certain proportion of DNA therein, thus
Finished product Vaccines DNA residual quantity is made to reach criterion of acceptability.
The technical scheme that the present invention is given is: this to utilize hollow-fibre membrane to remove in encephalitis B used for human being vaccine product residual
The method staying DNA, is characterized in: with encephalitis B used for human being vaccine harvest liquid as raw material, uses hollow-fibre membrane micro-filtration method,
Remove the foreign protein in encephalitis B used for human being vaccine harvest liquid and DNA, collect the liquid after filtering and make through ultrafiltration concentration system
Encephalitis B used for human being vaccine concentrated solution, uses sieve chromatography purification system to encephalitis B used for human being vaccine inactivation liquid after inactivation
It is purified.
This utilizing hollow-fibre membrane to remove the method for residual DNA in encephalitis B used for human being vaccine product, it is the most square
Case is:
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out micro-filtration
Process, hollow fibre membrane systems be GE company produce model be CFP-6-D-8A, aperture is 0.65 μm, and membrane area is 4.0m2,
Liquor inlet end pressure ＜ 10PSI, material liquid outlet end pressure is >=0PSI, and feed liquid backflow end pressure ＜ 3PSI, the people of process uses
Vaccinum Encephalitis B virus harvest liquid, cumulative volume is 230L-240L, and net cycle time is 1.5h-2.0h, and whole micro-filtration processes
During ensure feed temperature between 18 DEG C-26 DEG C.The liquid obtained after filtration enters into concentration technology program, passes through ultrafiltration
Concentration systems is made into Vaccinum Encephalitis B concentrated solution, and after inactivation, lot number is SA-03, purer by molecular sieve layer analysis system
Changing, the encephalitis B used for human being vaccine refined solution lot number collected is SA-04.
This utilizing hollow-fibre membrane to remove the method for residual DNA in encephalitis B used for human being vaccine product, it is the most square
Case is:
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out micro-filtration
Processing, the model of hollow fibre membrane systems is CFP-6-D-8A, and aperture is 0.65 μm, and membrane area is 4.0m2, liquor inlet side pressure
Power ＜ 10PSI, material liquid outlet end pressure is >=0PSI, feed liquid backflow end pressure ＜ 3PSI, the encephalitis B used for human being vaccine of process
Virus harvest liquid, cumulative volume is 110L-120L, and net cycle time is 0.8h-1.0h, ensures material in whole micro-filtration processing procedure
Liquid temp is between 18 DEG C-26 DEG C.The liquid obtained after filtration enters into concentration technology program, and the system that is concentrated by ultrafiltration is by it
Making Vaccinum Encephalitis B concentrated solution, after inactivation, lot number is SA-05, then by molecular sieve layer analysis system purification, the people collected
It is SA-06 with Vaccinum Encephalitis B refined solution lot number.
Hollow-fibre membrane has the open flow passage structure of fiber tubulose, and the tubulose flow passage structure without screen cloth avoids feed liquid
Random high turbulences, therefore there is lower shearing force, gentle operation can effectively prevent viral surface glycoprotein
Come off and the gathering of albumen, be conducive to the integrity of protection macromole virus, prevent virion from contributing to while assembling miscellaneous
Albumen and DNA through and remove.
Vaccine virus harvest liquid, after filtration treatment, enters into concentration technology program, and the system that is concentrated by ultrafiltration is by it
Make vaccine virus concentrated solution, in this process, in obtained ultrafiltration permeate can containing a number of foreign protein and
DNA. to vaccine virus harvest liquid, taking different filter types, in the ultrafiltration permeate that correspondence obtains, the content of DNA has bright
Significant difference is different.Concrete detection data are shown in Table 3.
Compared with prior art, the invention has the beneficial effects as follows:
1. hollow-fibre membrane has good physical chemistry toleration, and liquor inlet is pressure more than 4bar, can take the photograph 50
Below family name's degree, tolerating the thorough cleaning and sterilizing of 1mol/L NaOH solution, most of hollow-fibre membranes can be with autoclaving, the life-span
Long, low cost.
2. in separation process, there is not the change of matter carrying out solution in hollow-fibre membrane, and chemical compatibility is superior, power consumption
Low, technological process is simple, it is easy to operation management.
Accompanying drawing explanation
Fig. 1 SA-02 purification collection of illustrative plates
It is encephalitis B used for human being vaccine inactivation liquid (SA-01), directly through the collection of illustrative plates of sieve chromatography (SA-02) after purification,
Vertical coordinate (ultraviolet absorption value mAU) in figure, abscissa (volume ml).
