CN108342368A - A kind of low pH is incubated the verification method of inactivation of virus - Google Patents
A kind of low pH is incubated the verification method of inactivation of virus Download PDFInfo
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Abstract
The present invention relates to the verification methods that a kind of low pH is incubated inactivation of viruses, select indicator virus and its corresponding host cell；Indicator virus expands and purifying；The titer determination of Working viral stoste and sample；The optimization of inactivation of viruses confirmatory experiment pH value；Low ph value is incubated inactivation of viruses recruitment evaluation.Method provided by the present invention, it can realize that laboratory simulates the overall process of genetically engineered drug production technology inactivation of virus, optimize amplification, the purifying process of indicator virus, indicator virus working stock titre is set to improve, pH value is unified to 7.0, stable quality and beneficial to subsequent operation, particular technique details (such as optimal determination for being incubated pH value that low pH is incubated is optimized simultaneously, the optimization etc. of pH regulative modes), improve the validity and actual effect inactivated to common DNA virus, RNA virus, envelope virus and nonenveloped virus.
Description
Technical field
The invention belongs to biotechnologies, specifically, the present invention relates to one kind being used for genetically engineered drug/biological products
The low pH of production technology is incubated the verification method of inactivation of virus.
Technical background
With the development of 21 century biotechnology, animal derived tissue, cell, body fluid and genetic engineering eukaryotic cell expression
The biological products of preparation gradually increase, and the crowd used constantly expands, and animal derived product raw material quality control does not draw in addition
Enough attention is played, production technology is again basic without being verified by stringent virus removal/inactivating efficacy.Therefore, animal sources at present
The risk of the infections mankind is high, and potential pathogenic infection problems become to become increasingly conspicuous.Wherein animal tissue's source system
Product are since the healthy quarantine situation of animal itself is poor, and the viral exogenous factor that different animals carry is complicated and changeable, and controllable sexual factor is still
It is uncertain, to the potential risk of human body compared with by system identification and the genetic engineering eukaryotic cell expression product strictly controlled
Risk higher.And genetically engineered biological product is also vulnerable to virus pollution (as from the mankind, animal due to its raw material
Tissue, blood, cell etc.), in production process using or be added to may be containing the exogenous virus factor material (such as animal
Serum, pancreatin etc.), the viral quality control of product raw and auxiliary material does not cause the reasons such as enough attention, relevant risk not to be allowed yet
Ignore.
So far, the highest 6 class biotech drug of global marketing volume, be respectively tumor therapeutic agent, it is antitumor necrosis because
Sub- α drugs, hematopoietin, insulin, beta-interferon and coagulation factor.This 6 class bio-pharmaceutical is except beta-interferon is by big
Outside enterobacteria and Yeast expression, remaining 5 class is produced through mammalian cell expression.Zooblast large-scale culture is current
The main way of genetically engineered drug production.The attenuations such as the viral inactivation vaccines such as rabies viruses, hepatitis A virus, japanese encephalitis virus
The production of live vaccine also must all expand seed culture of viruses in animal cell line.Since cell is the work life entity for having life attribute,
Condition of culture is very harsh, and growth cycle is long, easily by all-pervasive microbial contamination.Especially cell culture needs to be added
Many culture substrates, such as culture medium, serum, growth factor, these matrix are varied, mostly from Ren Yuan, animal source material,
The risk of viral potential pollution is set to obviously increase.Oneself proves to be frequently found the viruses such as HBV, HCV, HIV, HTLV in blood product
Pollution.In order to improve the biology peace property of gene engineering drug, increase disease in genetically engineered drug/biological products production technology
Malicious inactivation step is inexorable trend.
According to《Drug registration management method》Requirement, the product extracted by people, animal tissue or body fluid, animal
The recombination product of source property monoclonal antibody and eukaryotic cell expression, need to increase virus inactivation technology verifying data.Therefore country's food
Product Drug Administration and drug evaluation center were successively issued in 2002 and 2005《Blood product removal/inactivation of viruses
Technical method and verification guide principle》With《The Viral safety of biological tissue extracted product and eukaryotic cell expression product is evaluated
Drug evaluation rule》, it is desirable that genetically engineered drug/biological products production technology must include having for virus removal/inactivation
Processing step is imitated, and has done the guidance of principle to its method and main points.Wherein, it is exactly a kind of effective virus that low pH, which is incubated method,
Inactivation/removal technique can be used in blood product and other drugs/biological products production.But the two policy papers are not
The technical detail that the confirmatory experiment of inactivation of viruses is incubated to low pH makes the specific range of strict specifications, especially pH, detection ginseng
Number, evaluation system；The selection etc. of indicator virus.The present invention is aiming at sick in genetically engineered drug/biological products production technology
Poison inactivation with verification new technology foundation and propose, have it is highly practical, the characteristics of being widely used, establish it is a set of it is complete,
Efficient low pH is incubated inactivation of virus and verification technique system.
Invention content
In view of this, the purpose of the present invention is to provide the verification methods that a kind of low pH is incubated inactivation of viruses.
In order to reach above-mentioned purpose, the present invention provides a kind of verification method of low pH incubations inactivation of viruses, including following
Step：
1) indicator virus and its corresponding host cell are selected：Selection Pseudorabies virus (Pseudorabies virus,
PRV), murine leukemia virus (MuLV), vesicular stomatitis virus (Vesicularstomatitis virus, VSV), pig are tiny
Viral (Porcineparvovirus, PPV) is indicator virus；Select the corresponding host cell of indicator virus, Pseudorabies virus
Corresponding is porcine kidney cell (PK-15), and corresponding murine leukemia virus is cat astrocytes (PG4), vesicular stomatitis virus pair
It is African green monkey kidney cell (Vero) cell to answer, and corresponding pig parvoviral is porcine kidney cell (IBRS-2)；
2) indicator virus amplification and purifying：It is molten to use fresh serum free MEM culture mediums instead for ultracentrifugation after conventional amplification receipts sample
Solution, makes its virus titer improve, and pH value is unified for 7.0；
3) titer determination of Working viral stoste and sample：Using TCID50Method or real-time fluorescence quantitative RT-PCR method carry out
Titer determination；
4) optimization of inactivation of viruses confirmatory experiment pH value：By preliminary experiment determine the optimal incubation pH. of indicator virus and
The best regulative mode of pH value；
5) low ph value is incubated inactivation of viruses recruitment evaluation：The virus titer compareed with positive domestic animal subtracts in real time sample sample
Virus titer obtains the inactivation of virus quantitative data (LRV, log/ml) of different sampling time points, if LRV >=4 at the time point,
Then it is effective to be incubated the inactivation technology inactivation of viruses of the time by low pH.
