Cytotechnology 9: 41-49, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Modified CelliGen-packed bed bioreactors for hybridoma cell cultures Guozheng Wang 1, Wenying Zhang 1, Corey Jacklin 1, David Freedman l, Lee Eppstein I and Avinoam Kadouri 2 1New Brunswick Scientific Co., Inc., 44 Talmadge Road, Edison, N J, 08818-4005, USA; 2Department of Membrane Research and Biophysics, The Weizmann hTstitute of Science, Rehovot, 76100, Israel Received 31 March 1992; accepted in revised form 24 August 1992
Key words: animal cell culture, hybridoma, monoclonal antibody, packed bed reactor, per'fusion
Abstract This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgGl monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen T M bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 108 cells per cm 3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.
Introduction Like most transformed cell lines, hybridoma cells are anchorage independent. However, they can be immobilized by immurement or entrapment methods. Examples of immurement techniques are hollow fiber (Hopkinson, 1985; Tharakan and Chau, 1988), membrane reactors (Scheirer, 1988) and encapsulation (Duff, 1985; Nilsson and Mos-
bach, 1987). These techniques allow very high unit cell densities to be maintained (about 108 cells ml -l) but scale-up of these methodologies are usually problematic. Entrapment methods include porous ceramic cartridge (Lyderson, 1987), sponge (Murdin et al., 1987; Lazar et al., 1988) and porous particles (Murdin et al., 1989). Although these methods do not permit as high a unit density (approximately 5 x 107 cells m1-1) they do
42 have good scale-up potential and require less sophisticated equipment and control. Packed bed reactors ( P B R ' s ) for the cultivation of anchorage-dependent animal cells have been in use for m a n y years. It has been shown in the past few years that hybridomas can be entrapped and grown within P B R ' s using polyester f o a m particles and glass beads as a support material (Murdin et al., 1989; Bliem et al., 1990). Recently, we have found that hybridomas can be successfully trapped and grown on the polyester carrier in a packed bed culture system (Wang et al., unpublished). This carrier is made of disks of polyester n o n - w o v e n fabric laminated to a polypropylene screen, surface treated and pre-coated with poly-D-lysine. This substrate (Fibra-cel carrier, Sterilin, England) has already been used for the growth of anchorage-dependent animal ceils such as normal and recombinant C H O cells, h u m a n and mouse fibroblasts (Kadouri and Ziper, 1989; Kadouri, 1990) and also for the growth o f insect cells ( K o m p i e r et al., 1991). In this article, two bioreactor designs were tested for the production of antibodies. The first design incorporated a packed-bed basket inside a CelliGen bioreactor whose vessel was modified to a c c o m o o d a t e the basket. The second design utilized a CelliGen as part of a closed system incorporating a p a c k e d - b e d column reactor. In this instance, the CelliGen was used as a media-
harvest
conditioning tank only, with the cells propagated in the external column. The goals of this study were to demonstrate that: 1. Polyester disks support the propagation of hybridomas and promote cell growth at a high cell density per unit volume of bioreactor. 2. H y b r i d o m a s can be retained within the packing material in a productive state, under perfusion, and with low levels o f serum or serumfree medium. 3. Scale-up is possible due to the low pressure drop across the basket and column, and oxygen up-take rate (OUR) can be calculated and used to optimize perfusion.
Materials and methods Cell line
M o u s e - m o u s e h y b r i d o m a D A 4 . 4 ( A T C C - 5 7 ) produces an IgG1 against h u m a n IgM. Medium
D M E M was used for maintenance and growth of cells in T-flasks and bioreactors. Additional supplements were added, such as primatone RL, oxalacetic acid, mercaptoethanol, insulin and antibiotics. Bovine calf serum was added to 5%, 3%
T probe
li
ps D.O.~ i--:
,
i
I :-
=='bed
packed column
ir; ;ti I fresh
m e d tum
Fig. 1. Schematic diagram of CelliGen-packed bed column cell culture system. Cells are immobilizedon the polyester non-woven fabric disks and the pinch valve controls the direction of medium flow through the bed column. The bed reactor was included in the incubator which can be set to hold a constant temperature (37~ The CelliGen vessel, serving as reservoir, was connected to the bed reactor by a silicone tubing.
43 or 1% volume. The serum-free medium was supplemented with 100 ml CHO-1 ~ o w t h additive per 10 1 volume. Final concentration of glucose was 4.5 g 1-~-9.0 g 1-].
immunodiffusion assay using rabbit and mouse antiserum in 1.5% agar.
Results and discussion
Equipment Cell attachment kinetics 1.5 1 CelliGen, 0.5 1 packed bed column reactor, perfusion system (New Brunswick Scientific Co.). The total volume of recirculating medium in this culture system is 1.6 1 (Fig. 1). 2.5 1 CelliGen, 1.0 1 packed bed basket reactor, perfusion system (New Brunswick Scientific Co.). The total working volume in this reactor is 1.8 1 (Fig. 2).