Fig. 2 SA-04 purification collection of illustrative plates
It is encephalitis B used for human being vaccine inactivation liquid (SA-03), after first using hollow-fibre membrane micro-filtration, then carries out molecular sieve
The collection of illustrative plates of (SA-04) after chromatography purification, vertical coordinate (ultraviolet absorption value mAU, abscissa (volume ml) in figure.
Fig. 3 SA-06 purification collection of illustrative plates
It is encephalitis B used for human being vaccine inactivation liquid (SA-05), after first using hollow-fibre membrane micro-filtration, then carries out molecular sieve
The collection of illustrative plates of (SA-06) after chromatography purification, vertical coordinate (ultraviolet absorption value mAU) in figure, abscissa (volume ml).
Detailed description of the invention
Following example are described in detail technical scheme, but are not used in and limit the scope of the invention.
Embodiment 1
Choose the Vaccinum Encephalitis B virus harvest liquid of a batch, use sleeve filter that it is carried out at clarification filtration
Reason, the model of sleeve biofilter system is TP012TV25TCTFELB, and aperture is 0.65 μm, and membrane area is about 4.0m2, liquor inlet
Pressure ＜ 10PSI, material liquid outlet pressure is about 0PSI, the encephalitis B used for human being vaccine harvest liquid of process, and cumulative volume is 230L-
240L, net cycle time is 1.5h-2.0h, and in whole clarification filtration processing procedure, guarantee feed temperature is between 18 DEG C-26 DEG C,
The liquid obtained after filtration enters into concentration technology program, and it is dense that the system of being concentrated by ultrafiltration is made into encephalitis B used for human being vaccine
Contracting liquid, after inactivation, lot number is SA-01, then by molecular sieve layer analysis system purification, the encephalitis B used for human being vaccine purification collected
Liquid lot number is SA-02.
In concentration technology program: ultrafiltration permeate lot number is SC-01.
Embodiment 2
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out micro-filtration
Processing, the model of hollow fibre membrane systems is CFP-6-D-8A, and aperture is 0.65 μm, and membrane area is about 4.0m2, liquor inlet end
Pressure ＜ 10PSI, material liquid outlet end pressure is about 0PSI, feed liquid backflow end pressure ＜ 3PSI, the encephalitis B used for human being epidemic disease of process
Seedling virus harvest liquid, cumulative volume is 230L-240L, and net cycle time is 1.5h-2.0h, ensures in whole micro-filtration processing procedure
Feed temperature is between 18 DEG C-26 DEG C.The liquid obtained after filtration enters into concentration technology program, and the system that is concentrated by ultrafiltration will
It makes Vaccinum Encephalitis B concentrated solution, and after inactivation, lot number is SA-03, then by molecular sieve layer analysis system purification, collects
Encephalitis B used for human being vaccine refined solution lot number is SA-04.
In concentration technology program: ultrafiltration permeate lot number is SC-02.
Embodiment 3
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out micro-filtration
Processing, the model of hollow fibre membrane systems is CFP-6-D-8A, and aperture is 0.65 μm, and membrane area is about 4.0m2, liquor inlet end
Pressure ＜ 10PSI, material liquid outlet end pressure is about 0PSI, feed liquid backflow end pressure ＜ 3PSI, the encephalitis B used for human being epidemic disease of process
Seedling virus harvest liquid, cumulative volume is 110L-120L, and net cycle time is 0.8h-1.0h, ensures in whole micro-filtration processing procedure
Feed temperature is between 18 DEG C-26 DEG C.The liquid obtained after filtration enters into concentration technology program, and the system that is concentrated by ultrafiltration will
It makes Vaccinum Encephalitis B concentrated solution, and after inactivation, lot number is SA-05, then by molecular sieve layer analysis system purification, collects
Encephalitis B used for human being vaccine refined solution lot number is SA-06.
In concentration technology program: ultrafiltration permeate lot number is SC-03.
By SA-01, SA-02, SA-03, SA-04, SA-05, SA-06 respectively through row antigenic content, DNA residues detection.
Detection data are shown in Table 1.
SC-01, SC-02, SC-03 are carried out respectively DNA content detection.Detection data refer to table 3.
Table 1:SA-01～SA-06 antigenic content, DNA residues detection result.
Table 2: different process detects data after processing for identical encephalitis B used for human being viral vaccine concentrated solution and compares
Table 3: in ultrafiltration permeate, DNA content detection data compare
SC-01 SC-02 SC-03
DNA content 0.8Ng/1ml 1.2Ng/1ml 1.3Ng/1ml
By table 1 it may be concluded that in the case of encephalitis B used for human being vaccinia virus antigens is almost without loss, hollow is fine
Dimension membranous system is removed the effect of residual DNA in vaccine product and is substantially better than sleeve filter filtration system.Hollow-fibre membrane has all
Even pore-size distribution, avoids the accumulation of foreign protein and DNA to a certain extent, desirably prevents the shape of Membrane surface proteins gel layer
Become, thus there is faster processing speed, be beneficial to improve later stage chromatography degree.In sum, unique knot of hollow-fibre membrane
Structure, has a certain proportion of removal ability for the DNA of residual in vaccine product, is fully able to reach to remove more residual DNA
Purpose.