Further, the amplification and purifying of the wherein step 2) indicator virus specifically comprise the following steps：
Each host cell is seeded in respectively in the T75 Tissue Culture Flasks of the MEM culture mediums containing 10% (v/v) FBS, is put
37 DEG C are placed in, 5%CO2It is cultivated in incubator；
When host cell degree of converging reaches 80%-90%, cell conditioned medium is discarded, is added 10-4-10-1Dilution it is corresponding
Virus liquid 3ml, 37 DEG C of 1~2h of absorption；
After absorption, virus liquid is discarded in Pasteur's liquid, cleans cell one time with the MEM culture mediums of serum-free, then use
MEM medium cultures 36-48h or so containing 2% (v/v) FBS, when 80% or more serious change (CPE) occurs for host cell
But when without the floating of complete lesion, cell is crushed, 4 DEG C of 3000g centrifugations 10min remove cell fragment, collect supernatant, then
As sterile viral seed stocks after being filtered with 0.22 μm of filter；
It takes 4 DEG C of above-mentioned supernatant, after 72,000g ultracentrifugation 4h, abandons supernatant, the 1/5- of original volume before precipitation ultracentrifugation
1/2 serum-free MEM (pH=7.0) dissolves so that the pH of Working viral stoste is uniformly adjusted to 7.0.
Further, wherein the breaking method of the cell is：When 80% or more serious change (CPE) occurs for host cell
But when without the floating of complete lesion, entire T75 bottles are frozen into -70 DEG C of ultra low temperature freezers, and -70 DEG C and 37 DEG C of water-baths repeatedly
Freeze thawing 3 times, each freeze thawing time are 30min-1h, and -70 DEG C of water-bath freeze thawing are stayed overnight for the first time；Or by host cell directly with super
Sound wave is crushed.
Further, wherein the wherein titer determination of step 3) the Working viral stoste and sample specifically includes following step
Suddenly：
The demand that the experiment of method Validation of Virus Inactivation in Human is incubated according to low pH dispenses Working viral stoste, while being dispensed with 1ml/ branch
And carry out titer determination；
It is significantly viral for CPE, using TCID50Method carries out titer determination, and not high for CEP unobvious or titre
Virus measures titre using real-time fluorescence quantitative RT-PCR method；
The TCID50When method measures virus titer, with reference to Reed-Muench methods, evaluation work stoste or sample virus
TCID50；
When the real-time fluorescence quantitative RT-PCR method measures virus titer, sample result phase is obtained by comparing standard curve
RNA copy numbers are answered, with formula 1000vRNA copy numbers/ml=1pfu infection titers/ml conversion working stocks or sample virus
Titre.
Further, wherein the TCID50Measuring method specifically comprises the following steps：
It is prepared by cell suspension：T75 bottles 2-3 bottles of host cell is taken, abandons supernatant, every bottle plus 3ml, 0.25% pancreatin, 37 DEG C
2~5min is digested, 6ml cell growth mediums, fully dispersed cell is added after the completely de- wall of cell；Microscopic count is sampled,
It is 2 × 10 to adjust cell concentration5/ ml is spare；
Cell inoculation and culture：Several pieces of 96 porocyte culture plates are taken, the type depending on measuring virus is inoculated with the place prepared
Chief cell suspension, the holes 0.1ml/；Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 24~36hr, work as cell
The single layer for growing up to 70% or so can be used to virus inoculation；
Viral dilution：By virus liquid to be measured after 3 DEG C of water-baths thaw rapidly, make continuous 10 in 15ml sterile centrifugation tubes
It dilutes again, i.e., is inhaled with 2ml suction pipes or pipettor takes 0.5ml virus liquids, be added to (10 in the 1st centrifuge tube equipped with 4.5mlMEM-1), after mixing well, suction pipe is replaced, 0.5ml is drawn and is added in the 2nd centrifuge tube, continuous so operation continues to dilute, so
Analogize, is diluted to the 8th pipe (10-8)；
Viruses adsorption：From CO296 porocyte plates are taken out in incubator, abandon supernatant, are washed 1 time with PBS or serum-free MEM,
The viral 0.1ml of different dilutions is added in every hole, 8 holes are repeated per dilution；Cell plates set 37 DEG C, 5% (v/v) CO2Training
It supports after adsorbing 1hr in case, abandons virus liquid, add fresh maintenance culture solution 0.1ml per hole, continue to cultivate；
Viral CPE observations：Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 3~4 days, record cell
CPE situations calculate sample virus TCID with reference to Reed-Muench methods50。
Further, wherein the real-time fluorescence quantitative RT-PCR measuring method specifically comprises the following steps：
It is prepared by cell suspension：1 T25 bottles of host cell is taken, supernatant is abandoned, adds 1ml, 0.25% (v/v) pancreatin, 37 DEG C disappear
Change 2~5min, the MEM cell growth mediums of 3ml10% (v/v) FBS are added after the completely de- wall of cell, it is fully dispersed thin
Born of the same parents；Microscopic count is sampled, adjustment cell concentration is 2 × 105/ ml is spare；
Cell inoculation and culture：Several pieces of 12 porocyte culture plates are taken, the type depending on measuring virus is inoculated with the place prepared
Chief cell suspension, the holes 1ml/；Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 24~36hr, when cell is long
It is used for virus inoculation at 60%~80% degree of converging；
Viruses adsorption：From CO212 porocyte plates are taken out in incubator, abandon supernatant, are washed 1 time with PBS or serum-free MEM,
The virus stock solution used (10 of acceptable diluent degree is added in every hole-1Or 10-2) 0.2ml, repeat 3 holes；It is simultaneously negative control with MEM, behaviour
Make to wait for gaging hole with virus；Cell plates set 37 DEG C, 5%CO2Incubator in after absorption 1hr, abandon virus liquid, add fresh dimension per hole
Culture solution 1ml is held, continues to cultivate；
Viral CPE observations and receipts sample：Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 3~4 days；With
Inverted microscope observes CPE situations, and sample can be received after there are CPE situations；The free and adherent all cells of supernatant are collected, per hole
Cell freezes after fully being cracked with 400 μ lTRIzol in -20 DEG C of refrigerators；
Sample RNA extractions：Use InvitrogenReagent extracts sample RNA；
Reverse transcription reaction：Reverse transcription is carried out using the M/MLV of Promegea, synthesizes the first chains of cDNA；
Real time fluorescent quantitative：Fluorescent quantitation reaction is carried out using specific primer probe and PremixEXTaq (qPCR) enzyme,
Measure Working viral stoste and sample RNA copy numbers, according to formula 1000vRNA copy numbers/ml=1pfu infection titers/
Ml conversion working stocks and sample virus titre.
