CN108570445A - PK15 cells tame suspension process and second order virus production technique - Google Patents
PK15 cells tame suspension process and second order virus production technique Download PDFInfo
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Abstract
The present invention relates to PK15 cells domestication suspension process and second order virus production techniques.Specifically, the present invention relates to domestication PK15 cells to adapt it to serum free suspension culture, and using it as host cell, malicious mode is connect using second order culture and produces and lesion or there can be the virus of sensibility on suspension PK15 cells, that is, cell is grown to 2.0~20.0 × 106When cell/mL, 1~5 times is diluted with production medium, makes density 1.0~10.0 × 106Between cell/mL, poison is then connect, or connect poison before dilution.The cell adapted suspension growths in the culture medium that serum-free and chemical composition determine of suspension PK15 of the present invention, second order virus production is simple for process, easily amplifies, and does not change liquid, and culture medium utilization rate is high；Production and growth medium may be the same or different, and the two proportionally mixes, you can makes cell diauxic growth and promotes expressing viral.
Description
Technical field
The present invention relates to PK15 cells domestication suspension process and suspension PK15 cell related vaccines production fields, and in particular to
The bioreactor culture suspension PK15 cells of 30L or more, with second order cultural method culture suspension PK15 cells to produce epidemic disease
The technology of seedling.
Background technology
Mammalian cell large-scale culture technology is one of Biopharmaceutical Enterprises downstream general technology, is sent out in the sector
Wave particularly important effect.The key of this technology is the suspension serum-free large-scale culture of realization mammalian cell, from
And production capacity is improved, cost is reduced, and be easy to amplify.Attached cell cultivate when usual way be add in the medium it is a certain amount of
Serum makes cell adherent growth, and serum chemistry ingredient is uncertain, and unstable quality, has difference between batch, is produced with such cell
Biological products, quality is difficult to control, it is difficult to by the examination ＆ approval of food and medicine Surveillance Authority, and suspension free serum culture is opposite
For common adhere-wall culture, unit volume interior energy grows more cells, so as to produce more biological products.
PK15 is porcine kidney cell system, and parent is originated from the adult porcine kidney cell PK- provided nineteen fifty-five U.S. Stice generations
2a, after by U.S. ATCC collections (collection number be ATCC-CCL33).PK15 cells have been widely used in pig circular ring virus, pig passes
The separation of metachromia marcy agent and pig parvoviral etc., the production of in-vitro multiplication and a variety of live vaccines.
The defect that existing PK15 cells domestication suspends is：1, some cannot achieve complete free serum culture；2, general
The method walked using two steps is first reduced to serum-free settling flux culture, due to adhere-wall culture use culture medium it is usual based on
Culture medium increase serum, basal medium nutritional ingredient is weaker, and adhere-wall culture surface area/volume ratio is usually smaller during domestication,
The intake of nutrition is limited, time-consuming for entire domestication process；3, it is tamed after genetic modification, i.e., is adherent thin by genetic modification
Born of the same parents' strain, which is inserted into, is unfavorable for adherent genetic fragment, changes original adhere-wall culture mode, makes cell suspension growth, then pass through again
Continue to optimize and the change of culture medium enable cell serum-free high-density growth, and such method is easy to implement, but inserts
Exogenous genetic fragment influences the quality of product.Therefore, it is necessary to PK15 cells suspension domestication and serum-free acclimation method into one
Step research, and overcome above tripartite's planar defect.
Malicious process aspect is connect in virus, after traditional viral vaccine production technology refers to cell growth to certain density,
Liquid is changed to connect poison again or directly connect poison.So far, it is not reported about the correlation of the second order cultural method of suspension PK15 cells
Road.
Invention content
The first aspect of the present invention is related to a kind of second order cultural method of suspension PK15 cell vaccines production, and it includes as follows
Step：
1) cell growth：Suspension PK15 cells grow to 2.0 × 10 in growth medium6Cell/mL~20.0 × 106
Cell/mL；
2) poison is connect：Poison amount is connect 10-1~10-5Between MOI, or poison amount is connect according to this after step 3) and connect poison；
3) dilute/add production medium：1~5 times is diluted with production medium, cell density is to 1.0 × 10 after dilution6
Cell/mL~10.0 × 106Cell/mL；
4) diauxic growth：Cell diauxic growth；
5) optionally, harvest virus liquid purifies seedling.
In some embodiments, suspension PK15 cell growths be suspension tank culture, training volume be 30L and with
On.
In some embodiments, in step 1) cell density 5.0 × 106Cell/mL~16.0 × 106Cell/mL,
Preferably, 6.0 × 106Cell/mL~15.0 × 106Cell/mL, most preferably, 8.0 × 106Cell/mL.
In some embodiments, cell connects poison amount 10 in step 2)-1~10-4It is more excellent to connect between MOI, 10-1~
10-3MOI, it is optimal to connect, 10-2MOI。
In some embodiments, suspension PK15 cell production mediums are diluted to 1~4 times in step 3), it is highly preferred that
1~3 times of dilution, most preferably 2 times dilutions.
In some embodiments, cell density is 1.0 × 10 after poison being connect in step 3)6Cell/mL~10.0 × 106Carefully
Born of the same parents/mL, it is preferable that 1.0 × 106Cell/mL~5.0 × 106Cell/mL.
