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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

yezhang@kth.se

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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/day

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Via

ble

ce

ll d

en

sity

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F/cm

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1st Run perfusion rate

200 MVC/mL

Trouble shooting Exponential growth

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Results – Total mAb production

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Acc

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Accumulated IgG production

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2nd Run

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IgG Concentration (mg/L)

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2nd Run- Harvest 1st Run- Harvest

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Shake flask 2nd Run 1st Run

DeltaEsp

Conductivity * 100

Biomass

Fc Results – Off-line cell measurements

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ble

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ells

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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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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