Further, further include with 10 before step 3) wherein after step 2)-4~10-1The virus kind of dilution
Sub- stoste carries out the step of next round expands to obtain indicator virus working stock.
Further, the optimization of wherein step 4) the inactivation of viruses confirmatory experiment pH value specifically includes：
Vesicular stomatitis virus is strongest to the tolerance of low pH in three kinds of indicator virus, selects it for the Optimal Experimental
Indicator virus, adjust pH=3.0 ± 0.1 of inactivated samples, 3.5 ± 0.1,4.0 ± 0.1,4.5 ± 0.1,5.0 ± 0.1, and
15-60min (preferably 30min) is kept the temperature in 37 DEG C of ± 1 DEG C of water-baths, when sample solution temperature rises to 37 DEG C ± 1 DEG C, by sample
Product：Virus=9:Ratio (the v of 1 (the Working viral stoste that 1/10 is added in total system):V) VSV viruses are added, 18~
25 DEG C are virus inactivated experiment at room temperature, are respectively sampled when being incubated 0min, 15min, 30min, 60min and 120min,
And measure each pH value and the sample virus titre at each time point.
Further, wherein the inactivated samples are sample/virus mixed liquor that indicator virus is added.
Further, wherein positive domestic animal control described in step 5) is Working viral stoste.
The present invention has the advantages that：
1, method provided by the present invention can realize that laboratory simulates the complete of genetically engineered drug production technology inactivation of virus
Process optimizes amplification, the purifying process of indicator virus, and indicator virus working stock titre is made to improve, pH value unification to 7.0,
Stable quality and beneficial to subsequent operation, while optimize particular technique details that low pH is incubated (such as optimal determination for being incubated pH value,
The optimization etc. of pH regulative modes), what raising inactivated common DNA virus, RNA virus, envelope virus and nonenveloped virus
Validity and actual effect.
2, since MuLV viruses are a kind of retrovirus in mouse source, it can infect and be usually used in genetically engineered drug/biology system
The CHO vehicles cells of product production, indicator virus strain amplification titre is relatively low, conventional method (such as TCID50) cannot be used to examine
It surveys；" virus virulence real time fluorescent quantitative measures new technology " that the present invention establishes, is different from conventional virus titration method, has
Quickly, feature efficiently, accurate, after being converted by copy number can before and after precise determination MuLV inactivation of virus sample titre, answer
It is tested for Validation of Virus Inactivation in Human.Meanwhile according to corresponding viral design primed probe after, this method can also be applied to other
The titer determination of indicator virus.
Description of the drawings
Fig. 1 is the column diagram that each pH value of VSV is incubated inactivation rate；
Fig. 2 is the column diagram that PRV low ph values are incubated inactivation rate；
Fig. 3 is the column diagram that VSV low ph values are incubated inactivation rate；
Fig. 4 is the column diagram that MuLV low ph values are incubated inactivation rate.
Specific implementation mode
The following example will be further illustrated other features and advantages of the present invention, but such embodiment it is merely illustrative and
With not limitation of the present invention.
Material or reagent used herein below are commercially available unless otherwise noted.
The present invention provides the verification method that a kind of low pH is incubated inactivation of viruses, the specific technical solution taken is as follows：
One, the amplification, purifying of indicator virus and titer determination
Select Pseudorabies virus (Pseudorabiesvirus, PRV), murine leukemia virus (MuLV), vesicular stomatitis disease
Malicious (Vesicularstomatitisvirus, VSV), pig parvoviral (Porcine parvovirus, PPV) are indicator virus
(being purchased from China typical culture collection center (CCTCC)).Its viral attribute is shown in Table 1, covers each kind and the DNA of host
Virus, RNA virus, envelope virus and nonenveloped virus.
