Journal of Biotechnology, 15 (1990) 129-146
Elsevier BIOTEC 00533
Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixed bed, porosphere culture system A.J. Racher, D. Looby and J.B. Griffiths Division of Biologics, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
(Received 12 January 1990; accepted 22 January 1990)
Summary The aim of this study was to investigate the potential of fixed beds of macroporous glass spheres as a production process for animal cell products. The growth, metabolism and monoclonal antibody expression of a mouse-mouse hybridoma cell line was investigated in order to both test the potential of and to optimise the system. After the initial growth phase, the culture went into a steady-state phase brought on by glutamine limitation. An event occurred after 120-160 h of steadystate operation which destabilised the culture, causing a decline in productivity, after which the culture recovered. This event was analysed in detail to determine its cause, and whether a major switch in metabolic function had occurred. The parameter which correlated most closely to antibody production rate was oxygen, but as this was kept constant in the void medium of the bed it has to be concluded that oxygen diffusion into the spheres was the regulatory factor. A comparison of the fixed bed and a flask culture identified interesting differences in glucose metabolism between the two systems. The data gave strong indications as to how the productivity of the fixed bed system can be further improved. This includes optimisation of the glutamine concentration and modifying the porous structure of the spheres to improve diffusion characteristics. Antibody production; Hybridoma; Immobilised culture; Physiology
Correspondence to: A.J. Racher, Div. of Biologics, PHLS Centre for Applied Microbiologyand Research,
Porton Down, Salisbury, Wiltshire SP4 0JG, U.K. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Immobilisation allows more efficient perfusion of animal cells and thus the potential for increasing unit cell density 20-50-fold from conventional batch densities. It also confers more stability to the culture system than free-suspension and provides the opportunity for longer-term culture periods. Most immobilised culture systems are very limited in volumetric scale-up potential and thus any system which is suitable for both high cell density and high volume application is of interest. Fixed beds of solid glass spheres have been widely used for growing anchorage dependent cells in both laboratory and industrial scale processes (Whiteside et al., 1979; Looby and Griffiths, 1987; Brown et al., 1988). However, glass sphere systems have certain limitations. They are not suitable for immobilising suspension cells due to excessive cell washout, and they are essentially low process intensity systems. These limitations have been overcome by substituting porous glass spheres (porospheres) for solid ones. Unit cell density increases significantly, by up to 20-fold for anchorage-dependent cells, and suspension cells can also be grown to similar densities without marked cell washout (Looby and Griffiths, 1988a,b) Siran porospheres are a very suitable matrix for immobilising suspension cells since no complicated immobilisation technique is required and the matrix is non-toxic to the cells. Due to the large pore size (60-300 #m) and open structure of interconnecting pores, cells are physically entrapped by filtration of medium containing cells. Up to 60% of the sphere is void volume. The cells growing in the internal structure of the spheres are protected from surface shear and can thus form dense aggregates. The combination of physical entrapment of cells and the loose adherence of cells in the sphere allows changes of medium without excessive loss of cells. The object of this study was to investigate the production of monoclonal antibody (mAb) and the physiology of hybridoma cells grown in a fixed bed, porosphere culture system. Cells grown in flask culture were also studied to improve our interpretation of the data from the fixed bed system. Flask culture has the advantage that cell densities can be readily monitored.
Materials and Methods
Cell line and medium The mouse-mouse hybridoma cell line CIE3, which secretes an IgG-class mAb against Toxoplasma gondii (Wright and Balfor, 1983), was obtained from Dr. S. Clark (PHLS CAMR). Cells were tested for mycoplasma infection and found to be negative by the Hoechst stain test. This cell line exhibits a pattern of both growth and non-growth associated mAb production (data not shown). The medium used was RPMI 1640 with 5% heat-inactivated FCS (56°C for 45 min) (Imperial Laboratories, Andover, U.K.). Glucose was included at 2 g 1-1.
Cell culture For both culture systems, cell seed was taken from mid-exponential phase cultures (4-5 x 105 cells per ml). In the flask culture (designated F2), 100 ml medium in a 175 cm 2 T-flask was inoculated to an initial density of about 105 viable cells per ml. Growth was allowed to continue at 37 o C, and samples were taken daily. At the points shown on the figures, the cells were removed from the conditioned medium by centrifugation and then resuspended in an equal volume of fresh medium, and growth allowed to continue. The carriers used in the fixed bed culture system were 5 mm diam., Siran porospheres (Schott Glaswerke, Mainz, F.R.G.). The design and operation of the fixed bed culture system have been previously described (Looby and Griffiths, 1988a,b). Briefly, the system consisted of a 1 1 (sphere bed volume) packed bed bioreactor for cell growth and a media reservoir vessel containing 10 1 medium. The cell seed was resuspended in sufficient medium to fill the void volume of the bed and introduced into the bottom of the bed of dried beads. Dry spheres absorb cells more fully into the internal structure of the spheres than do damp ones (Katinger, personal communication). The inoculation density was 2.0 x 106 viable cells per ml fixed bed volume. The bed was drained and refilled twice to promote even distribution of the inoculum. Medium perfusion was started immediately to give a linear flow rate of 2 cm min-1 increasing to 20 cm rain-1 with growth. In this study the system was operated in a repeated feed-and-harvest mode with replacement of medium in the reservoir (10 1) approximately every 24 h, after the first 72 h. The actual times of medium replacement are indicated in the figures. A list of the symbols used is given in Table 1.
