Cytotechnology 8:179-187. 1992. 9 1992KluwerAcademic Publishers. Printed in the Netherlands.
A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells B. Persson and C. Emborg
Center for Process Biotechnology, Deparonent of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark Received 3 December 1991; accepted in revised form 23 May 1992
Key words: cell culture equipment, simple bioreactors, hybridomas, monoclonal antibody production Abstract
Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors.
Introduction
Parallel to the increased use/need for mammalian cells as production vehicles for pharmaceutical and diagnostic products, many new culture systems for these cell types have emerged on the market. Some of these systems try to overcome the pronounced problem of cell shear sensitivity by using cell entrapment or membrane technology (Nilsson, 1987). However, it seems that not all cell lines are as shear sensitive as earlier assumed (Dodge and Hu, 1986; Lee et al., 1988; Marquis et al., 1989), and that many mammalian cells actually may be cultivated in only slightly modified traditional stirred tank fermentors. We report here on a comparison of three very simple culture vessels (built from standard laboratory equipment) and a specialized commercial mammalian cell bioreactor. The hybridoma
growth and antibody production supporting abilities of the culture vessels were tested in a series of standardized batch fermentations. Even though some disadvantages exist with this culture technique, i.e., changes in the homeostatic environment with time, etc., it is a very simple and reliable way of testing cell culture equipment if used with standard set-ups and culture conditions.
Materials and methods
Cells All investigations were carried out with a x63Ag8.653-derived mouse/mouse hybridoma cell line, NUC 1-4, a low-producer of monoclonal antibodies against the enzyme 'nuclease' (from Serratia marcescence). This cell line was estab-
180 lished during a collaboration between The Technical University of Denmark (DTH) and The Hybridoma Laboratory, The State Serum Institute (SSI) in 1987 (Andresen, 1987). The cells were maintained at an approximate cell density of 1 • 105 cells]ml in T-flasks (NUNC, Denmark) in a thermostated incubator (Queue Systems, Inc.) with a humidified atmosphere (92% relative humidity) of 5% CO2 in atmospheric air. For each experiment the cells were centrifuged and resuspended in 10-20 ml fresh medium. The reactors were inoculated with cells to a total density of approximately I x 105 cells/ml. The cells were regularly tested for mycoplasma (Mycotect-kit, Gibco) and no infections were ever found. At the end of the project period, the cell line was tested for changes in growth and production kinetics, and also these investigations turned out satisfactory (Sanfeliu, 1991).
Growth medium Standard DMEM powder medium (Biochrom, cat. no. T 043-10) containing 4.5 g/1 glucose and 4 mM L-glutamine was used. Additionally, NaHCO3 (3.7 gfl = 4.4 mM), pyruvate (0.122 g/l = 1.1 mM), ~-mercaptoethanol (0.556 ml/1 of a freshly prepared 0.125 M solution = 70 laM), HEPES (2.383 g/l = 10 mM), and extra L-glutamine (0.9086 g/1 = 6.2 mM) were added to the medium, and the solution was sterilized by filtration (0.22 p.m) and stored in a dark room at +4~ for no more than 4 weeks. No antibiotics were added to the medium at any time. Before use 10% FCS was added to the medium.
Equipment One of the simple bioreactors was a standard 1600 ml total volume Bellco spinner flask (Bellco, Inc.). Spinner flasks have already proven efficient for cell cultures, but have to our knowledge never been directly compared to a special mammalian cell bioreactor under standard conditions. The aeration was by surface aeration only, and agitation was by a magnetic spin,bar suspended in a
glass rod about 1/4 of the liquid height above the bottom of the flask, thus avoiding squeezing the cells between the stirrer and the vessel bottom. Temperature, DO (dissolved oxygen) and pH control were achieved by placing the spinner flask inside a CO2-incubator with a controlled, 37~ humid (92% relative humidity) atmosphere of 5% CO2 in atmospheric air. Another reactor was based on a commercially available plastic jar from Nalgene (art. no. 100.948, KEBO lab. laboratory supply, Sweden). The total volume was 332 ml (working volume = 200 ml), and like the spinner flask this bioreactor was operated inside a CO2-incubator under identical conditions. Aeration was by surface aeration and agitation was by a magnetic spin-bar mounted in a holder keeping it 0.5 cm above the bottom of the vessel. A standard 1500 ml Blue-cap bottle (Schott Glaswerke AG, Germany) was used as a basis for the third bioreactor. The bottle had a working volume of 1000 ml, and to improve the mass transfer an additional micro-sparging device was mounted in the vessel. This device was basically a piece of sintered glass and a piece of silicone tubing (as used in domestic aquaria) and placed on the bottom of the flask. A gas mixture of 5% CO2 in atmospheric air was used to control pH and DO, and agitation was by a standard magnetic spin-bar. The whole set-up was placed inside an incubator for optimal temperature control. A 2.5 1 total volume Biostat| MC (B. Braun Diessel Biotech, Melsungen, Germany) equipped with a variable-pitch paddle impeller was used to represent the group of specialized bioreactors for mammalian cells. For aeration, a membrane aeration system consisting of 6 m of silicone tubing (outer diameter: 3.35 mm and inner diameter of 3.0 ram) mounted on a specially designed holder and submerged into the medium is used (Kuhlmann, 1987). A gas mix unit provides the proper mixture of gasses for DO and pH control, and a temperature module connected to a full size water jacket controls the vessel temperature. All the culture vessels were autoclaved according to standard conditions before use.