In some embodiments, suspension PK15 cells are the suspension PK15 cells used in production of vaccine, or by adherent
The domestication of PK15 cells, the suspension PK15 cells for capableing of suspension growth production, it is preferable that suspension PK15 cells are by adherent
PK15 cells are tamed through low serum or serum-free domestication, and the serum-free domestication refers to that the PK15 cells after domestication can be in nothing
It grows, be proliferated in culture medium existing for serum, and/or, it is preferable that suspension PK15 cells are to be tamed and dociled by adherent PK15 cells through suspending
Change.
In some embodiments, virus for it is sensitive on corresponding suspension PK15 cells, corresponding vaccine can be produced
Virus.
In some embodiments, cell growth temperature is at 35 DEG C~37 DEG C, connect malicious restrovirus production temperature 30 DEG C~
37℃。
In some embodiments, cell growth inoculum density is 0.3 × 106Cell/mL~1.0 × 106Cell/mL, it is raw
Long number of days reached highest cell density at 2~8 days.
In other words, the technical problem that the present invention solves is to overcome the deficiencies of the prior art and provide a kind of adherent PK15
Low serum, serum-free domestication and the suspension acclimation method of cell, make cell growth state stabilization, good dispersion, are suitable for rule
Modelling culture, and be conducive to the duplication and expression of virus.
For this purpose, the present invention enables the cell strain of original adherent growth by the change of culture medium and the change of training method
Enough low serum or serum-free, suspension high-density growth.Wherein, low serum domestication includes during gradually reducing cell secondary culture
Culture medium serum-concentration, until the condition of culture under the cell adapted low-serum-concentrations of PK15.Starting for the domestication of low serum
PK15 cells in optimum state and stable growth can be from the adherent PK15 cell recoveries frozen, the PK15 of recovery
Cell.
Then, after PK15 cells have adapted to adherent low Serum Growth completely, 5~8 kinds of strong suitable biologies are selected
Serial serum free medium carries out serum-free domestication and the domestication that suspends, until PK15 cells adapt to suspension training method completely, and
And suspension PK15 cells can start stable passage growth, suspension PK15 cell densities can reach 6.0 × 106Cell/mL
~15.0 × 106Between cell/mL, motility rate maintains 95% or more.
The present invention also optimizes the ingredients such as amino acid in culture medium, vitamin, trace element, growth factor, hydrolysate, obtains
The suspension PK15 cell non-serum cell culture mediums that must optimize enable the density stabilized growth passage of suspension PK15 cell highers,
Suspension PK15 cells most high-density can reach 6.0 × 106Cell/mL~8.0 × 106Between cell/mL, motility rate maintains
98% or so.
The present invention also developed suspension PK15 cellsCD specific chemical components without albumen, animal origin-free
Serum-free cell culture medium, suspend full culture it is suitable for suspension PK15 high cell densities, suspension PK15 cell most high-densities
6.0 × 10 can be reached6Cell/mL~15.0 × 106Between cell/mL, motility rate maintains 98% or so.
The present invention also provides a kind of second order cultural methods of suspension PK15 cell related vaccines production, and it includes following steps
Suddenly：
(1) cell growth：Cell density is 2.0 × 106Cell/mL~20.0 × 106Cell/mL；
(2) poison is connect：According to production technology, in second day or third day of cell growth, cell density reaches 6.0 ×
106Cell/mL~10.0 × 106Cell/mL, selection connect poison amount 10-1~10-5The indirect poison of MOI, or press after the dilution
Poison amount is connect like this connects poison；
(3) dilute/add virus production culture medium：Give the dilution of 1~5 times of production medium, cell density after dilution
To 1.0 × 106Cell/mL~10.0 × 106Cell/mL；
(4) it harvests：Cell diauxic growth simultaneously purifies seedling according to production technology harvest virus liquid.
In some embodiments, harvest virus liquid purifying seedling refers to according to production technology harvest virus liquid purifying system
Seedling, wherein the production technology refers to that the known production technology of corresponding vaccine is produced using PK15 cells.
In some embodiments, PK15 cell growths are suspension tank culture, and training volume is 30L or more, example
Such as 30L, 35L, 40L, 50L, 60L, 70L, 80L, 90L, 100L or bigger.
In some embodiments, the growth medium of PK15 cells is the culture of any growth suitable for PK15 cells
Base, it is preferable that the culture medium is CD PK15 (JSB-LM17019) serum free medium.
In some embodiments, in step 1) cell density 5.0 × 106Cell/mL~16.0 × 106Cell/mL,
Preferably, 6.0 × 106Cell/mL~15.0 × 106Cell/mL, most preferably, 8.0 × 106Cell/mL.
In some embodiments, cell connects poison amount 10 in step 2)-1~10-4Between MOI, more excellent ground connection, 10-1~
10-3MOI, optimal ground connection, 10-2MOI。
In some embodiments, PK15 cells production medium is diluted to 1~4 times in step 3), it is highly preferred that 1~3
It dilutes again, most preferably 2 times dilutions.
In some embodiments, cell density is 2.0 × 10 after poison being connect in step 3)6Cell/mL~8.0 × 106Carefully
Born of the same parents/mL, it is preferable that 3.0 × 106Cell/mL~5.0 × 106Cell/mL.
