Journal of Biotechnology, 16 (1990) 67-86

67

Elsevier BIOTEC 00536

Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures M o h a m e d A1-Rubeai and A.N. Emery Centre for Biochemical Engineerin& School of Chemical Engineerin~ University of Birmingham, Birmingham, U.K.

(Received 5 December1989; accepted14 February 1990)

Summary The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures. Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G 1 / S phases. Secretion takes place through exocytotic release of vesicle contents. Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h. Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G 1 / S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress. The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody. DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases. Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase. The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control. Hybridoma; Synchronous cell culture; Monoclonal antibody

Correspondence to: M. A1-Rubeai,Centre for BiochemicalEngineering, Schoolof Chemical Engineering, University of Birmingham, P.O. Box 363, Birmingham, U.K.

0168-1656/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

68 Introduction

Although progress has been made in improving the growth and antibody production of hybridoma cells by manipulation of the physico-chemical environment, the precise mechanisms of cellular control of antibody synthesis and secretion and the relationship between growth and antibody production remain largely unknown. There are still many disadvantages to the use of in vitro cultures of hybridoma cells e.g., low cell density, expensive and complex culture media, and low rates of product formation. Some of these problems reflect physiological differences between the cells, but it seems likely that the lack of consistent understanding at the cellular level of the regulation of antibody synthesis and secretion is partly responsible. The nature of intracellular immunoglobulin (Ig) distribution in the population and whether Ig synthesis and secretion reflect cell cycle-dependent gene expression are essential elements, knowledge of which may allow a significantly higher production of antibody. The progress through the cell cycle may influence the ability of a cell to synthesize individual proteins, such as Ig, that are not directly involved in cycle events. In an extreme case the synthesis of each protein might be restricted within the cell cycle and many observations of protein accumulation in synchronous cultures of bacteria, yeasts, algae and mammalian cells can be taken to indicate that this is the case. We need to find out whether Ig, which is not involved in cycle events, does accumulate periodically as cells progress through the cell cycle. If it does, then by control of cell cycle events, the level of Ig accumulation could be increased. However, it has been questioned whether maintaining of cells in a non-growing yet product-generating state is actually due to cessation of cell division (Reuveny et al., 1986a, b) and if it is possible at all to arrest and maintain continuously dividing cells (like hybridomas) in interphase for extended periods of time. This question is critical to the major challenge in medium formulation, i.e., formulation of media which discourage growth and promote the specific kind of differentiation which yields most product (Spier, 1988). We perceive three problem areas that need to be brought to the fore: (1) What is the relationship between growth and antibody production? (2) Is the product secreted actively or released passively and how is the noted increase in antibody production during the decline phase explained? (3) What are the effects of nutrient depletion and accumulation of toxic metabolites on the rate of DNA and protein synthesis during the various growth phases of batch culture? Many authors have attempted to provide speculative answers to some of these problems (Reuveny et al., 1986a, b; Velez et al., 1986; Birch et al., 1987; Emery et al., 1987; Merten et al., 1987; Merten, 1988); however the results have been highly variable and the experiments not generally designed to study antibody synthesis and secretion as distinct events. Although it appears that the production of antibody occurs as long as the cells are in a viable state and the precursors needed for the synthesis are available, the synthesis and secretion are not necessarily continuous nor is the amount of antibody present in medium totally due to active secretion from viable cells (A1-Rubeai and

69 Emery, 1989; A1-Rubeai et al., 1989, 1990). In this paper we report our findings on (1) the kinetics of synthesis and secretion of antibody by viable cells; (2) release of antibody by necrotic cells; (3) cell-specific production of antibody by dividing and non-dividing cells; (4) environmental factors which limit the rates of DNA and protein synthesis.