1. low pH of table is incubated the indicator virus in Validation of Virus Inactivation in Human research
1, the amplification and purifying of indicator virus
The amplification flow of each virus is roughly the same, and the corresponding host cell of indicator virus is selected (to be purchased from Chinese Typical Representative
Culture collection (CCTCC)), if VSV is African green monkey kidney cell (Vero) cell, PRV is porcine kidney cell (PK-15),
MuLV is cat astrocytes (PG4), and corresponding pig parvoviral is porcine kidney cell (IBRS-2).When virus multiplication, by each host
Cell inoculation in the T75 Tissue Culture Flasks of the MEM culture mediums containing 10%FBS (a kind of host cell corresponds to a kind of virus, point
Not Jie Zhong), be positioned over 37 DEG C of 5%CO2It is cultivated in incubator.When cell confluency degree reaches 90%, cell conditioned medium is discarded, is added
Each virus liquid 3ml of acceptable diluent degree, 37 DEG C of 1~2h of absorption.After absorption, virus liquid is discarded in Pasteur's liquid (at disinfection
Reason), clean cell one time with the MEM culture mediums of serum-free, then left with the MEM medium cultures 36h containing 2% (v/v) FBS
The right side, when 80% or more serious change (CPE) occurs for cell but floats (cells float is caused by viral CPE) without complete lesion
When, entire T75 bottles are frozen into -70 DEG C of ultra low temperature freezers, multigelation 3 times (can be frozen overnight for the first time) also can be used directly
Ultrasonic disruption, 4 DEG C of 3000g centrifugations 10min remove cell fragment, collect supernatant, then are nothing after being filtered with 0.22 μm of filter
The viral seed stocks of bacterium.Viral seed stocks are dispensed with the sterile EP tube of 1.5ml, and often pipe 1ml is placed in ultra low temperature freezer long
Phase preserves.
And indicator virus working stock is then to carry out next round with appropriate diluted viral seed stocks to expand to obtain.Due to
PH when each virus harvest is different, and influence of 1/10 volume to whole system also each not phase is added when carrying out low pH and being incubated
Together, for the pH of unified each Working viral stoste and virus is further purified, improves the purity and quality of virus, at 4 DEG C 3,
After 000g centrifuges 10min removal cell fragments, after taking 4 DEG C of 72,000g ultracentrifugations 4h of supernatant, abandon supernatant, precipitation exceed the speed limit from
1/2 serum-free MEM (pH=7.0) dissolvings (MuLV and PPV titres are relatively low, original volume 1/5 can be used to dissolve) of original volume before the heart, most
The demand for being incubated the experiment of method Validation of Virus Inactivation in Human according to low pH afterwards dispenses Working viral stoste, while dispensing 1ml ramuscules and being dripped
Degree measures.
2, the titer determination of indicator virus
It is significantly viral for CPE, using TCID50Method carries out titer determination, and not high for CEP unobvious or titre
Viral (such as MuLV) measures Working viral stoste titre using real-time fluorescence quantitative RT-PCR method.The specific method is as follows for it：
2.1 TCID50Measuring method
2.1.1 prepared by cell suspension：T75 bottles 2-3 bottles of host cell is taken, supernatant is abandoned, every bottle adds 3ml0.25% pancreatin,
The MEM cell growth mediums that 6ml contains 10% (v/v) FBS are added in 37 DEG C of 2~5min of digestion after the completely de- wall of cell
(Gbico companies), fully dispersed cell.Microscopic count is sampled, adjustment cell concentration is 2 × 105/ ml is spare.
2.1.2 cell inoculation and culture：Several pieces of 96 porocyte culture plates are taken, the type inoculation depending on measuring virus prepares
Host cell suspension, the holes 0.1ml/.Tissue culture plate sets 37 DEG C, 5%CO2Incubator in cultivate 24~36hr, work as cell
The single layer for growing up to 70% or so can be used to virus inoculation.
2.1.3 viral dilution：By virus liquid to be measured after 3 DEG C of water-baths thaw rapidly, make in 15ml sterile centrifugation tubes
Continuous 10 times of dilutions, i.e., inhaled with 2ml suction pipes or pipettor take 0.5ml virus liquids, be added to the 1st centrifuge tube equipped with 4.5mlMEM
Interior (10-1), after mixing well, suction pipe to be replaced, 0.5ml is drawn and is added in the 2nd centrifuge tube, continuous so operation continues to dilute,
So analogize, is diluted to the 8th pipe (10-8)。
2.1.4 viruses adsorption：From CO296 porocyte plates are taken out in incubator, abandon supernatant, are washed with PBS or serum-free MEM
It 1 time, is added through 10 times of serial dilutions (10 in every hole-1-10-2...) different dilutions viral 0.1ml, per dilution repeat
8 holes.Cell plates set 37 DEG C, 5% (v/v) CO2Incubator in after absorption 1hr, abandon virus liquid, add containing 2% (v/v) per hole
The fresh MEM of FBS maintains culture solution 0.1ml, continues to cultivate.
2.1.5 virus CPE observations：Tissue culture plate sets 37 DEG C, 5%CO2Incubator in cultivate 3~4 days.It is aobvious with being inverted
Micro mirror observes cytopathy situation, records result.
2.2 real-time fluorescence quantitative RT-PCR measuring methods
2.2.1 prepared by cell suspension：1 T25 bottles of host cell is taken, supernatant is abandoned, adds 1ml0.25% pancreatin, 37 DEG C disappear
Change 2~5min, MEM cell growth mediums of the 3ml containing 10%FBS, fully dispersed cell is added after the completely de- wall of cell.It takes
Sample microscopic count, adjustment cell concentration are 2 × 105/ ml is spare.
2.2.2 cell inoculation and culture：Several pieces of 12 porocyte culture plates are taken, the type inoculation depending on measuring virus prepares
Host cell suspension, the holes 1ml/.Tissue culture plate sets 37 DEG C, 5%CO2Incubator in cultivate 24~36hr, when cell is long
Virus inoculation is can be used at 70% or so degree of converging.
2.2.4 viruses adsorption：From CO212 porocyte plates are taken out in incubator, abandon supernatant, are washed with PBS or serum-free MEM
1 time, the virus stock solution used (10 of acceptable diluent degree is added in every hole-1Or 10-2) 0.2ml, repeat 3 holes.It is simultaneously negative right with MEM
According to operation waits for gaging hole with virus.Cell plates set 37 DEG C, 5% (v/v) CO2Incubator in after absorption 1hr, virus liquid is abandoned, per hole
The fresh maintenance culture solution 1ml of MEM containing 2% (v/v) FBS are added, continue to cultivate.