As shown in Table 3, vaccine virus harvest liquid is after hollow fibre membrane systems micro-filtration processes, in ultrafiltration permeate
The content of DNA increases, hence it is evident that illustrate that in vaccine virus concentrated solution, the DNA of residual is reducing, and fully proves, hollow fibre membrane systems
For the DNA of residual in vaccine product, there is the removal effect of a capability, it is possible to reach effectively to remove the purpose of residual DNA,
Guarantee that final products reach country's acceptable quality.Above-mentioned data detection method all meets the Pharmacopoeia of the People's Republic of China
2010 editions.
Claims (1)
1. one kind utilizes hollow-fibre membrane to remove the method for residual DNA in encephalitis B used for human being vaccine product, it is characterised in that: with
Encephalitis B used for human being vaccine harvest liquid is raw material, uses hollow-fibre membrane micro-filtration method, removes encephalitis B used for human being vaccine and receives
Obtain the foreign protein in liquid and DNA, collect the liquid after filtering and make encephalitis B used for human being vaccine concentrated solution through ultrafiltration concentration system,
Use sieve chromatography purification system that encephalitis B used for human being vaccine inactivation liquid is purified after inactivation, specifically comprise the following steps that
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out at micro-filtration
Reason, hollow fibre membrane systems be GE company produce model be CFP-6-D-8A, aperture is 0.65 μm, and membrane area is 4.0, material
<10 PSI, material liquid outlet end pressure is>=0 PSI to liquid entrance point pressure, and<3 PSI, the people of process uses feed liquid backflow end pressure
Vaccinum Encephalitis B virus harvest liquid, cumulative volume is 230L-240 L, and net cycle time is 1.5h-2.0 h, at whole micro-filtration
During reason, guarantee feed temperature is between 18 DEG C-26 DEG C, and the liquid obtained after filtration enters into concentration technology program, passes through
Ultrafiltration concentration system is made into Vaccinum Encephalitis B concentrated solution, and after inactivation, lot number is SA-03, then by molecular sieve layer analysis system
Purification, that collects is encephalitis B used for human being vaccine refined solution, and lot number is SA-04；Or
Choose the encephalitis B used for human being vaccine virus harvest liquid of a batch, use hollow-fibre membrane that it is carried out at micro-filtration
Reason, the model of hollow fibre membrane systems is CFP-6-D-8A, and aperture is 0.65 μm, and membrane area is 4.0, liquor inlet end pressure
<10 PSI, material liquid outlet end pressure is>=0 PSI, feed liquid backflow end pressure<3 PSI, the encephalitis B used for human being vaccine of process
Virus harvest liquid, cumulative volume is 110L-120 L, and net cycle time is 0.8h-1.0 h, ensures in whole micro-filtration processing procedure
Feed temperature is between 18 DEG C-26 DEG C, and the liquid obtained after filtration enters into concentration technology program, is concentrated by ultrafiltration system
Being made into Vaccinum Encephalitis B concentrated solution, after inactivation, lot number is SA-05, then by molecular sieve layer analysis system purification, collects
Be encephalitis B used for human being vaccine refined solution, lot number is SA-06.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005080556A2 (en) * | 2004-02-23 | 2005-09-01 | Crucell Holland B.V. | Virus purification methods |
| CN101270350A (en) * | 2008-03-05 | 2008-09-24 | 辽宁依生生物制药有限公司 | Method for extracting rabies virus |
| CN102406933A (en) * | 2011-11-25 | 2012-04-11 | 成都康华生物制品有限公司 | Preparation method for measles attenuated live vaccine |
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- 2012-10-18 CN CN201210398655.XA patent/CN103768589B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005080556A2 (en) * | 2004-02-23 | 2005-09-01 | Crucell Holland B.V. | Virus purification methods |
| CN101270350A (en) * | 2008-03-05 | 2008-09-24 | 辽宁依生生物制药有限公司 | Method for extracting rabies virus |
| CN102406933A (en) * | 2011-11-25 | 2012-04-11 | 成都康华生物制品有限公司 | Preparation method for measles attenuated live vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 纯化病毒疫苗的通用方法;陈昕等;《色谱园》;20050531(第15期);第26页左栏第2-4段、右栏表格 * |
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