Materials and Methods TB/C3 murine hybridomas producing antibodies against human IgG were maintained in RPMI 1640 medium (Gibco, Paisley) supplemented with 5% newborn calf serum (NBSC) and used in all experiments. Cells were either grown in 2-1itre capacity stirred tank reactors (LSL) or in magnetically stirred 500-ml flasks. The bioreactor agitation speed was kept at 200 rpm. The culture medium was headspace gassed with 5% CO 2 in air mixture and temperature was controlled at 37°C. Antibodies were determined by the sandwich-type enzyme-linked immunosorbent assay (ELISA) (Desai, 1987) using human IgG-coated plates and sheep anti-mouse IgG peroxidase conjugate as the second antibody. To obtain the intracellular antibody the cell mass was pelleted, washed and resuspended back to its original culture volume with PBS. One-ml aliquots were dispensed into Eppendorf tubes and freeze-thawed twice, then centrifuged at 10 000 rpm for 15 min. The MTT assay was carried out as described by A1-Rubeai and Spier (1989). 0.1 ml culture samples (5 × 104 cells) were incubated with 0.01 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in 8 wells of a microtitre plate at 37°C for 3 h. Acid-isopropanol (0.1 ml of 0.04 M HC1 in isopropanol) was added to all wells and mixed thoroughly to dissolve the insoluble blue formazan crystals. Then plates were read at O.D. 540 nm. DNA synthesis was measured by pulsing 5 ml of 5 × 105 cells per ml with 1 #Ci ml -a of tritiated thymidine (Amersham) so as to give a final concentration of 40/~M thymidine. At this concentration thymidine incorporation is at equilibrium and the contribution of endogenous dTTP to DNA thymine is negligible (Adams, 1980). After 30 min the cells were collected on Whatman G F / C , washed with PBS, 10% trichloroacetic acid (TCA), 5% TCA and finally with 95% ethanol and dried for 30 rain. The amount of radioactive thymidine incorporated was determined by liquid scintillation counting. Protein synthesis in the intact cells was measured by the incorporation of [35S]methionine (Clemens et al., 1984). Incorporation of methionine into cell protein was determined by TCA precipitation of aliquots of cell suspension as described for DNA synthesis.

Labelling of synchronized cultures with [35S]methionine, cell extraction and immunoprecipitation On release from the thymidine block aliquots were removed periodically and labelled with 3 /~Ci/ml [35S]methionine (> 1000 Ci/mmol, Amersham) in methionine-free RPMI 1640 supplemented with 5% dialyzed foetal calf serum

70 (FCS). The cells were sedimented, washed 3 times and disrupted by resuspension in 1 ml of lysis buffer (10 mM Tris-HC1, pH 7.5, 1 mM MgC12, 0.5% Triton X-100) and kept at - 18°C until the end of the experiment. The mixtures were thawed and centrifuged in an Eppendorf microfuge at 10000 rpm for 15 min. The cell extracts were incubated with 200 /~1 of a 5% suspension of human IgG-agarose (Sigma, Poole, U.K.) for 18 h at 4°C on a roller shaker and centrifuged in an Eppendorf microfuge for 1 min. The pellets were washed 3 times with 1 ml of PBS and collected on filters. Quantitation was performed by counting the radioactivity in each filter in a liquid scintillation counter.

Cell synchronization Cells were synchronized by the double thymidine block as described by Volpe and Eremenko (1973). Cells from the exponential phase were adjusted to a concentration of 3 × 105 m1-1 and transferred into medium containing 2 mM thymidine. Thymidine was removed after 20 h and the cells transferred into fresh medium for 8 h; then thymidine was added to the culture for another 16 h. Thymidine was finally removed and the cells were allowed to grow in either 2-1 capacity stirred tank reactors (LSL) or stirred 500-ml flasks using 5% newborn serum-supplemented RPMI 1640 medium.

Fluorescence-activated cell sorting Cell samples were centrifuged, resuspended in cold 70% ethanol and fixed for at least 1 h. The fixed cells were washed in PBS and DNA was specifically stained with propidium iodide (PI) for 10 min (as described by Morasca and Erba, 1986). Samples were analysed in a Becton Dickinson Fluorescence Activated Cell Sorter (FACS 440) for DNA distribution.

Kinetics of secretion The TB/C3 cells plated in 25 cm2 flasks were labelled with [3~S]methionine (7.5 /~Ci m1-1 for 30 min and then chased for 22 h in complete RPMI 1640 supplemented with cold methionine 3 times in excess of the standard concentration in RPMI. Culture samples were taken at various intervals, centrifuged and both supernatant and cell lysates reacted separately with human IgG agarose, then washed and counted as described above.