Analysis Culture media was analyzed for glucose using pre-formed kits 16-UV (Sigma). Trypan blue cell counts by h e m o c y t o m e t e r were used to determine the cell numbers in the media during the course of the entire run. This method was also used to determine the final cell density in the packed bed. MAb concentration was determined by radial
f r ~ h __
A glass column, 14.6 cm high and 3.4 cm in diameter, was packed with 14.3 g of polyester disks to yield a void fraction of 90%. D M E medium was first placed into the packed bed. A suspension of 2.0 x 107 cells was then introduced into the column and the media flow rate was set at 45 ml min -l. Output medium was collected and reintroduced into the column for second pass cycle. The cells retained in the packed bed after the first three pass cycles were 39%, 54% and 60% of the inoculum, respectively. This indicates that the hybridomas were successfully trapped in the packed bed, and the attachment procedure needs more than one cycle in order to obtain complete entrapment. These experiments confirmed that continuous media circulation for one hour in the CelliGen-packed bed reactor resulted in the attachment of about 95% of the inoculum. The study further showed that use of polyester disks eliminate the need to immobilize cells before inoculating them into the reactor, as is normally required when using other materials such as agarose, alginate beads or polylysine capsules.
Effect of impeller speed on liquid flow rate ......
. . . . . . . .o.
............
D.O.
9* ~ . ~
#
packed bed basket
i/nlz:,;,]a~
!..L:I5~:,~,,I
--._._ impeller with st~r~er
A Pitot tubing method was used to determine liquid flow rate for various impeller speeds in the CelliGen-packed bed basket bioreactor. Flow rates were measured to be 45 ml s -l to 110 ml s -l at impeller speeds of 60 rpm to 130 rpm. See Fig. 3(A).
Pressure drop of the polyester disk bed Fig. 2. CelliGen-packed bed basket cell culture system. The polyester non-woven fabric disks were packed into the basket of the reactor. Cells are immobilized on the bed. Air is introduced from the sparger and oxygenates the medium and the bubbles disengage at the upper fluid surface. The CelliGen impeller can provide bubble-free medium to flow through the packed bed.
Pressure drop of the disk bed, Ap, was measured with a glass tube manometer at different liquid flow rates and was calculated by the equation: Ap = Ah(p L - Po)g = kgLu
44
MAb PRODUCTION
IN CELLIGEN-PACKED
BED COLUMN
Inoculation
SYSTEM
Medium Circulation
I
I
T-Flasks
> 1.5
CelliGen
> Cell Growth in Packed Bed Column
I I I
Perfusion with 5% Serum Ha~est MAb
High Cell Density
Modified CelliGen-packed bed bioreactors for hybridoma cell cultures Guozheng Wang 1, Wenying Zhang 1, Corey Jacklin 1, David Freedman l, Lee Eppstein I and Avinoam Kadouri 2 1New Brunswick Scientific Co., Inc., 44 Talmadge Road, Edison, N J, 08818-4005, USA; 2Department of Membrane Research and Biophysics, The Weizmann hTstitute of Science, Rehovot, 76100, Israel Received 31 March 1992; accepted in revised form 24 August 1992
Key words: animal cell culture, hybridoma, monoclonal antibody, packed bed reactor, per'fusion
Abstract This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgGl monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen T M bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 108 cells per cm 3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.
Introduction Like most transformed cell lines, hybridoma cells are anchorage independent. However, they can be immobilized by immurement or entrapment methods. Examples of immurement techniques are hollow fiber (Hopkinson, 1985; Tharakan and Chau, 1988), membrane reactors (Scheirer, 1988) and encapsulation (Duff, 1985; Nilsson and Mos-
bach, 1987). These techniques allow very high unit cell densities to be maintained (about 108 cells ml -l) but scale-up of these methodologies are usually problematic. Entrapment methods include porous ceramic cartridge (Lyderson, 1987), sponge (Murdin et al., 1987; Lazar et al., 1988) and porous particles (Murdin et al., 1989). Although these methods do not permit as high a unit density (approximately 5 x 107 cells m1-1) they do
42 have good scale-up potential and require less sophisticated equipment and control. Packed bed reactors ( P B R ' s ) for the cultivation of anchorage-dependent animal cells have been in use for m a n y years. It has been shown in the past few years that hybridomas can be entrapped and grown within P B R ' s using polyester f o a m particles and glass beads as a support material (Murdin et al., 1989; Bliem et al., 1990). Recently, we have found that hybridomas can be successfully trapped and grown on the polyester carrier in a packed bed culture system (Wang et al., unpublished). This carrier is made of disks of polyester n o n - w o v e n fabric laminated to a polypropylene screen, surface treated and pre-coated with poly-D-lysine. This substrate (Fibra-cel carrier, Sterilin, England) has already been used for the growth of anchorage-dependent animal ceils such as normal and recombinant C H O cells, h u m a n and mouse fibroblasts (Kadouri and Ziper, 1989; Kadouri, 1990) and also for the growth o f insect cells ( K o m p i e r et al., 1991). In this article, two bioreactor designs were tested for the production of antibodies. The first design incorporated a packed-bed basket inside a CelliGen bioreactor whose vessel was modified to a c c o m o o d a t e the basket. The second design utilized a CelliGen as part of a closed system incorporating a p a c k e d - b e d column reactor. In this instance, the CelliGen was used as a media-
harvest
conditioning tank only, with the cells propagated in the external column. The goals of this study were to demonstrate that: 1. Polyester disks support the propagation of hybridomas and promote cell growth at a high cell density per unit volume of bioreactor. 2. H y b r i d o m a s can be retained within the packing material in a productive state, under perfusion, and with low levels o f serum or serumfree medium. 3. Scale-up is possible due to the low pressure drop across the basket and column, and oxygen up-take rate (OUR) can be calculated and used to optimize perfusion.