181 Test set-up All experimental runs were batch fermentations carried out under following standard conditions. Culture medium: as mentioned above with 10% FCS; inoculum: approximately 1 x 105 cells/ml; temperature: +37~ (+ 0.2~ pH: 7.3 (+ 0. I); DO: 40% (with respect to max. dissolved atmospheric air); and CO2: 5%. The agitation was set to give almost identical mixing times during the cultivations. Therefore stirrer speeds in the range of 50-100 rpm (+ 10 rpm), depending on the system, were used. Assays and measurements Cell number. Cell viability was determined in a hemocytometer using the Trypan blue dye exclusion technique (Abu-Reesh and Kargi, 1989). The viable and dead cell numbers were mean values from two individual determinations. Glucose~lactate. The levels of glucose and lactate in the growth medium were determined using a YSI 2000 semi-automatic glucose/lactate analyzer (Yellow Spring Instruments). Product (lgG). The product concentration in the growth medium (the total amount of IgG) was determined by a three-layer sandwich ELISA (catching ELISA). The procedure is a modified version (The State Serum Institute (SSI), personal communication) of the 'capture ELISA" procedure described by Gardner and Eiteman (1989). Rabbit anti-mouse IgG and peroxidase conjugated rabbit anti-mouse antibodies (code Z 109 and code p 260, Dakopatts, Denmark) were used in layers 1 and 3, respectively, instead of the goat anti-mouse IgG antibodies used by Gardner. An IgG standard 'HAV 3 - 1 ' (20 /.tg/ml, SSI) was used as reference. The assays were carried out in Nunc immunoplates, Maxisorp F96 microtiter plates (NUNC, Denmark), and the absorbance in the wells were measured at 490 nm, using an Easy-reader (EAR 340 AT, SLT-Labinstruments, Austria).
Specific production rate. The specific production rate in the cultures was calculated according to the equation:
Spec.product.rate = ~ dt
-I
(Cp,_ - Cpj) ,11._____~). (n2 + (t2 - t I) 2
)
( 1)
where Up2 and Cp~ are the product concentrations at t2 and tl, respectively and n 2 and nl are the cell concentrations.
Mixing time. The mixing times of the different systems were reflected in the time to reach 95% homogeneity in the vessels following a pulse of acid. Tap water was used in the determinations and the pulse was the addition of 500-1000 lal (depending on the vessel size) of 37% HCI at one point to the liquid. A standard pH electrode (Ingold) was used in combination with the pHmeter from a Biostat| MC (B. Braun, Melsungen, Germany), and the signals were recorded on a SE 120 recorder (BBC, Goerz Metrawatt). kLa Detelwzination. The kLa values were determined by the gassing-in method derived by Bartholomew et al. (1950). The liquid in the vessel was a 0.15 M solution of NaCI in tap water mimicking the growth medium with respect to content of electrolytes and the gas used was atmospheric air. A polarografic electrode (Ingold) was used in combination with the O2-measuring device in a Biostat| MC (B. Braun, Melsungen, Germany) and the O2-values were recorded on a SE 120 recorder (BBC, Goerz Metrawatt). The kLa values can thus be estimated from the slope of a semi-logarithmic plot of the electrode response (change of O2-concentration) versus time (Bartholomew et al., 1950; Kim and Chang, 1989). As this method is based on a fast-responding electrode the electrode-response time was tested. Bringing the electrode from a N2-gassed chamber to atmospheric air, the readings were as followed. The response time, i.e., the time needed for the electrode to record the change from the 0% to the
182 95% oxygen level, was timed to be in the range of 2 0 - 2 5 seconds (+ 5 s). As this is significantly faster than even the fastest change in DO-concentrations during the kLa-measurements, the electrode was considered useful for these experiments. Oaygen transfer rate (OTR). Based on the kLavalues obtained from the individual culture systems the OTRs were calculated according to: OTR = kL a (C*g - CL )
(2)
A C*-value of 0.238 mM/1 (Schumpe et al., 1978) and a CL-value on 40% of the C*-value was used. Furthermore, to be able to directly compare the different systems, the OTR-values were normalized with respect to volume. This way it was possible to evaluate the total OTR for the different systems.