In some embodiments, the cell density in step 4) after diauxic growth is 2.0 × 106Cell/mL -20.0 ×
106Cell/mL, for example, 2.0 × 106Cell/mL, 3.0 × 106Cell/mL, 4.0 × 106Cell/mL, 5.0 × 106Cell/
mL、6.0×10 6Cell/mL, 7.0 × 106Cell/mL, 8.0 × 106Cell/mL, 9.0 × 106Cell/mL, 10.0 × 106Carefully
Born of the same parents/mL, 11.0 × 106Cell/mL, 12.0 × 106Cell/mL, 13.0 × 106Cell/mL, 14.0 × 106Cell/mL, 15.0
×106Cell/mL, 16.0 × 106Cell/mL, 17.0 × 106Cell/mL, 18.0 × 106Cell/mL, 19.0 × 106Cell/
mL、20.0×106Cell/mL.
In some embodiments, PK15 cells are the suspension cell used in production of vaccine, or from adherent domestication, energy
The suspension cell of enough suspension growth production.
In some embodiments, virus for it is sensitive on corresponding suspension PK15 cells, corresponding vaccine can be produced
Virus.In some embodiments, virus is selected from pig circular ring virus, transmissible gastro-enteritis virus and pig parvoviral.
In some embodiments, cell growth temperature is at 35 DEG C~37 DEG C, connect malicious restrovirus production temperature 30 DEG C~
37℃。
In some embodiments, cell growth inoculum density is 0.3 × 106Cell/mL~1.0 × 106Cell/mL, example
Such as 0.3 × 106Cell/mL, 0.4 × 106Cell/mL, 0.5 × 106Cell/mL, 0.6 × 106Cell/mL, 0.7 × 106Cell/
mL、0.8×106Cell/mL, 0.9 × 106Cell/mL or 1.0 × 106Cell/mL, it is thin that cell growth can reach highest in 3~8 days
Born of the same parents' density, such as 3 days, 4 days, 5 days, 6 days, 7 days or 8 days.
The novelty of the present invention is before cell growth to plateau, and cell density reaches highest, then dilutes original training
Volume is supported, the process of virus is inoculated, can be from low-density diauxic growth by diluted cell, virus also replicates therewith, finally
Improve viral yield.The advantage of the second order culture of the PK15 cell related vaccines production of the present invention is：Simple production process is held
Easily amplification；Liquid is not changed, culture medium utilization rate is high；Growth medium and production medium can be identical culture medium, can also
For different culture mediums.Growth medium and production medium proportionally mix, and can not only have the function that cell growth, but also
It can have the function that virus replication is proliferated.
Description of the drawings
Fig. 1 shows PK15 attached cells to replace low blood serum mediumBD004 (JSB-DP049) drops afterwards
The process of serum adhere-wall culture.PK15 stablizes 4 generations of culture in the basal medium DMEM suggested by ATCC companies, in T75
With 1 × 10 in culture bottle6Cell can grow to 16~20 × 10 after cell/15mL cultures 72h6Then cell is trained using low serum
Support baseBD004 (JSB-DP049) continuously decreases serum, first with 5% 3 generation of serum amount culture, is trained in T75
It supports in bottle with 1 × 106Cell can grow to 15~17 × 10 after cell/15mL cultures 72h6Cell, then trained with 2% serum amount
Supported for 3 generations, with 1 × 10 in T75 culture bottles6Cell can grow to 12~14 × 10 after cell/15mL cultures 72h6Cell, most
Afterwards with 0.5% 3 generation of serum amount culture its in T75 culture bottles with 1 × 106Cell can be grown to after cell/15mL cultures 72h
15~17 × 106Cell, during entirely drop serum, Cell viability is held at 98% or more.
Fig. 2 shows that the third day of PK15 cell growths, cell density reach 8.0 × 106When cell/mL, virus inoculation
And adsorb, the production medium of 1 times of original working volume is added, cell density becomes 4.0 × 10 at this time6Cell/mL, cell is again
Start diauxic growth, by growth in 2 days, cell density can reach 10.0 × 106Cell/mL.
Fig. 3：A-D shows PK15 cell growths to connecing before poison cell growth picture and connect cytopathy picture after poison, packet
Include 20X and 40X, picture can be seen that size is more uniform before cell connects poison, and bright, film is smooth, cell health, after cytopathy
Part cell cracking, cellular morphology change.
Specific implementation mode
The novelty of the present invention is：Low serum, serum-free acclimation method of the adherent growth PK15 cells using the present invention
After being tamed with suspension acclimation method, the domestication cell growth state that is obtained is stable, good dispersion, is suitable for high density, scale
Change culture, and is conducive to the duplication and expression of virus.Suspension PK15 cells reach certain cell density when growing into plateau,
Such as 8.0 × 106When cell/mL, production medium is added and dilutes original volume of culture, inoculates virus.PK15 cells are dilute
Start diauxic growth again after releasing low-density, virus also replicates therewith, finally improves viral yield.
In order to reach the above production advantage, present invention firstly provides a kind of adherent PK15 cell strains changing by culture medium
Become and the change of training method, enables the low serum of the cell strain of original adherent growth or serum-free, suspension high-density growth
Acclimation method, the low serum refer in culture medium serum-concentration less than without domestication adherent PK15 cell strains normal growth,
Serum-concentration needed for culture medium when proliferation, for example, low serum refer in culture medium serum-concentration less than 4%, 3%, 2%,
1%, 0.5%, 0.2% or lower, it is preferable that serum-concentration is less than 1%, it is highly preferred that serum-concentration is less than 0.5%, it is described
Serum-free refers to the serum or its ingredient that any animal origin is not contained in culture medium.A concentration of concentration of volume percent.