Labelling of unsynchronized cultures Aliquots of cells were taken from stirred flasks at various times after inoculation, adjusted to a concentration of 1 x 10 6 m1-1 and labelled with [35S]methionine (3 /~Ci ml-1) for 3 h in either the culture medium itself or methionine-free RPMI 1640 supplemented with 5% dialysed FCS. The labelled protein and cellular and secreted IgG were immunoprecipitated and counted as described above. Results and Discussion

Cells were synchronized by the double thymidine block. Using DNA synthesis, viable cell counts and FACS analysis as criteria, population synchrony and cell

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and then doubled in a short time reaching a new plateau at time 10 h. DNA distributions at time 0 showed a single peak which corresponded to late G 1 / S phases. The proportion of cells in G2 increased rapidly to a maximum at 8 h. At 14 h most of the cells have completed their first division and after that cells gradually began to lose synchrony. The lengths of the cell cycle and its phases were measured by combining the information from all these cases. The length of the G1, S, G2 and mitosis was approximately 4.5, 4, 5 and 1.5 h, respectively. For most mammalian cells grown at 37°C the lengths of S, G2 and M phases range in intervals of 6-9 h for S, 2-5 h for G2 and 1-2 h for M. G1 is the most variable phase of the cell cycle and can last 30 h or more in some cells and be entirely missing in others (Volpe and Eremenko, 1973). The relationship of cell cycle phase to the synthesis of IgG was examined (Fig. 2). Experiments were initiated with synchronized cells at late G1/S phases which after resuspension in fresh medium progressed through the S phase of the cell cycle. The rate of IgG synthesis was maximal at the start decreasing rapidly to a minimum rate (approximately 30% of the maximum) during mitosis. Immediately after mitosis the rate of synthesis increased rapidly as the cells entered the G1 phase. The synthesis of IgG clearly preceded the onset of DNA synthesis which began by the release from the block. The kinetics of IgG secretion were followed by measuring secreted IgG within 3 h of pulse labelling cells removed at time intervals (Fig. 3). It must be mentioned that

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time (h) Fig. 3. Kineticsof IgG secretionin synchronizedcultures of TB/C3 hybridomacells. cells during the chase period were progressing through the cell cycle, therefore, the secretion rate was not absolutely related to the phase of cell cycle at which the pulsing occurred. Secretion rate during the cell cycle showed continuous decrease for 10 h followed by an increase at 14 h. Taking into account a delay of about 1-2 h, changes in the rate of secretion during cell cycle generally matched those occurring in the synthesis. The maximum rate (at G1) was double the minimum rate (at M). Both periodic and continuous synthesis of protein have been noted in a number of mammalian cell types (for review see Hochhauser et al., 1981; Denhardt et al., 1986). Our results are in agreement with others using a C3H mouse derived X5563 myeloma line (Byars and Kidson, 1970); a human lymphoid cell line WIL (Buell and Fahey, 1969; Lerner and Hodge, 1971); MPC-11 myeloma (Garatun-Tjeldsto et al., 1976) and Peyer Patch B cells (Beagley et al., 1988). However, other investigators have not observed such periodic production of Ig during the cell cycle (Cowan and Milstein, 1972; Liberti and Baglioni, 1973; Damiani et al., 1979). Such variation in the pattern of Ig synthesis and secretion is probably due to the different methods of synchronization being employed and to some extent to cell type and origin. Killander et al. (1977) used cytophotometric techniques to analyse the cytoplasmic levels of IgE of human myeloma cells in an asynchronously proliferating cell population and found in 4 out of 7 experiments an increase in IgE concentration during G1 followed by a decrease in S, sometimes with a recovery during G2. In the other 3 experiments no periodicity was found during the cell cycle. Flow cytometry was used to analyse the relation between the cell cycle and IgG distribution in asynchronously proliferating mouse hybridoma cells (Altshuler et al., 1986). They found no change in IgG distribution throughout the entire range of DNA contents. However, they have not considered the amount of IgG in relation to the cellular