Results and discussion Despite a considerable amount of newly developed/designed culture systems for mammalian cell cultivation, the tank type bioreactors are still the systems o f choice in industry (Phillips et al., 1985; T y o and Spier, 1987). Research in this area can therefore still be justified and especially the shear problems need to be addressed. A general attitude has been that the cell shear sensitivity could only be surmounted by changes in bioreactor design or culture mode. W e believe, though, that cells can be adapted to increased shear stress environments and thus cultured in reactors of very simple designs.
capabilities. This primarily because the spin-bar is a radial type impeller that merely creates vortices in the liquid with only a small amount o f axial mixing. As soon as (bubble-) aeration (the Blue-cap bioreactor) or impeller type agitators (the Biostat| MC) are used, significant effects on the mixing is seen as an axial component is added to the flow patterns in the vessels. A b o v e 150 rpm almost identical mixing times were found with the systems, but considering the hydrodynamic shear in the tanks stirrer speeds of 100 rpm (50 rpm for the Biostat| MC) were used. In this range, reasonable and comparable mixing times were found without detrimental effects to the cells. M a s s transfer rates
Mass transfer rates (kLa) were determined for the systems. In the aerated vessels (Blue-cap reactor and Biostat| MC) a flow rate of 500 ml/min was used. As expected the surface aerated bioreactors displayed very low kca-values due to the diffusive nature of mass transfer connected to surface aeration (Table 1). Also predictable is the difference in mass transfer rates between the micro-sparged Blue-cap reactor and the Biostat| M C system caused by the variation in mass transfer areas.
! .2 0
L I 9 0 7-
t ~'![ •--
t-
\
90
\
\
'
40
\
! '\ _
M i x i n g times
\
9
\, ~'
20
\\
~--------~,
,,
T,]
A good starting parameter in a comparison of bioreactors is the mixing times, since homogeneity is rather important in mammalian cell cultures. From Fig. 1 it can be seen that a large scattering of mixing times exist at low stirrer speeds. As could be expected the spin-bar agitated vessels display some very poor mixing
2, i
~
0
:
50
,
i
l
~Cv
150
L
239
250
,
r
~'-,'~
35C
43,3
rpm vo
Spin he!" flask 9
Plastic-jar Biostat
reactor
,>
Elue-eap
reactor
fllue-cnp
reactor
/aera'.ion /aeration
MC
Fig. 1. Mixing times in the bioreactors vs. stirrer speed.
183 Table 1. Comparison of the different cell culture systems
Spinner flask Plastic-jar bioreactor Blue-cap bioreactor Biostat| MC bioreactor
Working volume
Maximum obtained cell density
Total IgGproduction
Mass transfer rate (kLa)
Oxygen transfer rate (OTR)
Maximum specific production rate
(ml)
(x 105/ml)
(mg/l)
(h-1)
(mM/I/h)
(xl0-10 mg/cell/h)
15
13.2
0.42
0.060
2.49
none
B, SC
30.0
0.52
0.075
5.08
none
B, SC
800 200
7.3
Control facilities (basis)
Operation modes:
B: batch; SC: semicontinuous; C: continuous; P: perfusion
1000
17
10.0
14.66
1.540
2.16
none
B, SC, C
2000
19
13.7
3.00
0.419
1.61
many
B, SC, C, pa
~ell retention device needed (extra).