2.2.5 virus CPE observations and receipts sample：Tissue culture plate sets 37 DEG C, 5%CO2Incubator in cultivate 3~4 days.With
Inverted microscope observes CPE situations, and sample can be received after there are CPE situations.The free and adherent all cells of supernatant are collected, per hole
Cell freezes after fully being cracked with 400 μ lTRIzol in -20 DEG C of refrigerators.
2.2.6 sample RNA extractions：Use InvitrogenReagent extracts sample RNA.Specific method is such as
Under：
A. trypsin digestion cell, 1000r/min centrifuge 10min and collect 5*105~5*106Cell is filled with 400 μ l Trizol
Divide dissolving cell precipitation, be transferred in the 1.5mlEP pipes of new no RNA enzyme, 80 μ l chloroform (chloroforms are added：Trizol=1:5) it fills
Divide mixing, acutely concussion 15 seconds, are placed at room temperature for 5min, the liquid of mixing is made tentatively to be layered.
B. sample is put into 4 DEG C of 12000g centrifugation 15min in refrigerated centrifuge, carefully takes out upper layer and moves to new no RNA
Enzyme EP pipes will avoid being drawn onto middle protein layering or lower layer's chloroformic solution, and would rather take less can not be drawn onto other compositions more.
C. isopropanol (the isopropanol of 200 μ l precoolings is added：Trizol=1:2) it, pitches and mixes well for several times, room temperature
(or -20 DEG C) staticly settle 10min.
D.4 DEG C 12000g centrifuges 10min, carefully sops up supernatant, precipitation is fully washed with 75% absolute ethyl alcohol of precooling.
E.4 DEG C 7500g centrifuges 5min, fully removes supernatant, the inversion of EP pipes is placed on to be precipitated close to complete in super-clean bench
When dry (there are no being completely dried, precipitation color has just begun to change into bright), it is heavy that 20 μ lRNase-freeWater dissolvings are added
It forms sediment.
F.60 DEG C water-bath 10min is fully to dissolve RNA, after spectrophotometer Biophotemeter measured concentrations, rapidly
RNA is put into ultra low temperature freezer (- 70 DEG C of refrigerators) to preserve, spare carry out follow-up test.2.2.7 reverse transcription reaction：It uses
The M/MLV of Promegea carries out reverse transcription, synthesizes the first chains of cDNA.It is carried out in two steps, reaction system is first added：
1 μ g of total serum IgE
RP-MuLV(50μM) 1μl
ddH2O is until 13 μ l
It is quickly placed on ice, adds after 70 DEG C of reaction 10min after mixing well：
Reaction system mix well after in 37 DEG C react 60min, after in 70 DEG C inactivate 5min, be stored in -20 DEG C with
For subsequent reactions.
2.2.8 real time fluorescent quantitative
PCR instrument：U.S. Bio-Rad
Reaction system:20ul
Reaction condition：
Used primer, probe sequence are respectively in real time fluorescent quantitative reaction：
FP-MuLV:ACCTCTCATTGACCTTCT
RP-MuLV:GGAATCTCAGAGTGGAG
Q-MuLV probes:FAM-CTTCTCTGTCGCCATCTCCG-BHQ1
Using TCID50Method measures virus titer, then with reference to Reed-Muench methods, calculates sample virus TCID50。
Virus titer is measured using real-time fluorescence quantitative RT-PCR method, then obtains sample result phase by comparing standard curve
RNA copy numbers are answered, with formula 1000vRNA copy numbers/ml ≈ 1pfu infection titers/ml conversion sample virus titres.Each batch
The titre of indicator virus working stock is as shown in table 2, show we expand, purifying virus titer it is equal>6log/ml meets medicine
Prison office and the requirement for evaluating hub file can be used for follow-up low pH and be incubated inactivation of viruses confirmatory experiment.
2. each batch Working viral stoste technical parameter of table
Two, low pH is incubated inactivation indicator virus condition optimizing and confirmatory experiment effect
1, the optimization of inactivation of viruses confirmatory experiment pH value
In the confirmatory experiment, most crucial experiment condition is the pH value after sample and indicator virus mixing.PH value in principle
It is lower, it is better to the inactivating efficacy of virus, but in practical biological industry production technology, many tolerable pH of biological products
There is certain limit in the time that value and low pH are incubated, it is necessary to not influence gene engineering drug/biological products molecular characterization
With technique is virus inactivated under the premise of biological function.Therefore, it is necessary to contrived experiment finds the upper limit of a pH value, it can be really
The indicator virus that all kinds are inactivated within the regular hour is protected, so as to the incubation that the property reselection according to different samples is optimal
PH value scheme.
VSV viruses are strongest to the tolerance of low pH in three kinds of indicator virus, select it for the instruction of the Optimal Experimental
Virus, adjusts pH=3.0 ± 0.1 of inactivated samples, and 3.5 ± 0.1,4.0 ± 0.1,4.5 ± 0.1,5.0 ± 0.1, and in 37 DEG C
30min is kept the temperature in ± 1 DEG C of water-bath, when sample solution temperature rises to 37 DEG C ± 1 DEG C, by sample：Virus=9:1 is added VSV diseases
Poison is being incubated 0min respectively in the inactivation of virus experiment of (18~25 DEG C) progress genetically engineered drug production technologies of room temperature,
It is sampled when 15min, 30min, 60min and 120min, and measures each pH value and the sample virus titre at each time point, result is such as
Shown in table 3, the sample of 0min samplings has virus in blending process and is inactivated, therefore the drop of available VSV Working viral stostes
Degree (log/ml) subtracts the virus titer (log/ml) in real time sample sample, and the inactivation of virus for obtaining different sampling time points is fixed
Data (LRV, log/ml) are measured, according to Bureau of Drugs Supervision and evaluate hub file requirement, as LRV >=4.0, then the inactivation technology is effective.