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mass which is continuously changing from G1 to G2 (Killander et al., 1977; Altshuler et al., 1986). Moreover, their hybridoma cell population was not homogenous because of the existence of a substantial non-producing subpopulation. The cell cycle does not therefore commonly restrict the synthesis of immunoglobulin relative to other proteins but there is evidence from our and previous work that it does have effects on general rates of immunoglobulin accumulation. The results presented thus far indicate that merely by manipulating the amount of thymidine in the culture medium, cell growth, in effect, can be encouraged or discouraged. This opens up the possibility of maintaining the cells, especially when they are immobilized at high cell densities, in a non-dividing yet product generating state for a prolonged period. However, the following experiments were shown to indicate that this possibility is not easy to accomplish. The effect of various concentrations of excess thymidine on cell growth is shown in Fig. 4. The data reported here establishes again the optimum concentration of thymidine which inhibits the hybridoma cell growth in suspension. However, the viability could not be maintained at a high level for more than 50 h. It is clear that death occurs because cells are prevented from dividing rather than from starvation or accumulation of toxic metabolites. Attempts were made to maintain cells for prolonged periods in a non-dividing state under conditions where the medium is changed at intermittent periods (Fig. 5). The results are not encouraging as the cell viability continues to drop. After 4 d, cell numbers began to increase gradually at a low rate until day 10 when the cell concentration experienced a sudden exponential increase with a concomitant increase in viability. The explanation for such an increase in cell concentration following refeeding appears to involve a process of adaptation a n d / o r selection. In this regard, cells maintained for a long period in medium containing 20 mM lactic acid exhibited a similar decrease in cell number and viability followed by

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a 'steady state', however, no increase in cell numbers was observed after that (unpublished observation). Two plausible explanations for the inability of cells to survive in a non-dividing state can be offered. First, when cells are held in the blocked state for more than one cell doubling time they become subject to 'thymineless death' presumably as a result of their 'unbalanced growth' (Stubblefield, 1968). Secondly, hybridoma cells possess the ability to grow infinitely from the malignant myeloma cell. One vital feature of such malignant cells is their inability to survive for a long period if prevented from continuous division. The cells rapidly senesce, then die and disappear. One important feature of the continuous thymidine treatment is the higher specific antibody production rate (QA) obtained in the arrested cultures (Fig. 6). This result appears to be in agreement with the previous demonstration (Fig. 2) that IgG synthesis occurs primarily in G 1 / S phases of the cell cycle. The observation that protein synthetic rate continued at the control rate for at least 19 h after transfer to excess thymidine medium and at a time when the DNA synthesis rate was completely shut off (Fig. 7), suggests that although cells are stopped in their reproductive cycle by the DNA inhibitor, they continue to make protein, part of it secretory protein (i.e. monoclonal antibody). The low [3H]thymidine uptake in the presence of extra cold thymidine might be explained simply as a dilution effect. However, there is ample evidence in the literature indicating that at concentrations above 1 mM inhibition of DNA synthesis is almost complete (Adams, 1980). Higher QA was also obtained at lower dilution rate in continuous culture. Table 1 presents the effect of two dilution rates on viable cell number, viability and

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specific antibody production rate. Although the result clearly demonstrates that higher QA was not growth-associated, it also shows that higher numbers of dead cells are associated with higher QA. It is possible, therefore, that at low dilution rate the increase in death rate leads to the apparent increase in QA. Ray et al. (1989) observed such low viability at the reduced dilution rate but preferred to explain the increase in QA as due to the increase of G1 fraction. Their argument was based on an observation reported by Sen et al. (1988, Ref. from Ray et al., 1989) that in a batch hybridoma culture the G1 fractions in flow cytometry histograms increase significantly as the population-specific growth rate decreases. Interpretation of DNA distributions measured b y flow cytometry is difficult because no information is available about the absolute rate of DNA synthesis (Dolbeare et al., 1983).

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77 TABLE 1 Specific antibody production rate (QA) as a function of dilution rate and viability in continuous hybridoma culture

Dilution rate h - 1

Cell number × 10-5

Viability (%)

(S.D.) 0.012 0.026

2.37 (0.23) 5.12 (0.17)

45.8 92.8

Antibody conc. (gg ml-1)

QA (pg per cell per h)

(S.D.)

(S.D.)

20.5 (2.51) 16.08 (1.42)

1.05 (0.021) 0.82 (0.011)