T h e d i f f e r e n c e s in k L a - v a l u e s are o f c o u r s e r e f l e c t e d in the o x y g e n t r a n s f e r rates ( O T R ) o f the s y s t e m s . O n this basis it is i n t e r e s t i n g to see that, d e s p i t e large v a r i a t i o n s in the abilities to t r a n s f e r o x y g e n to the cells ( T a b l e 1), a l m o s t i d e n t i c a l m a x i m u m cell n u m b e r s are a c h i e v e d in the m a j o r ity o f the vessels. T h i s m e a n s that o x y g e n is n o t a
l i m i t i n g factor in (our) s m a l l - s c a l e b a t c h s y s t e m s , an o b s e r v a t i o n that has also b e e n m e n t i o n e d b y other g r o u p s w o r k i n g w i t h s m a l l - s c a l e s y s t e m s . M o r e likely are the l i m i t a t i o n s d u e to l o w n u t r i e n t or h i g h waste c o n c e n t r a t i o n s . R e c e n t l y Jo e t al. (1990) r e p o r t e d o n the use o f fortified g r o w t h m e d i a as the m e a n s to e x c e e d the - for b a t c h
Table 2. Flow and shear characteristics
Reynolds numbera
Power numberb
(Dimensionless) Spinner flask Plastic-jar bioreactor Blue-cap bioreactor
2672 2672 2672
0.143 0.373 0.113
Biostat| MC bioreactor
3000
0.120
Impeller power inputc
Terminal eddy sized
Eddy velocitye
(x 10-2 W/l)
(x 10-6 m)
(x 10-3 m/s)
6.764 17.693 5.360 (31.426)g 2.697
330 184 369 (75) 439
3.03 5.45 2.71 (13.31) 2.28
Maximum turbulent stressf (x 10-2 N/m2) 0.92 2.97 0.73 (17.70) 0.52
aD2Np/la, where D is impeller diameter, N is rev. per min., p is liquid density, and I.t is liquid viscosity (103 Pa.s); baccording to Nagata, 1975, p. 35 (equ. 1.53); Cderived from the power number equation Np = p/N3D5p; p = 103 kg/m3 normalized to volume; dbased on rI = (~3/E0-25, 9 being the kinematic viscosity of the medium and E the rate of turbulent energy dissipation (e =(NpN3D5/V) (Cherry and Papoutsakis, 1988); ebased on v = (~e) 0.25 (Cherry and Papoutsakis, 1988); fbased on x = p(v)2, v being the terminal eddy velocity (Kunas and Papoutsakis, 1989; Oh et al., 1989); gderived from the gas power number PG/V = pgv s (Chisti, 1989), PG being the power number, V the liquid volume, p the liquid density and Vs is the superficial gas velocity and an assumed 40% conversion to hydrodynamic stress (Katinger et al., 1979).
184 cultures - almost magic cell density limit of 2 • 10 6 cells/ml, and even though the concept has not been tried in this work our glucose/lactate measurements (data not shown) support the idea and it seems a reasonable way to go. The low cell number found with the Plastic-jar bioreactor might on the other hand be related to the high level of hydrodynamic shear stress developed in this vessel.
Power input To further characterize the culture systems the Reynolds numbers and power inputs were calculated (Table 2). Further the gas-power input was determined for the Blue-cap reactor (added in brackets). From the Reynolds number it is obvious that all the reactors are operated with turbulent liquid flow patterns, as the Reynolds numbers exceed 1000 (Oldshue, 1983). Due to differences in geometry and impeller configurations in the vessels it is not possible to assume identical power numbers for all the bioreactors. Therefore the power number was calculated in each case according to Nagata (1975): h
A
N" = v e + 8 (10 -, +
3.2Re~176
(,;',0) ' 2
(3) where ,)
A--14+
.)
(4)
,, +4/ 1
0 /2
and b = the width of the impeller blades; d = impeller diameter; D = vessel diameter; and 0 = angle of the impeller blades.
Hydrodynamic shear Several types of detrimental effects are introduced into the culture systems, but basically they are all derived from two sources: the effects from bubbles (in the sparger regions and in the burstingzone at the liquid-gas interface), and the influence of the agitator and other internals on the liquid flow in the vessels. Much work have shown that the most important damaging mechanism for animal cells are related to the bursting of bubbles at the liquid-gas interface (Handa-Corrigan et al., 1989; Oh et al., 1989; Passini and Goochee, 1989), but since only one of the reactors in this investigation is bubble-aerated, the bubble effects can not be used as a comparative parameter. Therefore we have chosen to use the hydrodynamic shear (and 'The K o l m o g o r o v Eddy T h e o r y ' ) as a standard of reference in this work. Certainly, this approach will underestimate the total amount o f detrimental effects in the 'Blue-cap system', but as shear data based on the gas power input are added to the hydrodynamic data in Table 2 (in brackets), it is possible to get an idea o f the size o f the bubble effect. These data support the general view on bubble-bursts as the major detrimental source in sparged bioreactors since the damaging effects from bursting bubbles are directly related to the gas power input. Based on the power input and following the ' K o l m o g o r o v Eddy T h e o r y ' terminal eddy sizes and eddy velocities were calculated according to Cherry and Papoutsakis (1988), and the maximum turbulent stress levels, z, were determined from: z = p(v) 2 (Kunas and Papoutsakis, 1989; Oh et al., 1989) (see Table 2). The differences in vessel size and geometry is reflected in the power input numbers, and thus in the terminal eddy sizes and velocities, and turbulent stress numbers. As would be expected the high gas-flow rate (0.5 vvm) used with the Bluecap system should give very small and fast m o v i n g terminal eddies, and thus a (quite) high turbulent stress level, compared to the non-sparged systems. However, as can be seen from Table 2, despite this high power input the terminal eddy size is still much larger than the hybridoma cell
185 2~ Z_ ~
J .J
r _
/i
15~
E
,
'k \
/'
~O
!4 L
•
!