Materials and methods Cell line
M o u s e - m o u s e h y b r i d o m a D A 4 . 4 ( A T C C - 5 7 ) produces an IgG1 against h u m a n IgM. Medium
D M E M was used for maintenance and growth of cells in T-flasks and bioreactors. Additional supplements were added, such as primatone RL, oxalacetic acid, mercaptoethanol, insulin and antibiotics. Bovine calf serum was added to 5%, 3%
T probe
li
ps D.O.~ i--:
,
i
I :-
=='bed
packed column
ir; ;ti I fresh
m e d tum
Fig. 1. Schematic diagram of CelliGen-packed bed column cell culture system. Cells are immobilizedon the polyester non-woven fabric disks and the pinch valve controls the direction of medium flow through the bed column. The bed reactor was included in the incubator which can be set to hold a constant temperature (37~ The CelliGen vessel, serving as reservoir, was connected to the bed reactor by a silicone tubing.
43 or 1% volume. The serum-free medium was supplemented with 100 ml CHO-1 ~ o w t h additive per 10 1 volume. Final concentration of glucose was 4.5 g 1-~-9.0 g 1-].
immunodiffusion assay using rabbit and mouse antiserum in 1.5% agar.
Results and discussion
Equipment Cell attachment kinetics 1.5 1 CelliGen, 0.5 1 packed bed column reactor, perfusion system (New Brunswick Scientific Co.). The total volume of recirculating medium in this culture system is 1.6 1 (Fig. 1). 2.5 1 CelliGen, 1.0 1 packed bed basket reactor, perfusion system (New Brunswick Scientific Co.). The total working volume in this reactor is 1.8 1 (Fig. 2).
Analysis Culture media was analyzed for glucose using pre-formed kits 16-UV (Sigma). Trypan blue cell counts by h e m o c y t o m e t e r were used to determine the cell numbers in the media during the course of the entire run. This method was also used to determine the final cell density in the packed bed. MAb concentration was determined by radial
f r ~ h __
A glass column, 14.6 cm high and 3.4 cm in diameter, was packed with 14.3 g of polyester disks to yield a void fraction of 90%. D M E medium was first placed into the packed bed. A suspension of 2.0 x 107 cells was then introduced into the column and the media flow rate was set at 45 ml min -l. Output medium was collected and reintroduced into the column for second pass cycle. The cells retained in the packed bed after the first three pass cycles were 39%, 54% and 60% of the inoculum, respectively. This indicates that the hybridomas were successfully trapped in the packed bed, and the attachment procedure needs more than one cycle in order to obtain complete entrapment. These experiments confirmed that continuous media circulation for one hour in the CelliGen-packed bed reactor resulted in the attachment of about 95% of the inoculum. The study further showed that use of polyester disks eliminate the need to immobilize cells before inoculating them into the reactor, as is normally required when using other materials such as agarose, alginate beads or polylysine capsules.
Effect of impeller speed on liquid flow rate ......
. . . . . . . .o.
............
D.O.
9* ~ . ~
#
packed bed basket
i/nlz:,;,]a~
!..L:I5~:,~,,I
--._._ impeller with st~r~er
A Pitot tubing method was used to determine liquid flow rate for various impeller speeds in the CelliGen-packed bed basket bioreactor. Flow rates were measured to be 45 ml s -l to 110 ml s -l at impeller speeds of 60 rpm to 130 rpm. See Fig. 3(A).
Pressure drop of the polyester disk bed Fig. 2. CelliGen-packed bed basket cell culture system. The polyester non-woven fabric disks were packed into the basket of the reactor. Cells are immobilized on the bed. Air is introduced from the sparger and oxygenates the medium and the bubbles disengage at the upper fluid surface. The CelliGen impeller can provide bubble-free medium to flow through the packed bed.
Pressure drop of the disk bed, Ap, was measured with a glass tube manometer at different liquid flow rates and was calculated by the equation: Ap = Ah(p L - Po)g = kgLu
44
MAb PRODUCTION
IN CELLIGEN-PACKED
BED COLUMN
Inoculation
SYSTEM
Medium Circulation
I
I
T-Flasks
> 1.5
CelliGen
> Cell Growth in Packed Bed Column
I I I
Perfusion with 5% Serum Ha~est MAb
High Cell Density