Furthermore, DNA distribution is unreliable if viability is lower than 95% due to the fact that the peaks of dead cells or debris and G1 cells are either coincidental or so close that any attempt to separate them is futile. If a lower growth rate is a result of higher death rate then an increase in QA is more likely to be due to an increase of Ig release by the increasing fraction of necrotic cells than to decrease of the fraction of proliferating cells. This is exactly what we were able to demonstrate in cultures agitated at various rates while keeping the sparging rate constant (Table 2). Previously we have shown (Oh et al., 1989) that growth was unaffected by relatively high agitation rates. However, once the culture was continuously sparged with air the net cell growth rate and the maximum cell number fell markedly, that fall increasing with increasing agitator speed. Examination of the relation between the apparent growth rate with QA shows a negative correlation but as the reduction in growth rate in this case is actually due to the increasing cell death which is caused by the combined effects of sparging and agitation, then it is essential to reach once again the same conclusion, i.e., increasing death rate leads to increase in QA. Moreover if we plot the values of QA against growth rates of cells grown in batch cultures for a period of 100 h (Fig. 8) similarly a negative correlation is obtained with QA values in the death phase much higher than those in the growth phase. It is therefore important to examine cell viability whenever an examination of QA is required. Electron-microscopic (EM) studies of immunogold anti-mouse IgG labelling indicated that cells store the synthesized IgG in vesicles which are increased in number and enlarged in size with time (A1-Rubeai et al., 1989). To investigate

TABLE 2 Specific antibody production rate as a function of growth rate in hybridoma batch culture with various rates of agitation and sparging at 100 ml min - 1

Condition of growth

Apparent growth rate (h -1)

Antibody productivity (pg per cell per h)

400 rpm (no sparging) 100 rpm+ sparging 200 rpm + sparging 300 rpm + sparging

0.048 0.030 0.021 0.0

0.54 0.56 0.66 1.29

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further such phenomena the synthesis and secretion rates of IgG were followed during a period of 21 h by tracing labelled methionine into intracellular and extracellular antibody (Fig. 9). The rate of appearance of labelled IgG into the medium was non-linear, being maximal during the first 4 h with 71% of the synthesized IgG secreted within 8 h of the pulsing period and only a further 4% secreted by 22 h. The synthesized IgG remaining intracelhilarly decreased to an apparent minimum level after 8 h from pulsing and was maintained for a further 14 h. The negligible rate of labelled antibody appearance extracellularly while a steady-state concentration was maintained intracellularly over a long period after peaking is consistent with our previous EM observation (A1-Rubeai et al., 1989) and in agreement with the finding of Walker et al. (1987). These results together with the association between IgG secretion and cell cycle reflect a time-dependent regulation of IgG secretion. However, these findings are in contrast with the popular belief that secretion rates in non-regulated cells, such as hybridoma and plasma cells, are maintained at relatively constant levels (Bienkowski, 1983). The newly synthesized Ig in hybridoma cells appears to leave the Golgi apparatus in rather long-rived membrane vesicles that fuse continuously with the plasma membrane. This indicates that the secretory process differs from the typical non-regulated pathway which suggests certain complex control mechanisms may be involved.

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Batch growth and antibody production kinetics Batch cell growth is summarised in Fig. 10 where it can be seen that the culture cycle begins with a lag phase, proceeding through the exponential phase to a decline phase. The growth curve is typical for an inoculum obtained from the late exponential phase when the cells are at their maximum concentration (Baker, 1986, Ref. from Dodge et al., 1987). Tritiated thymidine incorporation and viable cell number profiles indicate a rapid flow of cells into the S-phase of the cell cycle during the lag period followed by a burst of cell division resulting in the increase of viable cell numbers from about 2 x 105 m1-1 at 24 h to about 9 x 105 m1-1 at 78 h. DNA synthesis continued but at a lower rate during the exponential phase and ceased at the end of it. Specific metabolic activity as measured by the MTT assay peaked up to 20 h after the peak in DNA synthesis. However, it similarly declined during the death phase. Antibody accumulation in the medium (Fig. 11) continued after the maximum in viable cell concentration had been reached. Intracellular antibody also increased during the lag phase followed by gradual decrease to about 80% of the maximum. However, as the cell concentration increased, the total intracellular antibody concentration rose to reach about 146% of the secreted antibody concentration at the end of the exponential phase. Nevertheless, during the death phase this value fell to about 86%, which suggests that a substantial increase in the extracelhilar antibody resulted from the release of antibody from dead or dying cells. The accumulated intracellular antibody at the completion of the growth phase was about 13.4 /xg m1-1, more than sufficient to account for the 6.6 /~g ml -~ increase in extracellular antibody observed during the death phase. This raises the question of where the rest of the accumulated intracellular antibody has gone? One apparent explanation for the disappearance of antibody is the cellular degradation