--
C. , , ~
[]
M
IC'~
///
'
'/" .
I 2 ~ ..z~,
,'7" 9
A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells B. Persson and C. Emborg
Center for Process Biotechnology, Deparonent of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark Received 3 December 1991; accepted in revised form 23 May 1992
Key words: cell culture equipment, simple bioreactors, hybridomas, monoclonal antibody production Abstract
Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors.
Introduction
Parallel to the increased use/need for mammalian cells as production vehicles for pharmaceutical and diagnostic products, many new culture systems for these cell types have emerged on the market. Some of these systems try to overcome the pronounced problem of cell shear sensitivity by using cell entrapment or membrane technology (Nilsson, 1987). However, it seems that not all cell lines are as shear sensitive as earlier assumed (Dodge and Hu, 1986; Lee et al., 1988; Marquis et al., 1989), and that many mammalian cells actually may be cultivated in only slightly modified traditional stirred tank fermentors. We report here on a comparison of three very simple culture vessels (built from standard laboratory equipment) and a specialized commercial mammalian cell bioreactor. The hybridoma
growth and antibody production supporting abilities of the culture vessels were tested in a series of standardized batch fermentations. Even though some disadvantages exist with this culture technique, i.e., changes in the homeostatic environment with time, etc., it is a very simple and reliable way of testing cell culture equipment if used with standard set-ups and culture conditions.
Materials and methods
Cells All investigations were carried out with a x63Ag8.653-derived mouse/mouse hybridoma cell line, NUC 1-4, a low-producer of monoclonal antibodies against the enzyme 'nuclease' (from Serratia marcescence). This cell line was estab-
180 lished during a collaboration between The Technical University of Denmark (DTH) and The Hybridoma Laboratory, The State Serum Institute (SSI) in 1987 (Andresen, 1987). The cells were maintained at an approximate cell density of 1 • 105 cells]ml in T-flasks (NUNC, Denmark) in a thermostated incubator (Queue Systems, Inc.) with a humidified atmosphere (92% relative humidity) of 5% CO2 in atmospheric air. For each experiment the cells were centrifuged and resuspended in 10-20 ml fresh medium. The reactors were inoculated with cells to a total density of approximately I x 105 cells/ml. The cells were regularly tested for mycoplasma (Mycotect-kit, Gibco) and no infections were ever found. At the end of the project period, the cell line was tested for changes in growth and production kinetics, and also these investigations turned out satisfactory (Sanfeliu, 1991).
Growth medium Standard DMEM powder medium (Biochrom, cat. no. T 043-10) containing 4.5 g/1 glucose and 4 mM L-glutamine was used. Additionally, NaHCO3 (3.7 gfl = 4.4 mM), pyruvate (0.122 g/l = 1.1 mM), ~-mercaptoethanol (0.556 ml/1 of a freshly prepared 0.125 M solution = 70 laM), HEPES (2.383 g/l = 10 mM), and extra L-glutamine (0.9086 g/1 = 6.2 mM) were added to the medium, and the solution was sterilized by filtration (0.22 p.m) and stored in a dark room at +4~ for no more than 4 weeks. No antibiotics were added to the medium at any time. Before use 10% FCS was added to the medium.
Equipment One of the simple bioreactors was a standard 1600 ml total volume Bellco spinner flask (Bellco, Inc.). Spinner flasks have already proven efficient for cell cultures, but have to our knowledge never been directly compared to a special mammalian cell bioreactor under standard conditions. The aeration was by surface aeration only, and agitation was by a magnetic spin,bar suspended in a
glass rod about 1/4 of the liquid height above the bottom of the flask, thus avoiding squeezing the cells between the stirrer and the vessel bottom. Temperature, DO (dissolved oxygen) and pH control were achieved by placing the spinner flask inside a CO2-incubator with a controlled, 37~ humid (92% relative humidity) atmosphere of 5% CO2 in atmospheric air. Another reactor was based on a commercially available plastic jar from Nalgene (art. no. 100.948, KEBO lab. laboratory supply, Sweden). The total volume was 332 ml (working volume = 200 ml), and like the spinner flask this bioreactor was operated inside a CO2-incubator under identical conditions. Aeration was by surface aeration and agitation was by a magnetic spin-bar mounted in a holder keeping it 0.5 cm above the bottom of the vessel. A standard 1500 ml Blue-cap bottle (Schott Glaswerke AG, Germany) was used as a basis for the third bioreactor. The bottle had a working volume of 1000 ml, and to improve the mass transfer an additional micro-sparging device was mounted in the vessel. This device was basically a piece of sintered glass and a piece of silicone tubing (as used in domestic aquaria) and placed on the bottom of the flask. A gas mixture of 5% CO2 in atmospheric air was used to control pH and DO, and agitation was by a standard magnetic spin-bar. The whole set-up was placed inside an incubator for optimal temperature control. A 2.5 1 total volume Biostat| MC (B. Braun Diessel Biotech, Melsungen, Germany) equipped with a variable-pitch paddle impeller was used to represent the group of specialized bioreactors for mammalian cells. For aeration, a membrane aeration system consisting of 6 m of silicone tubing (outer diameter: 3.35 mm and inner diameter of 3.0 ram) mounted on a specially designed holder and submerged into the medium is used (Kuhlmann, 1987). A gas mix unit provides the proper mixture of gasses for DO and pH control, and a temperature module connected to a full size water jacket controls the vessel temperature. All the culture vessels were autoclaved according to standard conditions before use.