The low serum domestication of the adherent PK15 cells includes to gradually reduce culture medium serum-concentration during cell secondary culture, up to
Condition of culture under the serum-concentration of the cell adapted low-serum-concentrations of PK15 such as 0.5%.Specifically, the low serum domestication includes
Make in optimum state and stablizes the PK15 cells of growth in low blood serum mediumBD004 (JSB-DP049) adds
Passage 1~4 time in 3.5~6.5% (such as 5%) newborn bovine serum (NBCS), such as 3~4 times, make cell be restored to optimum state simultaneously
Stablize growth, then in low blood serum mediumBD004 (JSB-DP049) plus 1.5~3% (such as 2%) new born bovines
Passage 1~4 time in serum (NBCS), such as 3~4 times make cell be restored to optimum state and stablize growth, are then trained in low serum
Support base1~4 is passed in BD004 (JSB-DP049) plus 0.3~1% (such as 0.5%) newborn bovine serum (NBCS)
It is secondary, such as 3~4 times, until cell restores state, it being capable of stability passage and doubling time stabilization.For rising for low serum domestication
What is begun can be from the adherent PK15 cell recoveries frozen, restore in the PK15 cells of optimum state and stable growth
PK15 cells.Specifically, after the recovery, recovery are related to the adherent PK15 cell recoveries for making to freeze, in the blood containing normal concentration
Clearly, such as 10% fetal calf serum or newborn bovine serum, culture medium (such asDMEM (high glucose) (JSB-
66001) basal medium) in culture, passed on when cell reaches 90% converging state, continuous passage 2~5 times, until cell
It is restored to optimum state and stablizes growth.
Then, after PK15 cells have adapted to adherent low Serum Growth completely, 5~8 kinds of strong suitable biologies are selected
Serial serum free medium carries out serum-free domestication and the domestication that suspends.Specifically, the serum-free domestication includes that adaptation is made to paste
The PK15 cells of the low Serum Growth of wall are good at 5~8 kinds along biologyIt grows and passes in serial serum free medium
(being passed on when 90% converging state) abandons directly adapting to domestication PK15 cell growths by sampling to countNothing
Blood serum medium, other can adapt to the PK15 cells and correspondence of domesticationSerum free medium carries out irregular biography
In generation, repeats the step, and until PK15 cells adapt to suspension training method completely, and the PK15 cells that suspend can start stabilization
Passage growth, suspension PK15 cell densities can reach 6.0 × 106Cell/mL~15.0 × 106Between cell/mL, motility rate dimension
It holds 95% or more.Initially higher inoculum density, such as 0.5X10 are usually required when domestication6Cell/mL~1.0X106Cell/
mL.If PK15 cells are slow-growing in initial domestication or growth arrest, motility rate remain relatively low, can select 3~5 days
It is passed on, the method that liquid is changed using centrifugation when passage.If cell growth is extremely slow during domestication, can suitably adjust
Then volume of culture irregularly carries out fluid infusion.
Optionally, excellent by the ingredients such as amino acid, vitamin, trace element, growth factor, hydrolysate in Optimal Medium
Change suspension PK15 cell non-serum cell culture mediums, enables the density stabilized growth passage of suspension PK15 cell highers, suspend
PK15 cells most high-density can reach 6.0 × 106Cell/mL~8.0 × 106Between cell/mL, motility rate maintains 98% left side
It is right.
Optionally, suspension PK15 cells are developedThe nothing without albumen, animal origin-free of CD specific chemical components
Serum cell culture medium suspends culture entirely it is suitable for suspension PK15 high cell densities, and suspension PK15 cell most high-densities can
Reach 6.0 × 106Cell/mL~15.0 × 106Between cell/mL, motility rate maintains 98% or so.
In some embodiments, the low serum or the domestication of its serum-free and/or the culture domestication that suspends include following step
Suddenly：
(1) adherent PK15 cell recoveries (the cell strain source that will be frozen：U.S. ATCC, CCL-33, Lot:61284198)；
(2) the adherent PK15 cells of recovery are placed in T75 culture bottles, 15mL is addedDMEM (high grapes
Sugar) (JSB-66001) plus 10% fetal calf serum, 37 DEG C are put in, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90%
Converging state；
(3) reject culture medium, and 2mL 0.25%Trypsin-EDTA are added, make PK15 cell dissociations be detached from culture dish,
Then 5mL is added in vessel surfaceThe complete medium of DMEM (high glucose) plus 10% fetal calf serum is with end
Only pancreatin digests；
(4) above-mentioned postdigestive cell is dispelled with pipette, with the cell density of cell counter unit of account volume
And Cell viability, according to 14000 cells/cm2Inoculum density be inoculated in T75 culture bottles again, 15mL is added
DMEM (high glucose) plus 10% fetal calf serum, are put in 37 DEG C, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90%
Converging state；
(5) cell that incubator is put into step (4) often cultivates 3 days repetition steps (3) and (4), repeats 2~3 times, until
Cell is restored to optimum state and stablizes growth；Herein it should be noted that if using optimum state and stabilization has been in
The PK15 cells of growth, then step (1) be dispensed to step (5)；
(6) after digesting the cell for having stablized growth according to the method for step (4), according to 14000 cells/cm2Connect
Kind density is placed in the strong low blood serum medium along biological independent researchBD004 (JSB-DP049) plus 5% is newborn
Cow's serum (NBCS), is put in 37 DEG C, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90% converging state；
(7) cell that incubator is put into step (6) often cultivates 3 days repetition steps (3) and (4), repeats 2~3 times, until
Cell is restored to optimum state and stablizes growth；
(8) after digesting the cell for having stablized growth according to the method for step (4), according to 14000 cells/cm2Connect
Kind density is placed in the strong low blood serum medium along biological independent researchBD004 (JSB-DP049) plus 5% is newborn
