study of perfusion process of cho cells with celltank 2202
TRANSCRIPT
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Study of perfusion process of CHO cells with CellTank 2202 prototype, bench-top Single-Use-Bioreactors (SUB) with 150 cm3 CellCore matrix
Introduction Materials and Methods Major advantages of perfusion are high cell numbers and high total production in a relatively small
size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the cell
line product yield is low. On the other hand, disadvantages are for example technical challenges
originating from non-robust cell separation devices as well as sterility concerns from the more
complex set-up needed.
Recently, CerCell® (Denmark) has developed a perfusion integrated Single-Use-Bioreactor named
CellTank operated by magnetic stirring. The cells stay harboured inside a revolutionary matrix for cell
densities beyond 100 million cells/ml is integrated in the CellTank.
The EVO200 Biomass System (FOGALE nanotech) uses the dielectric properties of living cells and is
capable to measure the live cell density independently of cell size variations.
In the present work, CellTank 2202 prototype, bench-top Single-Use-Bioreactor (SUB) with 150cm3
CellCore matrix (CerCell®) was investigated. Perfusion cultivations were performed using a
recombinant CHO cell line producing a monoclonal antibody as a model system.
Conclusions
Very high viable cell density of 200 x 106 viable cells/ml achieved at perfusion rate 10 RV/day.
Very high viable cell density of 130 x 106 viable cells/ml maintained for 11 days at perfusion rate 8-10
RV/day with temperature lowered gradually from 37°C to 29°C.
The use of a single-use-bioreactor equipped with a real-time cell mass sensor offers a solution
alleviating technical and sterility challenges occurring in perfusion processes. Operation using
CellTank SUB much easier and handy compared with traditional perfusion technologies with robust
integrated perfusion device.
IgG accumulated nicely with time and increasing cell density with a cell specific productivity
comparable or higher than batch culture. No retention of IgG in the polymer matrix.
Similar concentrations of metabolites were measured in the bioreactor and harvest line indicating a
very good fluid homogeneity in the CellTank (data not shown).
2nd Run 1st Run
Cell density kept ≈ 130x106 viable cells/ml at
perfusion rate of 8/10 RV/day for over 10 days
Temperature lowered from 37ºC to 32ºC on day 10,
to 31ºC on day 11 and to 30ºC on day 14, resulting in
only partial growth arrest.
Temperature decreased to 29ºC on day 16,
resulting in complete cell growth arrest.
Cell density maintained then at a stable level for the
following days.
One pF/cm read on the EVO biomass sensor
system is equivalent with 1x106 viable cell/ml
First run (medium and setting adjustments
days 0 to 14) followed by exponential growth
Cell density up to 200x106 viable cells/ml
after 25 days of cultivation at a perfusion rate
10RV/day
Cell specific productivity in
perfusion mode comparable to
shake flask productivity except at
30˚C where it was ≈ 40 % higher
Fc: Fc is an indicator of average cell size. Between day 16 and 21, Fc was stable; it was then decreasing
indicating a larger cell diameter. After day 24, it decreases again, coinciding with a smaller diameter.
Alpha: Alpha is an indicator of the size homogeneity. During the run, alpha was increasing, indicating an
increasing inhomogeneity of the cell sizes (or different radii if the cells were not spherical anymore).
DeltaEps: Due to the very high viable cell density (200 x 106 viable cells/mL), the limit for DeltaEps signal
measured by the Fogale EVO 200 system was reached – it seems to be 1000 pF/cm.
Conductivity: From day 19 the conductivity was dropping to almost half value at the end, which might be
due to the fact that some ions have been consumed while not fully replaced by the perfusion medium.
There is a very nice correlation between biomass (2 frequencies) and DeltaEps (all frequencies)
This work was financed the Swedish Governmental Agency for Innovation Systems (VINNOVA).
We acknowledge Bent Svanholm Hansen, supplier of the iBiomass System, EVO 200 (Fogale
nanotech) for technical inputs. Thank you to Lars Mörtsell and Tomas Lindblom from Belach.