181 Test set-up All experimental runs were batch fermentations carried out under following standard conditions. Culture medium: as mentioned above with 10% FCS; inoculum: approximately 1 x 105 cells/ml; temperature: +37~ (+ 0.2~ pH: 7.3 (+ 0. I); DO: 40% (with respect to max. dissolved atmospheric air); and CO2: 5%. The agitation was set to give almost identical mixing times during the cultivations. Therefore stirrer speeds in the range of 50-100 rpm (+ 10 rpm), depending on the system, were used. Assays and measurements Cell number. Cell viability was determined in a hemocytometer using the Trypan blue dye exclusion technique (Abu-Reesh and Kargi, 1989). The viable and dead cell numbers were mean values from two individual determinations. Glucose~lactate. The levels of glucose and lactate in the growth medium were determined using a YSI 2000 semi-automatic glucose/lactate analyzer (Yellow Spring Instruments). Product (lgG). The product concentration in the growth medium (the total amount of IgG) was determined by a three-layer sandwich ELISA (catching ELISA). The procedure is a modified version (The State Serum Institute (SSI), personal communication) of the 'capture ELISA" procedure described by Gardner and Eiteman (1989). Rabbit anti-mouse IgG and peroxidase conjugated rabbit anti-mouse antibodies (code Z 109 and code p 260, Dakopatts, Denmark) were used in layers 1 and 3, respectively, instead of the goat anti-mouse IgG antibodies used by Gardner. An IgG standard 'HAV 3 - 1 ' (20 /.tg/ml, SSI) was used as reference. The assays were carried out in Nunc immunoplates, Maxisorp F96 microtiter plates (NUNC, Denmark), and the absorbance in the wells were measured at 490 nm, using an Easy-reader (EAR 340 AT, SLT-Labinstruments, Austria).
Specific production rate. The specific production rate in the cultures was calculated according to the equation:
Spec.product.rate = ~ dt
-I
(Cp,_ - Cpj) ,11._____~). (n2 + (t2 - t I) 2
)
( 1)
where Up2 and Cp~ are the product concentrations at t2 and tl, respectively and n 2 and nl are the cell concentrations.
Mixing time. The mixing times of the different systems were reflected in the time to reach 95% homogeneity in the vessels following a pulse of acid. Tap water was used in the determinations and the pulse was the addition of 500-1000 lal (depending on the vessel size) of 37% HCI at one point to the liquid. A standard pH electrode (Ingold) was used in combination with the pHmeter from a Biostat| MC (B. Braun, Melsungen, Germany), and the signals were recorded on a SE 120 recorder (BBC, Goerz Metrawatt). kLa Detelwzination. The kLa values were determined by the gassing-in method derived by Bartholomew et al. (1950). The liquid in the vessel was a 0.15 M solution of NaCI in tap water mimicking the growth medium with respect to content of electrolytes and the gas used was atmospheric air. A polarografic electrode (Ingold) was used in combination with the O2-measuring device in a Biostat| MC (B. Braun, Melsungen, Germany) and the O2-values were recorded on a SE 120 recorder (BBC, Goerz Metrawatt). The kLa values can thus be estimated from the slope of a semi-logarithmic plot of the electrode response (change of O2-concentration) versus time (Bartholomew et al., 1950; Kim and Chang, 1989). As this method is based on a fast-responding electrode the electrode-response time was tested. Bringing the electrode from a N2-gassed chamber to atmospheric air, the readings were as followed. The response time, i.e., the time needed for the electrode to record the change from the 0% to the
182 95% oxygen level, was timed to be in the range of 2 0 - 2 5 seconds (+ 5 s). As this is significantly faster than even the fastest change in DO-concentrations during the kLa-measurements, the electrode was considered useful for these experiments. Oaygen transfer rate (OTR). Based on the kLavalues obtained from the individual culture systems the OTRs were calculated according to: OTR = kL a (C*g - CL )
(2)
A C*-value of 0.238 mM/1 (Schumpe et al., 1978) and a CL-value on 40% of the C*-value was used. Furthermore, to be able to directly compare the different systems, the OTR-values were normalized with respect to volume. This way it was possible to evaluate the total OTR for the different systems.