Using sample time as abscissa, virus titer (log/ml) is ordinate, draws the column diagram that the low pH of VSV are incubated inactivation rate
(Fig. 1), in conjunction with table 3 and Fig. 1, when pH=3.0 ± 0.1 and ± 0.1 pH=3.5, LRV >=4.0 can be made by being incubated 60min, when
PH=4.0 ± 0.1, then to be incubated 120min could inactivate effectively.The data to it is follow-up we be customization experimental program tool
There is directive significance, that is, the pH value highest of sample is recommended to control in pH=4.0 ± 0.1.
Table 3. is respectively incubated pH value to VSV inactivation of virus effects
2, the optimization of pH value adjusting method
Even by the result of table 3 and Fig. 1 it is found that in the 0min that indicator virus is just added, the drop of virus is measured by sampling
Degree comparison VSV working stocks all decreases to some degree.In actual verification experiment, we want it is confirmed that final sample adds
Enter pH value when incubation after indicator virus, since the low pH of the biological sample buffer systems being incubated are generally glycine or citric acid is molten
Liquid, buffer capacity is poor, and 1/10 indicator virus (pH=7.0) is added and necessarily increases the pH value of total system, therefore must survey
PH value after fixed addition virus recalls to specified pH value with conditioning agent again, and ordinary operation mode takes time and effort, and the sample of 0min takes
Sample can also be affected, PRV especially sensitive to low pH and MuLV viruses, can strong influence measurement result.Therefore, we
By taking sample is glycine buffer system as an example, step pH value adjusting preliminary experiment is added before formal verification is tested, to ensure result
Accuracy, every batch of sample are tested in triplicate, and the results are shown in Table 4：If being pre-adjusted pH value the incubating less than setting of sample
PH value about 0.3~0.4 is educated, then the Working viral stoste of 1/10 volume pH=7.0, which is added, can directly make the pH value liter of total system
The pH value up to set need not then measure and adjust pH, to save time and behaviour again in formal confirmatory experiment
Make step, and the sampling error to 0min samples is preferably minimized.
4. sample pH of table adjusts preliminary result
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
| Sample volume | 18ml | 18ml | 18ml | 18ml | 18ml | 18ml |
| PH before addition virus | 3.0±0.1 | 2.7±0.1 | 3.5±0.1 | 3.2±0.1 | 4.0±0.1 | 3.6±0.1 |
| Viral volume (pH=7.0) is added | 2ml | 2ml | 2ml | 2ml | 2ml | 2ml |
| Low pH is incubated total volume | 20ml | 20ml | 20ml | 20ml | 20ml | 20ml |
| PH after addition virus | 3.4±0.1 | 3.0±0.1 | 3.8±0.1 | 3.5±0.1 | 4.4±0.1 | 4.0±0.1 |
3, low pH is incubated inactivation of viruses confirmatory experiment effect
The low pH of affinity chromatography main peak sample is incubated (pH=3.7 ± 0.1) inactivation of viruses process certification experiment, selects PRV,
VSV and MuLV is indicator virus, and PK15, Vero and PG4 cells are virus host cells.Chromatography main peak sample is derived from production process
In, before low pH is incubated, 18ml/ batches, in -20 DEG C of preservations, thaw before experiment, measure and adjust its pH value to pH=3.4 ±
0.1, and it is temporary spare in 2~8 DEG C.Sample 9ml is taken, three kinds of 1ml indicator virus working stocks are added, mixing, keeps sample and (take immediately
Tris neutralizers are added after sample and adjust pH=7.0), it is 0min samples.It is another to take sample 18ml, 2ml is added and indicates three kinds of viral works
Make stoste, immediately mixing, sample is placed in Biohazard Safety Equipment under the conditions of 18~25 DEG C of P2 grades of bio-safety>15h, therebetween
Respectively at 15min, 30min, 60min, 2h, 3h, 6h, 12h and>15h, which keeps sample, (to be added Tris neutralizers and adjusts pH=after sampling
7.0).Three batches of samples operate on an equal basis.Samples taken is stored in -20 DEG C of standby surveys after being neutralized to neutrality.Viral drop is carried out to the above sample
Degree measures, and every part of sample replication is twice.
PRV and VSV titer determinations use 96 well culture plate micromethods, and observation in 3 days is cultivated simultaneously after virus infection host cell
Pathological changes caused by virus degree is recorded, calculating virus titer (log/ml) with Karber methods assesses inactivation of viruses efficiency.MuLV drops
Degree measures and then uses real-time fluorescence quantitative RT-PCR measuring method, is cultivated 6 days after virus infection host cell and observes and records virus
Cytopathogenic effect degree receives sample and extracts cell total rna, and after reverse transcription synthesizes the first chains of cDNA, real time fluorescent quantitative measures virus
Titre obtains the corresponding RNA copy numbers of sample result, with formula 1000vRNA copy numbers/ml ≈ 1pfu by comparing standard curve
Infection titer/ml conversion sample virus titres, and calculate virus titer (log/ml) assessment inactivation of viruses efficiency.Inactivation of virus
The sample of three batches of validation test, if untreated Working viral stoste is as positive control, sour neutralizer system blank
Compare (virus-free), host cell culture negative control.Each test experience is repeated 2 times, and each dilute sample sets 8 repetitions
Hole.Verification testing result is shown in Table 5 respectively, table 6 and table 7.
Detect low pH be incubated (pH=3.7 ± 0.1) 0min, 15min, 30min, 60min, 2h, 3h, 6h, 12h and>15h samples
The PRV virus titers (log/ml) of product, because low pH incubations are rapid to the inactivation of PRV viruses, the sample of 0min samplings is in mixing mistake
Have virus in journey to be inactivated, therefore the virus titer (log/ml) of available positive domestic animal control subtracts the virus in real time sample sample
Titre (log/ml) obtains the inactivation of virus quantitative data (LRV, log/ml) of different sampling time points, is cross with sample time
Coordinate, virus titer (log/ml) are ordinate, draw the column diagram (Fig. 2, Fig. 3 and Fig. 4) of three kinds of viral inactivation rates.As a result
Show that the virus inactivation technology that the low pH of the affinity chromatography main peak sample is incubated (pH=3.7 ± 0.1) is effective inactivation instruction disease
The method of malicious PRV, VSV and MuLV.