Cow's serum is put in 37 DEG C, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90% converging state；
(9) cell that incubator is put into step (8) often cultivates 3 days repetition steps (3) and (4), repeats 2~3 times, until
Cell is restored to optimum state and stablizes growth；
(10) after digesting the cell for having stablized growth according to the method for step (4), according to 14000 cells/cm2Connect
Kind density is placed in the strong low blood serum medium along biological independent researchBD004 (JSB-DP049) plus 2% is newborn
Cow's serum is put in 37 DEG C, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90% converging state；
(11) cell that incubator is put into step (10) often cultivates 3 days repetition steps (3) and (4), repeatedly 2~3 times, directly
Restore state to cell and stablizes growth；
(12) after digesting the cell for having stablized growth according to the method for step (4), according to 14000 cells/cm2Connect
Kind density is placed in the strong low blood serum medium along biological independent researchBD004 (JSB-DP049) plus 0.5% is new
Raw cow's serum, is put in 37 DEG C, 5%CO2Incubator in cultivate 3 days, so that cell is reached 90% converging state；
(13) cell that incubator is put into step (12) often cultivates 3 days repetition steps (3) and (4), repeatedly 2~3 times, directly
Restore state to cell, can stablize stability passage and doubling time, microscopically observation cell morphological characteristic waits for adherent
After PK15 cells integrally restore optimum growh state, larger culture vessel is expanded to covering with single layer；
(14) after cell has adapted to adherent low Serum Growth completely, 5~8 kinds of strong suitable biologies are selectedSeries is without blood
Clear culture medium (specifying information is shown in Table 1), directly carries out screening domestication；Adherent PK15 cells in step (13) are repeated into step
(3) and (4) digestion method is prepared into individual cells suspension, and sampling counts, and 800rpm, 5min centrifugation abandon supernatant, it is thin to retain PK15
Born of the same parents are precipitated；
Table 1 is strong along biologySerial serum free medium
(15) by the PK15 cell precipitations in step (14) respectively with strong along biologySerial serum free medium
Resuspension is set in Shaker shaking flasks, and CO is placed on2Suspend culture on incubator shaking table, and the shaking speed that race way diameter is 25mm is arranged
In 120rpm/min~130rpm/min.In this case, since the cell of only part inoculation can adapt to a new life
Long mode, so usually requiring higher inoculum density, such as 0.5X10 when initially domestication6Cell/mL~1.0X106Cell/mL,
This is conducive to increase the number of the living cells in later passages.It is recommended that under small-scale condition of culture (such as：125mL~
250mL shaker) it is tamed, it needs periodically to sample counting during domestication, monitors cell density and Cell viability in time,
Cell viability may drop to very low level during this, this is a necessary stage of domestication, embodies the adaptation of cell
Property, also will appear cell can not adapt at some completelyIt is grown in serum free medium and dead, this is also initial
One necessary process of screening and culturing medium；
(16) the PK15 cells cultivated that step (15) suspend judge according to regular count result, abandon directly adapting to
Tame PK15 cell growthsSerum free medium, other can adapt to the PK15 cells and correspondence of domesticationSerum free medium carries out irregular passage, such as the slow-growing or stagnation in initial domestication of PK15 cells
Growth, motility rate remain relatively low, can select passed within 3~5 days, the method for changing liquid using centrifugation when passage；
(17) leniently (800rpm/min, 5min are centrifuged to remove former culture collection step (16) suspension PK15 cells
Base), supernatant is abandoned, is gently resuspended in centrifugation gained PK15 cells correspondingIn serum free medium, inoculation is close
Degree can tame growing state according to PK15 cells and suitably adjust to 0.3 × 106Cell/mL~1.0 × 106Between cell/mL；
(such as cell density is higher, reaches 6.0 × 10 under some cases6Cell/mL~15.0 × 106When cell/mL) it can also select
The mode of direct diluted passage is not centrifuged.If cell growth is extremely slow during domestication, volume of culture can be suitably adjusted,
Then fluid infusion is irregularly carried out, that is, is added freshSerum free medium adjusts cell density 0.3 × 106Cell/
ML~1.0 × 106Cell/mL cultures, daily timing sampling count；
(18) the irregular suspension PK15 cells passage of step (17) is repeated, sampling daily counts, in this way until PK15
Cell adapts to suspension training method completely, and the PK15 cells that suspend can start stable passage growth, suspension PK15 cells
Density can reach 6.0 × 106Cell/mL~15.0 × 106Between cell/mL, when motility rate maintains 95% or more, need solid
Determine inoculating cell density and passage period, such as：According to 0.5 × 106Cell/mL~1.0 × 106Cell/mL inoculating cell density,
It was passed on every 3~4 days, daily timing sampling counts；
(19) it is PK15s cells to name the suspended culture cell, with 10.0 × 106The density of cell/mL freezes；
(20) optimize suspension PK15 cell non-serum cell culture mediums, by amino acid in Optimal Medium, vitamin, micro-
The ingredients such as secondary element, growth factor, hydrolysate can be highly denser by the suspension PK15 cells that can stablize passage in step (18)
Degree stablizes growth passage, and suspension PK15 cells most high-density can reach 6.0 × 106Cell/mL~8.0 × 106Cell/mL it
Between, motility rate maintains 98% or so；
(21) the suspension PK15 cells developedCD specific chemical components without albumen, animal origin-freeCD PK15 (JSB-LM17019) serum-free cell culture medium, hangs entirely suitable for suspension PK15 high cell densities
Floating culture, suspension PK15 cells most high-density can reach 6.0 × 106Cell/mL~15.0 × 106Between cell/mL, motility rate
Maintain 98% or so.