Results and discussion Despite a considerable amount of newly developed/designed culture systems for mammalian cell cultivation, the tank type bioreactors are still the systems o f choice in industry (Phillips et al., 1985; T y o and Spier, 1987). Research in this area can therefore still be justified and especially the shear problems need to be addressed. A general attitude has been that the cell shear sensitivity could only be surmounted by changes in bioreactor design or culture mode. W e believe, though, that cells can be adapted to increased shear stress environments and thus cultured in reactors of very simple designs.
capabilities. This primarily because the spin-bar is a radial type impeller that merely creates vortices in the liquid with only a small amount o f axial mixing. As soon as (bubble-) aeration (the Blue-cap bioreactor) or impeller type agitators (the Biostat| MC) are used, significant effects on the mixing is seen as an axial component is added to the flow patterns in the vessels. A b o v e 150 rpm almost identical mixing times were found with the systems, but considering the hydrodynamic shear in the tanks stirrer speeds of 100 rpm (50 rpm for the Biostat| MC) were used. In this range, reasonable and comparable mixing times were found without detrimental effects to the cells. M a s s transfer rates
Mass transfer rates (kLa) were determined for the systems. In the aerated vessels (Blue-cap reactor and Biostat| MC) a flow rate of 500 ml/min was used. As expected the surface aerated bioreactors displayed very low kca-values due to the diffusive nature of mass transfer connected to surface aeration (Table 1). Also predictable is the difference in mass transfer rates between the micro-sparged Blue-cap reactor and the Biostat| M C system caused by the variation in mass transfer areas.
! .2 0
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A good starting parameter in a comparison of bioreactors is the mixing times, since homogeneity is rather important in mammalian cell cultures. From Fig. 1 it can be seen that a large scattering of mixing times exist at low stirrer speeds. As could be expected the spin-bar agitated vessels display some very poor mixing
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reactor
,>
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MC
Fig. 1. Mixing times in the bioreactors vs. stirrer speed.
183 Table 1. Comparison of the different cell culture systems
Spinner flask Plastic-jar bioreactor Blue-cap bioreactor Biostat| MC bioreactor
Working volume
Maximum obtained cell density
Total IgGproduction
Mass transfer rate (kLa)
Oxygen transfer rate (OTR)
Maximum specific production rate
(ml)
(x 105/ml)
(mg/l)
(h-1)
(mM/I/h)
(xl0-10 mg/cell/h)
15
13.2
0.42
0.060
2.49
none
B, SC
30.0
0.52
0.075
5.08
none
B, SC
800 200
7.3
Control facilities (basis)
Operation modes:
B: batch; SC: semicontinuous; C: continuous; P: perfusion
1000
17
10.0
14.66
1.540
2.16
none
B, SC, C
2000
19
13.7
3.00
0.419
1.61
many
B, SC, C, pa
~ell retention device needed (extra).