5. low pH of table is incubated inactivation Pseudorabies virus (PRV) process certification testing result
6. low pH of table is incubated inactivation vesicular stomatitis virus (VSV) process certification testing result
7. low pH of table is incubated inactivation murine leukemia virus (MuLV) process certification testing result
In conclusion the foregoing is merely presently preferred embodiments of the present invention, the patent for not thereby limiting the present invention is protected
Shield range, therefore description of the invention and the made equivalence changes of attached drawing etc. such as, should all be included in protection scope of the present invention.
Claims (10)
1. a kind of low pH is incubated the verification method of inactivation of viruses, which is characterized in that include the following steps：
1) indicator virus and its corresponding host cell are selected：Select Pseudorabies virus, murine leukemia virus, vesicular stomatitis disease
Poison, pig parvoviral are indicator virus；The corresponding host cell of indicator virus is selected, corresponding Pseudorabies virus is that pig kidney is thin
Born of the same parents, corresponding murine leukemia virus is cat astrocytes, and corresponding vesicular stomatitis virus is African green monkey kidney cell cell, pig
Corresponding parvovirus is porcine kidney cell；
2) indicator virus amplification and purifying：Ultracentrifugation uses the dissolving of fresh serum free MEM culture mediums instead after conventional amplification receives sample, makes
Its virus titer improves, and pH value is unified for 7.0；
3) titer determination of Working viral stoste and sample：Using TCID50Method or real-time fluorescence quantitative RT-PCR method carry out titre
It measures；
4) optimization of inactivation of viruses confirmatory experiment pH value：The optimal incubation pH. and pH value of indicator virus is determined by preliminary experiment
Best regulative mode；
5) low ph value is incubated inactivation of viruses recruitment evaluation：The virus titer compareed with positive domestic animal subtracts the virus in real time sample sample
Titre obtains the inactivation of virus quantitative data (LRV, log/ml) of different sampling time points, if LRV >=4 at the time point, low
The inactivation technology inactivation of viruses that pH is incubated the time is effective.
2. low pH as described in claim 1 is incubated the verification method of inactivation of viruses, which is characterized in that step 2) the instruction disease
The amplification and purifying of poison specifically comprise the following steps：
Each host cell is seeded in respectively in the T75 Tissue Culture Flasks of the MEM culture mediums containing 10% (v/v) FBS, is positioned over
37 DEG C, 5%CO2It is cultivated in incubator；
When host cell degree of converging reaches 80%-90%, cell conditioned medium is discarded, is added 10-4-10-1The corresponding virus of dilution
Liquid 3ml, 37 DEG C of 1~2h of absorption；
After absorption, virus liquid is discarded in Pasteur's liquid, cell is cleaned one time with the MEM culture mediums of serum-free, then with containing
The MEM medium cultures 36-48h or so of 2% (v/v) FBS, when 80% or more serious change (CPE) occurs for host cell but does not have
When having complete lesion to float, cell is crushed, 4 DEG C of 3000g centrifugations 10min remove cell fragment, collect supernatant, then use
As sterile viral seed stocks after 0.22 μm of filter filtering；
It takes 4 DEG C of above-mentioned supernatant, after 72,000g ultracentrifugation 4h, abandons supernatant, the 1/5-1/2 of original volume before precipitation ultracentrifugation
Serum-free MEM (pH=7.0) dissolves so that the pH of Working viral stoste is uniformly adjusted to 7.0.
3. low pH as claimed in claim 2 is incubated the verification method of inactivation of viruses, which is characterized in that the broken side of the cell
Method is：When 80% or more serious change (CPE) occurs for host cell but is floated without complete lesion, entire T75 bottles are frozen
Enter -70 DEG C of ultra low temperature freezers, and in -70 DEG C and 37 DEG C of water-bath multigelations 3 times, each freeze thawing time is 30min-1h, and first
Secondary -70 DEG C of refrigerator overnights；Or host cell is directly crushed with ultrasonic wave.
4. low pH as described in claim 1 is incubated the verification method of inactivation of viruses, which is characterized in that the wherein step 3) disease
The titer determination of malicious working stock and sample specifically comprises the following steps：
The demand that the experiment of method Validation of Virus Inactivation in Human is incubated according to low pH dispenses Working viral stoste, while being gone forward side by side with the packing of 1ml/ branch
Row titer determination；
It is significantly viral for CPE, using TCID50Method carries out titer determination, and for the not high disease of CEP unobvious or titre
Poison measures titre using real-time fluorescence quantitative RT-PCR method；
The TCID50When method measures virus titer, with reference to Reed-Muench methods, evaluation work stoste or sample virus
TCID50；
It is corresponding by comparing standard curve acquisition sample result when the real-time fluorescence quantitative RT-PCR method measures virus titer
RNA copy numbers are dripped with formula 1000vRNA copy numbers/ml=1pfu infection titers/ml conversion working stocks or sample virus
Degree.