In addition, in order to reach the above production advantage, the present invention also provides one kind to give birth to about suspension PK15 cell related vaccines
The second order culture process method of production, detailed process and steps are as follows：
(1) cell growth：Suspension PK15 cells growth medium such as CD PK15(JSB-LM17019)
Middle growth, density reach 2.0 × 106Cell/mL~20.0 × 106Between cell/mL；Preferred cell density is 6.0 × 106Carefully
Born of the same parents/mL~15.0 × 106Cell/mL, it is highly preferred that 9.0 × 106Cell/mL~15.0 × 106Cell/mL, most preferably cell
Density is 8.0 × 106Cell/mL；
(2) poison is connect：According to production technology, cell connects poison amount 10-1~10-5Between MOI, poison amount is preferably connect 10-1~
10-4MOI, the more excellent poison amount that connects is 10-1~10-3MOI, the optimal poison amount that connects is 10-2MOI；
(3) fluid infusion：Production medium is added, the fresh production medium of 1~5 times of original growth medium volume is added,
Preferably, 1~4 times, it is highly preferred that 1~3 times, most preferably 2 times, make cell density reach 2.0 × 106Cell/mL~10.0
×106Cell/mL, it is preferable that 1.0 × 106Cell/mL~5.0 × 106Cell/mL, most preferably, 4.0 × 106Cell/mL；
(4) diauxic growth：Cell continued growth,
In some embodiments, after the cell density of step 4) diauxic growth reaches technological requirement, including according to life
Production. art harvests the step of virus liquid purifying seedling, wherein the production technology refers to producing corresponding vaccine using PK15 cells
Known production technology.
Cell growth temperature is at 35 DEG C~37 DEG C wherein in step 1), for example, 35 DEG C, 35.5 DEG C, 36 DEG C, 36.5 DEG C, 37
℃.The suspension PK15 cells are low serum as described above or the suspension PK15 of the domestication of its serum-free and/or the culture domestication that suspends
Cell.
Poison amount is wherein connect in step 2) 10-1~10-5Between MOI, specifically by between production technology and virus and cell
Relationship control, virus production temperature between 30 DEG C~37 DEG C, such as 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36
℃、37℃。
1~5 times that productive culture base unit weight is original growth medium is wherein added in step 3).
Wherein " growth medium " refers to growing into highdensity low serum or serum-free cell from low-density for cell
Culture medium.
" production medium " refers to the virus production culture medium that can meet virus and effectively replicate.
" growth medium " and " production medium " of the present invention can be known in the art, any a or a few moneys
It can be used for the culture medium of suspension PK15 cell growths and production.
In some embodiments of the present invention, described " growth medium " and " production medium " can be same trainings
Base is supported, such as is all the strong commercialization along biological independent researchCDPK15 cell culture mediums, number JSB-
LM17019.In other embodiments of the present invention, " growth medium " is different culture with " production medium "
Base, such as " growth medium " and " production medium " ratio can be 1:1、1:2、1:3、1:4、1:5.
The present invention relates to the low serum free culture system and free serum culture of PK15 cells, the virus for production include pig annulus,
All viruses that can be infected on PK15 cells including transmissible gastroenteritis of swine and the tiny vaccine of pig.
Wherein, described " low serum free culture system " refers to the cow's serum of the addition 0.2%~3% in incubation to meet cell
The training method of growth, such as 0.5% or 1%.
" free serum culture " refers to that animal blood serum such as cow's serum is not added in incubation, the battalion only provided by culture medium
Form point training method for meeting cell growth.
The wherein described production technology is the bioreactor of 30L or more, including stirring type bioreactor and rip current type
Bioreactor, for example, 50L, 100L, 200L, 300L, 400L, 500L, 1000L, 2000L, 3000L, 4000L, 5000L,
The even greater bioreactors of 10000L.
Second order cultural method refers to after PK15 cells are according to certain inoculum density inoculating cell, passing through life in 2~8 days
Long, cell density can reach 2.0 × 106Cell/mL~20.0 × 106Between cell/mL, then virus inoculation adsorbs 0.5h
~2.0h, preferably 0.6h~1.5h, more preferable 0.7h~1.2h, more preferable 0.8h~1.1h, most preferably 1.0h, then add original
The production medium that 1~5 times of working volume, makes cell density be reduced to 2.0 × 106Cell/mL~10.0 × 106Cell/mL it
Between, by 1~7 day virus production, harvest virus liquid and seedling.It is of course also possible to adding the 1~5 of original working volume
Poison is connect again after production medium again.The technology is suitable for the production of vaccine for man and live vaccine.
Wherein, " inoculum density " typically refers to, and when cell growth to high density, medium nutrient content cannot meet cell
Growth when, then highdensity cell is proportionally diluted to the process of low-density, general inoculum density is 0.1 × 106Carefully
Born of the same parents/mL~2.0 × 106Cell/mL.
" plateau " of the present invention refers to the time that cell reaches highest stand density, and " before plateau " refers to cell
Before reaching highest stand density.
" cell density reaches highest " of the present invention refers to that culture medium disclosure satisfy that the highest growth of cell growth is close
Degree.