Exponential growth after trouble shooting
Exponential growth before temperature decrease
Product accumulated nicely
with time and increasing cell
density (after day 14 for 1st run)
No retention of IgG in the
polymer matrix.
Glucose
Glutamine
Medium
Feed
Gas out
Matrix
Mixed gas in Impeller
Reservoir
Harvest
Gas in
Back pressure
Alkali
Symbols Gas filter Pump
Zhang, Y.1, Stobbe, P.2, Orrego, C.S.3, Jing, F.4 and Chotteau, V.1 1School of Biotechnology, Cell Technology Group (CETEG), Royal Institute of Technology (KTH), 10691 Stockholm, Sweden 2CerCell ApS, Malmmosevej 19C, DK-2840 Holte, Denmark 3Belach Bioteknik AB, Dumpervägen 8, 14250 Skogås, Stockholm, Sweden 4Fogale nanotech Inc. 1714 Lombard Street San Francisco, CA 94123 - United States
Results – Cell density, viability and perfusion rate
Results – Fogale iBiomass sensor EVO 200 signals for the 1st Run
Parameters 1stRun 2ndRun
Cellline mAbproducingDHFR-CHO(DP12)
Inoculationviablecelldensity 1MVC/mL(=1*106viablecells/mL)
DO 45% 40%
pH 7.1 7.0
TemperatureA)àshiftedtoB)B)àshiftedtoC)
37˚C A)37˚CàB)32˚Cat100MVC/mLàC)furtherdecreasedto29˚Cprogressively
Recirculationflowrate 1.0L/min 1.6L/min
Celldensityspecificperfusionrate ≥0.05nL/cell/day(or1ReactorVolume/dayfor20MVC/mL)
Perfusionrate ≥1RV/day(=Reactorvolume/day)
Bioreactorandseparationdevice CellTankwithmatrix
Realcultureworkingvolume 150mL
Volumeofreservoir 1280mL
Cultivationmedium animal-componentfreeISCHOCDXPmediumwithhydrolysate(IrvineScientific)+3%ofIS-CHOFeed-CDXP(IrvineScientific)
Alkali 0.5MNa2CO3
AnalysesbyNovaBioprofileFLEX celldensity,viability,celldiameter,pH,pCO2,osmolality,concentrationsofglucose,glutamine,lactateandammonia
AnalysisofmAbconcentration proteinAHPLC
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EVO
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1st Run EVO (Viable cell density)
1st Run perfusion rate
200 MVC/mL
Trouble shooting Exponential growth
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2nd Run EVO (Viable cell density) 2nd Run Perfusion rate 2nd Run Temperature
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EVO
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1st Run Viable cell density 2nd Run Viable cell density
Results – Total mAb production
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Acc
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IgG
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Accumulated IgG production
1st Run
2nd Run
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IgG
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Time (days)
IgG Concentration (mg/L)
2nd Run - Bioreactor 1st Run- Bioreactor
2nd Run- Harvest 1st Run- Harvest
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Ce
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Cell specific productivity (pg/cell/day)
Shake flask 2nd Run 1st Run
DeltaEsp
Conductivity * 100
Biomass
Fc Results – Off-line cell measurements
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Via
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ells
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Time (days)
Viable cell number in the matrix vs. viable cell number lost in the harvest
1st Run Accumulated viable cells lost in harvest
2nd Run Accumulated viable cells lost in harvest
1st Run viable cells in the matrix
2nd Run viable cells in the matrix
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Via
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Viable cell number (EVO), dead cell number removed daily in the harvest and viability by LDH analysis
1st Run Viable cells in the matrix
1st Run daily dead cells
2nd Run viable cells in the matrix
2nd Run daily dead cells
1st Run Viability
2nd Run Viability
The accumulated viable cell number lost
in harvest was analyzed by daily off-line
samples in the harvest line.
The viability of the cultivations and the
dead cell number generated daily in the
harvest line were calculated based on LDH
measurement (Lactate Dehydrogenase).
The viability were high (>95%) through
out the whole cultivations for both runs.
Alpha
Biomass
Acknowledgements
CCE XIII, Scottsdale, AZ,
USA, Apr. 22-27 2012