T h e d i f f e r e n c e s in k L a - v a l u e s are o f c o u r s e r e f l e c t e d in the o x y g e n t r a n s f e r rates ( O T R ) o f the s y s t e m s . O n this basis it is i n t e r e s t i n g to see that, d e s p i t e large v a r i a t i o n s in the abilities to t r a n s f e r o x y g e n to the cells ( T a b l e 1), a l m o s t i d e n t i c a l m a x i m u m cell n u m b e r s are a c h i e v e d in the m a j o r ity o f the vessels. T h i s m e a n s that o x y g e n is n o t a
l i m i t i n g factor in (our) s m a l l - s c a l e b a t c h s y s t e m s , an o b s e r v a t i o n that has also b e e n m e n t i o n e d b y other g r o u p s w o r k i n g w i t h s m a l l - s c a l e s y s t e m s . M o r e likely are the l i m i t a t i o n s d u e to l o w n u t r i e n t or h i g h waste c o n c e n t r a t i o n s . R e c e n t l y Jo e t al. (1990) r e p o r t e d o n the use o f fortified g r o w t h m e d i a as the m e a n s to e x c e e d the - for b a t c h
Table 2. Flow and shear characteristics
Reynolds numbera
Power numberb
(Dimensionless) Spinner flask Plastic-jar bioreactor Blue-cap bioreactor
2672 2672 2672
0.143 0.373 0.113
Biostat| MC bioreactor
3000
0.120
Impeller power inputc
Terminal eddy sized
Eddy velocitye
(x 10-2 W/l)
(x 10-6 m)
(x 10-3 m/s)
6.764 17.693 5.360 (31.426)g 2.697
330 184 369 (75) 439
3.03 5.45 2.71 (13.31) 2.28
Maximum turbulent stressf (x 10-2 N/m2) 0.92 2.97 0.73 (17.70) 0.52
aD2Np/la, where D is impeller diameter, N is rev. per min., p is liquid density, and I.t is liquid viscosity (103 Pa.s); baccording to Nagata, 1975, p. 35 (equ. 1.53); Cderived from the power number equation Np = p/N3D5p; p = 103 kg/m3 normalized to volume; dbased on rI = (~3/E0-25, 9 being the kinematic viscosity of the medium and E the rate of turbulent energy dissipation (e =(NpN3D5/V) (Cherry and Papoutsakis, 1988); ebased on v = (~e) 0.25 (Cherry and Papoutsakis, 1988); fbased on x = p(v)2, v being the terminal eddy velocity (Kunas and Papoutsakis, 1989; Oh et al., 1989); gderived from the gas power number PG/V = pgv s (Chisti, 1989), PG being the power number, V the liquid volume, p the liquid density and Vs is the superficial gas velocity and an assumed 40% conversion to hydrodynamic stress (Katinger et al., 1979).
184 cultures - almost magic cell density limit of 2 • 10 6 cells/ml, and even though the concept has not been tried in this work our glucose/lactate measurements (data not shown) support the idea and it seems a reasonable way to go. The low cell number found with the Plastic-jar bioreactor might on the other hand be related to the high level of hydrodynamic shear stress developed in this vessel.
Power input To further characterize the culture systems the Reynolds numbers and power inputs were calculated (Table 2). Further the gas-power input was determined for the Blue-cap reactor (added in brackets). From the Reynolds number it is obvious that all the reactors are operated with turbulent liquid flow patterns, as the Reynolds numbers exceed 1000 (Oldshue, 1983). Due to differences in geometry and impeller configurations in the vessels it is not possible to assume identical power numbers for all the bioreactors. Therefore the power number was calculated in each case according to Nagata (1975): h
A
N" = v e + 8 (10 -, +
3.2Re~176
(,;',0) ' 2
(3) where ,)
A--14+
.)
(4)
,, +4/ 1
0 /2
and b = the width of the impeller blades; d = impeller diameter; D = vessel diameter; and 0 = angle of the impeller blades.
Hydrodynamic shear Several types of detrimental effects are introduced into the culture systems, but basically they are all derived from two sources: the effects from bubbles (in the sparger regions and in the burstingzone at the liquid-gas interface), and the influence of the agitator and other internals on the liquid flow in the vessels. Much work have shown that the most important damaging mechanism for animal cells are related to the bursting of bubbles at the liquid-gas interface (Handa-Corrigan et al., 1989; Oh et al., 1989; Passini and Goochee, 1989), but since only one of the reactors in this investigation is bubble-aerated, the bubble effects can not be used as a comparative parameter. Therefore we have chosen to use the hydrodynamic shear (and 'The K o l m o g o r o v Eddy T h e o r y ' ) as a standard of reference in this work. Certainly, this approach will underestimate the total amount o f detrimental effects in the 'Blue-cap system', but as shear data based on the gas power input are added to the hydrodynamic data in Table 2 (in brackets), it is possible to get an idea o f the size o f the bubble effect. These data support the general view on bubble-bursts as the major detrimental source in sparged bioreactors since the damaging effects from bursting bubbles are directly related to the gas power input. Based on the power input and following the ' K o l m o g o r o v Eddy T h e o r y ' terminal eddy sizes and eddy velocities were calculated according to Cherry and Papoutsakis (1988), and the maximum turbulent stress levels, z, were determined from: z = p(v) 2 (Kunas and Papoutsakis, 1989; Oh et al., 1989) (see Table 2). The differences in vessel size and geometry is reflected in the power input numbers, and thus in the terminal eddy sizes and velocities, and turbulent stress numbers. As would be expected the high gas-flow rate (0.5 vvm) used with the Bluecap system should give very small and fast m o v i n g terminal eddies, and thus a (quite) high turbulent stress level, compared to the non-sparged systems. However, as can be seen from Table 2, despite this high power input the terminal eddy size is still much larger than the hybridoma cell
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