Obviously, the above according to the present invention is not departing from this hair according to the general knowledge and conventional process of this field
Under the premise of bright basic fundamental thought, the modifications and changes of diversified forms can also be made.
It will be further illustrated the present invention below by following non-limiting embodiments, it is well known to those skilled in the art, not
In the case of spirit of that invention, many modifications can be made to the present invention, such modification also falls into the scope of the present invention.
Following experimental methods are conventional method unless otherwise instructed.Institute is had using cell strain is accredited as U.S. typical case
The feature for the cell line that culture collection (ATCC) number is CCL-33, it is commercially available.Used medium is strong along raw
The product of object independent research.Other experiment materials used unless otherwise instructed, can be obtained from commercial company.
Embodiment 1PK15 cell non-serums tame suspension process and culture medium exploitation
(1) experiment material
Cell strain：From the adherent PK15 of ATCC adherent PK15 cell strains (U.S. ATCC, CCL-33, Lot:
61284198)
Culture medium： BD004(JSB-DP049)
(2) serum-free tames suspension process
Include the following steps：
1) recovery, passage of attached cell：
A. the preparation of culture medium：10L fluid nutrient mediums are prepared to specifications BD004(JSB-
DP049), after aseptic filtration 4 DEG C be kept in dark place；
B. the recovery of cell：Freeze-stored cell strain is taken out from liquid nitrogen container, is added to containing 15mL after 37 DEG C of thawingsIn BD004 (JSB-DP049) and the T75 culture bottles of 5% newborn bovine serum (NBCS), 37 DEG C are placed in, 5%CO2
Incubator is cultivated；
C. it passes on：The cell newly recovered is primary every passage in 3 days, according to 1.0x106Cell/T75 inoculations, continuous passage 2
It is secondary, proceed by experiment；
D. drop serum passage：As shown in Figure 1, after the cell restoration ecosystem recovered, cell is reached strong along the low of biology
In blood serum medium BD004-DP049, then add 5% newborn bovine serum (NBCS) passage, it is new to shift to 2% after cell growth stabilization
Raw cow's serum continues to pass on, then serum is down to 0.5% passage, and usual 2~3 generation can grow stabilization；
E. domestication suspends：After above-mentioned cell dissociation and centrifugal treating, supernatant is abandoned, uses PK15 cell precipitations and respectively table
It is strong along biology listed by 1Serial serum free medium resuspension is set in Shaker shaking flasks, 37 DEG C, 5%CO2, 120rpm/
Min incubators are cultivated, daily sampling record cell density and motility rate；
F. suspension cell passes on：According to count results, abandon directly adapting to domestication PK15 cell growthsSerum free medium, other can adapt to the PK15 cells and correspondence of domesticationSerum free medium every
It 3~5 days, is passed on using the method that liquid is changed in centrifugation.Through passage after a period of time, cell is suitable for suspension growth；
G. serum free medium is developed：By the ingredients such as amino acid, vitamin, trace element in Optimal Medium, make
The suspension PK15 cells of passage can be stablized after optimization by stating in stepCD PK15 (JSB-LM17019) are real
Existing high density stablizes passage.
The second order culture process of 2 suspension PK15 cells of embodiment is tested
(1) experiment material
A. cell strain：Suspension has been tamed in embodiment 1 and energy high density stablizes the PK15 cells of growth；
B. culture medium：10L growth mediums are prepared to specificationsCD PK15 (JSB-LM17019),
It is kept in dark place for 4 DEG C after aseptic filtration；
(2) experimental procedure
A. as shown in Fig. 2, in the third day of cell growth, cell density reaches 8.0 × 106When cell/mL, add original
Cell density is diluted to 4.0 × 10 by the growth medium that 1 times of working volume6Cell/mL is inoculated with pig circular ring virus, connects malicious amount
For 0.5MOI, cell starts diauxic growth again after connecing poison, and 10.0 × 10 are grown by 2 days cell densities6Cell/mL；
B. from Figure 2 it can be seen that the superiority of PK15 cell production technology second order culture techniques：High-density growth, low-density connect
Poison, cell can diauxic growth, save production cost, improve density titre.
Tests of the embodiment 3PCV on PK15 cells
(1) experiment material
A. cell strain：From the PK15 cell strains of ATCC, through it is strong along the autonomous domestication of biology can serum-free or low serum (such as
0.5% newborn bovine serum) suspension growth, acclimation conditions are as described above, can be summarized as follows：Adherent PK15 cell serums are reduced
To 0.5% passage 3 times, the domestication culture that suspends, acclimation conditions 5%CO are then carried out2, shaking speed 120rpm/min, sieve
Select the PK15 cell strains for capableing of high density suspension growth；
B. culture medium： CD PK15(JSB-LM17019)。
C. viral：PCV (offer of Hua Nong (Zhaoqing) biological industry Institute for Research and Technology)
(2) experimental method
Include the following steps
A. the preparation of culture medium：10L fluid nutrient mediums are prepared to specificationsCD PK15, aseptic filtration
It is kept in dark place for 4 DEG C afterwards；
B. the recovery, passage of cell：Freeze-stored cell strain is taken out from liquid nitrogen container, and 30mL is added to after 37 DEG C of thawingsIn CD PK15 culture mediums, 1000rpm/min centrifuges 5min, abandons supernatant, and cell is resuspended in freshIn CD PK15 culture mediums, 37 DEG C are placed in, 5%CO2Incubator is cultivated；
C. cell passes on：Passage 3~4 times are needed before the cell experiment newly recovered, it is primary every passage in 3 days, according to 1.0 ×
106The amount inoculating cell of cell/mL, continuous passage 3 times；
D. poison is connect：When cell is passaged to the 3rd day the 4th generation, cell density reaches 8.0 × 106Cell/mL, plan cell connect
Malicious density is 4.0 × 106Cell/mL is 8.0 × 10 in cell density6The PCV of malicious 0.5MOI is met when cell/mL, after adsorbing 1h
It is 4.0 × 10 to add production medium to cell density6Cell/mL；
E. poison valency measures：The TCID of infective dose Test Virus is cultivated according to median tissue (cell)50；
F. malicious result is connect：From Figure 2 it can be seen that PCV viruses are given birth to using suspension PK15 cells production second order technique culture technique
The virus titer of production, TCID50For log106.6/mL, and the TCID of the suspension culture virus of general technology50For log105.5/mL
It can be seen that virus titer is significantly higher than general technology.Fig. 3 A-D show PK15 cell growths to connecing cell growth picture before poison and connect
Cytopathy picture after poison, including 20X and 40X, picture can be seen that size is more uniform before cell connects poison, and bright, film is smooth,
Cell health, part cell cracking after cytopathy, cellular morphology change.This second order cultivation viral yield is higher, superior
Property is that high-density growth, low-density connect poison, and cell can continue diauxic growth, save production cost, improve virus titer.