TABLE 1 List of symbols Symbol
PATe Q Qo~y Q'~y. Y Y'
mmol d - 1 mmol 1-a d - l
Total ATP produced by the culture per day Volumetric uptake rate Total oxygen consumed by the culture per day Oxygen consumption rate pre-medium change Estimated volumetric m.Ab production rate Apparent yield of product from substrate (equals the ratio of product formation and substrate utifisation rates)
mrnol d - 1 mmol d- I
/~g m1-1 d-1 tool tool- 1
Subscripts ATP Gin lac
ATP Glucose Glutamine Lactate
ATP produced by oxidative phosphorylation
Assays Cell numbers were determined in an Improved Neubauer chamber and the viability by trypan blue exclusion. Ammonium was assayed enzymatically using glutamate dehydrogenase (Bergmeyer and Beutler, 1984). Glucose was determined using a Beckman Glucose Analyser 2 (Beckman Instruments Inc., CA, U.S.A.). Glutamine was determined using glutamine synthetase as described by Mecke (1984). Lactate was assayed enzymatically using a commercial kit (Sigma Chemical Co., cat. no. 826-A). Protein was determined by the dye-binding assay of Bradford (1976). Cells were recovered from the porospheres by washing with PBS. The cells were lysed with ice-cold PBS + 0.5% v / v Triton X-100 (30 min incubation on ice). The washed spheres were treated in the same way to lyse any remaining cells. The lysates were pooled and cell debris removed by centrifugation. Lactate dehydrogenase (EC 18.104.22.168: LDH) activity was assayed spectrophotometrically at 30°C (Vassault, 1983) in either cell-free medium or cell lysates. One unit (U) of LDH activity was defined as 1/~mol NADH consumed per minute. Oxygen was monitored using polarographic oxygen electrodes (Ingold Ltd.) placed in the media inlet and outlet. The pH was monitored using a pH electrode (Russell Ltd.). The concentration of mAb secreted into the culture supernatants was assessed using the ELISA assay described by Clark et al. (1988), except that C1E3 antibody was used instead of the OKT3 one.
Calculations The oxygen consumption rates were calculated according to the method described by Lydersen (1987). The total ATP produced per day by the culture was estimated from the total lactate produced and oxygen consumed per day by the method of Miller et al. (1988a). A value of 2.0 was used for the P / O ratio while the glycolytic correction factor fglycolysis had the value of 1.0 (Miller et al., 1988a). Sample means were compared by an ANOVA test. Linear and stepwise regression analyses were done using the Statgraphics statistical package (Statistical Graphics Corp.).
Definition of optimum medium change regime The fixed bed culture system was operated with either a variable percentage (50, 75 and 100%) (designated I1) or 100% (designated I2) change of the medium in the reservoir. When the bioreactor bed was drained at the end of each experiment (358 h for I1 and 382 h for I2), a greater proportion of cells recovered from culture I2 were viable (63%) compared to I1 (46%). The total cell density for I2 was also higher (2.82 X 107 and 3.92 × 107 cells per ml bed for I1 and I2, respectively). The cumulative mAb yield at 358 h for culture I2 was 183.4/xg m1-1 but only 113.4/~g
133 m1-1 for 11 (for the flask culture F2, the cumulative mAb yield was 72.3/tg ml-1). The difference between I1 and I2 is greater than can be accounted for by differences in cell numbers at the end of the experiment. These results clearly show that 100% replacement of the medium in the reservoir gives higher yields of both mAb and cells, with a greater proportion of viable cells, compared to replacement of a variable and lower proportion of the medium. Consequently, further studies in both the flask and fixed bed cultures used a regime of 100% replacement of the medium in the reservoir. Culture growth Culture F2. The graph of viable cell density for culture F2 (Fig. la) shows that the culture was in the exponential growth phase between 24-48 h, in the deceleration phase between 48-94 h, in the stationary phase between 94-290 h, and in the decline phase between 290-360 h. In the stationary growth phase, although the percent viability of the culture exhibited a downward trend, the viable cell density remained relatively constant (Fig. la). This indicates that replacement growth was occurring. This is supported by the observation of positive specific growth rates in the stationary growth phase (Fig. la). In culture F2, the volumetric glucose uptake rate (Fig. lb) increased during the deceleration growth phase to a relatively constant value. The increase in the volumetric glucose uptake rate parallelled the increase in viable cell density. When the viable cell density was relatively constant (between 94-290 h), the volumetric glucose uptake rate was also constant. The decline in the viable cell density at 290 h was accompanied by a decrease in the volumetric glucose uptake rate. The rate of release of LDH activity by culture F2 (Fig. lb) tended to zero in the exponential growth phase, but started to increase upon entry to the deceleration growth phase. In the stationary phase, the rate of release continued to rise, but at a lessening rate. In this period, the rate of release generally increased as the percent viability of the culture declined. The rate of LDH release increased markedly at the end of the stationary growth phase and entry to the decline phase, and was coincident with the decrease in volumetric glucose uptake rate. Culture 12. The glucose uptake profile of culture 12 (Fig. lc) shows that the uptake rate increased rapidly between 40-80 h, after which it slowed but continued to increase at a steady rate for the rest of the culture. The oxygen uptake profile of culture I2 (Fig. lc) shows a steady rise in uptake rate to a plateau at 210-280 h, before declining and then increasing again at the end of the culture. The profile of LDH release by culture 12 (Fig. lc) shows that the rate tended to zero during the first 80 h of the culture. Between 80-190 h, the rate of release of activity increased markedly, but the rate of increase gradually declined. Subsequently the rate decreased between 190-240 h, before increasing to a peak at 290 h and again decreasing.
=~'zi o g'~=
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o \'~, ",o "~,~,,
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mmol 1-1 d "1
U m1-1 d -1
10 5 m1-1