5. low pH as claimed in claim 4 is incubated the verification method of inactivation of viruses, which is characterized in that the wherein described TCID50It surveys
The method of determining specifically comprises the following steps：
It is prepared by cell suspension：T75 bottles 2-3 bottles of host cell is taken, abandons supernatant, every bottle plus 3ml, 0.25% pancreatin, 37 DEG C digest 2
6ml cell growth mediums, fully dispersed cell is added after the completely de- wall of cell in~5min；Microscopic count is sampled, adjustment is thin
Born of the same parents a concentration of 2 × 105/ ml is spare；
Cell inoculation and culture：Several pieces of 96 porocyte culture plates are taken, the host that the type inoculation depending on measuring virus prepares is thin
Born of the same parents' suspension, the holes 0.1ml/；Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 24~36hr, when cell grows up to
70% or so single layer can be used to virus inoculation；
Viral dilution：By virus liquid to be measured after 3 DEG C of water-baths thaw rapidly, make in 15ml sterile centrifugation tubes continuous 10 times it is dilute
It releases, i.e., is inhaled with 2ml suction pipes or pipettor takes 0.5ml virus liquids, be added to (10 in the 1st centrifuge tube equipped with 4.5ml MEM-1),
After mixing well, suction pipe is replaced, 0.5ml is drawn and is added in the 2nd centrifuge tube, continuous so operation continues to dilute, such as such
It pushes away, is diluted to the 8th pipe (10-8)；
Viruses adsorption：From CO296 porocyte plates are taken out in incubator, abandon supernatant, are washed 1 time with PBS or serum-free MEM, in every hole
The viral 0.1ml of different dilutions is added, 8 holes are repeated per dilution；Cell plates set 37 DEG C, 5% (v/v) CO2Incubator in
After adsorbing 1hr, virus liquid is abandoned, fresh maintenance culture solution 0.1ml is added per hole, continues to cultivate；
Viral CPE observations：Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 3~4 days, record cell CPE shapes
Condition calculates sample virus TCID with reference to Reed-Muench methods50。
6. low pH as claimed in claim 4 is incubated the verification method of inactivation of viruses, which is characterized in that the real time fluorescent quantitative
RT-PCR measuring methods specifically comprise the following steps：
It is prepared by cell suspension：1 T25 bottles of host cell is taken, supernatant is abandoned, adds 1ml, 0.25% (v/v) pancreatin, 37 DEG C of digestion 2
The MEM cell growth mediums of 3ml 10% (v/v) FBS, fully dispersed cell is added in~5min after the completely de- wall of cell；
Microscopic count is sampled, adjustment cell concentration is 2 × 105/ ml is spare；
Cell inoculation and culture：Several pieces of 12 porocyte culture plates are taken, the host that the type inoculation depending on measuring virus prepares is thin
Born of the same parents' suspension, the holes 1ml/；Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 24~36hr, when cell grows up to
60%~80% degree of converging is used for virus inoculation；
Viruses adsorption：From CO212 porocyte plates are taken out in incubator, abandon supernatant, are washed 1 time with PBS or serum-free MEM, in every hole
The virus stock solution used (10 of acceptable diluent degree is added-1Or 10-2) 0.2ml, repeat 3 holes；It is simultaneously negative control with MEM, operates with disease
Poison waits for gaging hole；Cell plates set 37 DEG C, 5%CO2Incubator in after absorption 1hr, abandon virus liquid, fresh maintenances added per hole and is cultivated
Liquid 1ml continues to cultivate；
Viral CPE observations and receipts sample：Tissue culture plate sets 37 DEG C, 5% (v/v) CO2Incubator in cultivate 3~4 days；With inversion
Micro- sem observation CPE situations can receive sample after there are CPE situations；The free and adherent all cells of supernatant are collected, per hole cell
It is frozen in -20 DEG C of refrigerators after fully being cracked with 400 μ l TRIzol；
Sample RNA extractions：Use InvitrogenReagent extracts sample RNA；
Reverse transcription reaction：Reverse transcription is carried out using the M/MLV of Promegea, synthesizes the first chains of cDNA；
Real time fluorescent quantitative：Fluorescent quantitation reaction is carried out using specific primer probe and Premix EX Taq (qPCR) enzyme, is surveyed
The RNA copy numbers for determining Working viral stoste and sample, according to formula 1000vRNA copy numbers/ml=1pfu infection titers/ml
Convert working stock and sample virus titre.
7. low pH as described in claim 1 is incubated the verification method of inactivation of viruses, which is characterized in that wherein step 2) it
Afterwards, further include with 10 before step 3)-4~10-1The viral seed stocks of dilution carry out next round and expand to obtain indicator virus
The step of working stock.
8. low pH as described in claim 1 is incubated the verification method of inactivation of viruses, which is characterized in that step 4) the inactivation disease
The optimization of malicious confirmatory experiment pH value specifically includes：
Vesicular stomatitis virus is strongest to the tolerance of low pH in three kinds of indicator virus, selects it for the finger of the Optimal Experimental
Show virus, adjusts pH=3.0 ± 0.1 of inactivated samples, 3.5 ± 0.1,4.0 ± 0.1,4.5 ± 0.1,5.0 ± 0.1, and in 37
15-60min is kept the temperature in DEG C ± 1 DEG C of water-bath, when sample solution temperature rises to 37 DEG C ± 1 DEG C, by sample：Virus=9:1(v:v)
Ratio VSV viruses are added, be virus inactivated experiment at room temperature at 18~25 DEG C, respectively in incubation 0min, 15min,
It is sampled when 30min, 60min and 120min, and measures each pH value and the sample virus titre at each time point；
Preferably, the time kept the temperature in the water-bath is 30min.
9. low pH as claimed in claim 8 is incubated the verification method of inactivation of viruses, which is characterized in that the inactivated samples are to add
Enter sample/virus mixed liquor of indicator virus.
10. low pH as described in claim 1 is incubated the verification method of inactivation of viruses, which is characterized in that positive described in step 5)
Domestic animal control is Working viral stoste.
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| CN112359139A (en) * | 2020-11-06 | 2021-02-12 | 苏州药明检测检验有限责任公司 | Method for determining heterophilic murine leukemia virus titer by tissue median infection staining |
| CN113151593A (en) * | 2020-12-31 | 2021-07-23 | 肇庆大华农生物药品有限公司 | Method for determining content of virus difficult to observe whether tissue cells are infected or not |
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| CN115181816A (en) * | 2022-08-10 | 2022-10-14 | 杭州纽创生物检测有限公司 | Method for detecting virus titer in ultralow virus titer system and application thereof |
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