Claims (10)
1. a kind of second order cultural method of suspension PK15 cell vaccines production, it includes following steps：
6) cell growth：Suspension PK15 cells grow to 2.0 × 10 in growth medium6Cell/mL~20.0 × 106Cell/
mL；
7) poison is connect：Poison amount is connect 10-1~10-5Between MOI, or poison amount is connect according to this after step 3) and connect poison；
8) dilute/add production medium：1~5 times is diluted with production medium, cell density is to 1.0 × 10 after dilution6Cell/
ML~10.0 × 106Cell/mL；
9) diauxic growth：Cell diauxic growth；
10) optionally, harvest virus liquid purifies seedling.
2. the second order cultural method of suspension PK15 cell vaccines production according to claim 1, wherein suspension PK15 cells
It is grown to suspension tank culture, training volume is 30L or more.
3. the second order cultural method of suspension PK15 cell vaccines production according to claim 1 or 2, thin wherein in step 1)
Born of the same parents' density is 5.0 × 106Cell/mL~16.0 × 106Cell/mL, it is preferable that 6.0 × 106Cell/mL~15.0 × 106Carefully
Born of the same parents/mL, most preferably, 8.0 × 106Cell/mL.
4. the second order cultural method of suspension PK15 cell vaccines production according to any one of claims 1 to 3, wherein step
2) cell connects poison amount 10 in-1~10-4It is more excellent to connect between MOI, 10-1~10-3MOI, it is optimal to connect, 10-2MOI。
5. the second order cultural method of suspension PK15 cell vaccines production according to any one of claims 1 to 4, wherein step
3) suspension PK15 cell production mediums are diluted to 1~4 times in, it is highly preferred that 1~3 times of dilution, most preferably 2 times dilutions.
6. the second order cultural method of suspension PK15 cell vaccines production according to any one of claims 1 to 5, wherein step
3) cell density is 1.0 × 10 after connecing poison in6Cell/mL~10.0 × 106Cell/mL, it is preferable that 1.0 × 106Cell/mL~
5.0×106Cell/mL.
7. the second order cultural method of suspension PK15 cell vaccines production according to any one of claims 1 to 6, wherein suspending
PK15 cells be production of vaccine used in suspension PK15 cells, or by adherent PK15 cells domestication from, being capable of suspension growth
The suspension PK15 cells of production, it is preferable that suspension PK15 cells are to be tamed through low serum by adherent PK15 cells or serum-free is tamed and dociled
To change, the serum-free domestication, which refers to the PK15 cells after domestication, can grow in the culture medium existing for serum-free, be proliferated,
And/or, it is preferable that suspension PK15 cells be by adherent PK15 cells through suspend domestication from.
8. the second order cultural method of suspension PK15 cell vaccines production according to any one of claims 1 to 7, wherein virus
For the virus sensitive, that corresponding vaccine can be produced on corresponding suspension PK15 cells.
9. according to the second order cultural method that claim 1 to 8 any one of them suspension PK15 cell vaccines produce, wherein cell
Growth temperature connects malicious restrovirus and produces temperature at 30 DEG C~37 DEG C at 35 DEG C~37 DEG C.
10. according to the second order cultural method that claim 1 to 9 any one of them suspension PK15 cell vaccines produce, wherein carefully
Intracellular growth inoculum density is 0.3 × 106Cell/mL~1.0 × 106Cell/mL, growth number of days reached highest cell at 2~8 days
Density.
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| CN111040986A (en) * | 2019-12-25 | 2020-04-21 | 乾元浩生物股份有限公司 | Suspension culture cell monoclonal screening culture method |
| CN111235089A (en) * | 2018-11-28 | 2020-06-05 | 成都天邦生物制品有限公司 | Method for producing porcine circovirus type 2 (PCV 2) by cell pure suspension process |
| CN111454910A (en) * | 2020-03-13 | 2020-07-28 | 成都天邦生物制品有限公司 | Suspension PK15 cell line expressing porcine CD163 and CD169 molecules |
| CN115322955A (en) * | 2022-09-22 | 2022-11-11 | 金宇保灵生物药品有限公司 | Domestication method of full-suspension serotype-free PK-15 cells |
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