SCALE-UP OF A FLUIDIZED B E D REACTOR OPERATED

WITH POROUS GLASS CARRIERS

F. Unterluggauer, 0. Doblhoff, M. Reiter, G. Blüml, N. Zach

G. Kral, Ch. Schmatz and H. Katinger.

Institute of Applied Microbiology (IAM), University of Agriculture

Nußdorfer Lände 11, A-I 190 Vienna, Austria

ABSTRACT

The IAM modular iluidized bed reactor was scaled up from laboratory size (7 1) to pilot
scale (80 1). Hardeware was designed to fit the modular IAM pilot plant structure and
software was adapted to the new reactor design. The new reactor was tested using porous
glass beads and CHO cells as model cell fine. After inoculation and batch phase a
continuous perfusion system using protein free-medium was set up and operated over a
period of three weeks. At the final dilution rate (approx. 12 times the carrier volume) a
cell density up to 5 x 10 ' cells/ml carrier bed could be achieved.
Fermentation control was achieved using an industrial direct digital process control system
(Honeywell TDC 3000).

INTRODUCTION

Since van WezeFs* pioneering innovation and use of microcarriers, these, as well as
macroporous carriers are finding increasing application for the suspension culture of
anchorage-dependent cells (microcarriers are solid beads to which cells attach,
macroporous carriers are spherical, but porous and allow cells to enter and multiply
within the bead)^. Recently several groups have demonstrated the practical potential of
packed bed and fluidized bed technologies in mass propagation and production of
mammalian cells. In this communication, we deal with the scale-up of a recently
developed bioreactor, based on fluidized bed and packed bed technology, that is
especially designed for mammalian cell production processes.

MATERIAL AND METHODS

Culture conditions

A recombinant CHO cell fine was cultivated in protein-free DMEM/HAM'S F 12 medium

containing 4mM glutamine. The cells were precultivated in roller bottles and in a 10 1
stirred tank reactor respectively. Culture temperature was 37° C, set point for pH was 7.2
and the dissolved oxygen level was maintained at 25 %.

Computer control

Fermentation control was achieved using an industrial direct digital control system
(Honeywell 3000)3. Parameters such as temperature, pH, p02 could be adjusted with
minimum operational deviations. pH and p02 was controlled by a software driven
continuous gas blending unit (oxygen, air, CO2 and nitrogen).

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Reactor system

The hard- and software of the existing modular pilot plant fermentor was adapted to a 80 1
fluidized bed reactor (Fig. 1).
Basically the system is a basic reactor vessel equipped with an adapter and a liquid
distrubator plate. Instead of an external recirculation loop a low shear stress impeller is
used as the circulation purnp^. This new configuration provides a series of advantages: 1)
no shear stress; 2) no break down of pump; 3) oxygenator fouling is limited; 3) in-situ
steam sterilization.

Fig.l Modular integrated

fluidized bed bioreactor
system.

1 fluidized bed
2 distributor
3 oxygenator
4 stirrer ( = low shear pump)
Hpg liquid hight of fluidized bed
Hpg liquid hight of packed bed

Sample analyses

Cell count:
Total cell numbers were measured by a Coulter Counter (Coulter Elektronics FRG). The
released nuclei were counted after treatment with citric acid containing 1 % Triton X-100.
Chemical analysis:
Carbohydrate substrates and secreted metabolic endproducts in the cell culture
supernatant were determined by HPLC on a strong cation exchange column in H + -form.

RESULTS AND DISCUSSION

The culture was initially started at a density of 1.9 x 10" cells/ ml of carrier bed. As
porous matrix a SIKUG 012/300 A (Schott FRG) glass carrier with a diameter between
1.0 and 2.0 mm was used (Fig. 2).

Fig. 2 Microphotograph of the porous glass carriers. (Magnif.: 50 x)

A native carrier B CHO cells grown on the carrier

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The cells were transfered to the bioreaclor containing the carriers and the conditioned
medium. The stirrer speed was adjusted to 300 RPM and 100 RPM in alternative order
(lmin:30 min) and after 4 hours 90 % of the cells were immobilized on the carrier beads.
The final stirrer speed was then adjusted to 300 RPM. Data from the culture run are
given in Fig. 3.
β
•5 P
c 5
8
4, rr
t^
f>
cr cr
LU
3
3
o
3 ■2 £

■1 W
1
°" υ

( 100
200
300
400
500
TIME (h)
A

Fig. 3 Growth kinetics of

recombinant CHO cells cultured on
glas carriers.
A) cell density (4) and
product titer (n)
B) glucose (r?) and lactate (A) in the
culture supernatant
C) perfusion rate

CONCLUSION

The results (e.g. cell growth and density, specific product formation, isoprotein patterns,
etc.) achieved in the scale-up of this novel fluidized bed reactor comparable to those at
bench scale. Improved fluidization of the carrier bed with scale indicates an enormous
scale-up potential of this reactor.

REFERENCES
1 Van Wezel, A.L. and Van der Velden-De Groot, C.A.M. Large scale cultivation of
animal cells in microcarrier culture. Process Biochemistry 1978, 3^ 6-8.

2 Nilson, K., Buzsaky, F., and Mosbach, K. Growth of anchorage dependent cells on
macroporous microcarriers. Bio/Technology 1986, fL 989-990.

3 Doblhoff-Dier, 0., Huss, S., Litos, R., Plail, R., Reiter, M., Unterluggauer, F., A
modular computer controlled fermentation pilot plant. Process Biochemistry 1991, in
press

4 Reiter, M., Blüml, G., Gaida, T., Zach, N., Unterluggauer, F., Doblhoff-Dier, 0.,
Noe, M., Plail, R., Katinger, H., Modular integrated fluidized bed bioreactor technology.
Bio/Technology 1991, in press

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