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which can occur extensively within the vesicles and as a result of fusion of these vesicles with lysosomes (Bienkowski, 1983). Secretion-coupled degradation has been demonstrated in many cell types, e.g., some myeloma IgM #-chains (Sidman et al., 1981) and fibrinogen (Grieninger et al., 1984) are destroyed in transit to the cell surface. The rate of proteolysis is probably enhanced during the decline phase of batch culture since it has been shown that the chemical environment such as hormones, nutrients and oxygen radicals can modify the proteolytic process (Dean,

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81 1980; Dean and Pollak, 1985). Amino acids are consumed at a high rate during growth which leads to the depletion of most of them at the decline phase (Reid et al., 1988; Sammon, 1989). Amino acids have also been found to inhibit protein degradation (Cleplinski et al., 1985; MacLennan et al., 1988). Such studies suggest that amino-acid depletion in culture media may have a role to play in the regulation of proteolysis. Increase in antibody concentration during the decline phase has been reported in many hybridoma cell lines (Velez et al., 1986; Birch et al., 1987; Merten, 1988). Nevertheless, a pattern of production kinetics was reported by Merten (1988) in which the product release was more or less growth-associated and production completely stopped when the cells entered the stationary and death phases. The antibody production characteristics of hybridoma cell lines during the stationary and decline phases vary greatly and range from continuous rise to actual decline (Boraston et al., 1984; Williams, 1984; Lavery et al., 1985; Merten et al., 1985; Reuveny et al., 1985, 1986a,b; Velez et al., 1986; Birch et al., 1987; Merten, 1988; Miller et al., 1988). These differences have been attributed to the influence of the culture system used, type of hybridoma cell line and the species of the fusion partners, and to nutrients, waste products and p O E (Merten, 1988). Another factor widely acknowledged but hardly mentioned in the literature is the inconsistency of cell count and ELISA tests both of which are highly predisposed to errors. Finally the rate of intracellular degradation of secretory product, although not enough data is available now to draw a final conclusion, is probably the most crucial factor which influences the production patterns. Figure 12 shows the results of [3H]thymidine and [35S]methionine incorporations during hybridoma batch culture. There are basically no differences in the incorporation patterns of the two labelled compounds. It may be seen that the cells reach their peak specific activity values for both types of incorporation at the same time. However, the specific activity value of [35S]methionine incorporation varies over about a 3-fold range while that of [3H]thymidine varies over about a 5-fold range. It must be noted that the zero-time values shown here were observed with a culture derived from a 2 d old inoculum culture; it shows DNA synthesis to be at a much lower end of the activity range than protein synthesis. These results appear to be in contrast to our earlier findings on the [35S]methionine incorporation patterns during batch culture in which we found that the synthetic and secretory activities of cells removed from batch culture at the lag, mid-exponential and death phases were surprisingly similar (unpublished observation). However, in this case the cells were incubated in fresh methionine-free medium during the labelling period. This may explain the difference as the cells in the fresh medium demonstrated that their potential synthetic machinery was unimpaired by the changing environment while in the case of using the same culture medium the cells were subjected in situ during the labelling period to the changes in pH, nutrients and waste metabolite concentrations. Recently Morenkov et al. (1989) reported a decrease in the protein synthesis rate in the stationary phase of mouse hybridoma cell growth and were able to relate such a decrease to 3 main factors - a 15.25% decrease in ribosome content per cell, a

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3

time (d) Fig. 12. DNA and protein synthesis rates during batch culture of TB/C3 hybridoma cells.

two-fold decrease in the ribosome protein involved in mRNA translation and a 5-15% decrease in the rate of mRNA translation. Temporal changes seen in this study and in many previous ones, in the growth cycle of hybridoma cells, i.e., the cyclic synthetic and metabolic variation, is a reflection of the dynamics of the culture and its response to an ever changing environment. Specific synthetic and metabolic activity is more flexible than the classical growth curve and perhaps may offer a degree of sensitivity in the specificity of reaction to stimuli not realised before in mammalian cell culture. The reduction in the synthetic and metabolic activities of cells after a few population doublings in batch culture is usually accompanied by loss of viability. It has been generally accepted that the cause of this decreasing viability is either accumulation of toxic products or the depletion of nutrients (Dodge et al., 1987; Glacken et al., 1988; Geaugey et al., 1989). It is therefore plausible to presume that the fall-off in incorporation of labelled thymidine and methionine, which may be taken as an indication of the loss of reproductive viability and protein metabolism, can lead to cellular death. Feeding of culture during the late growth phase with nutrients like amino acids improves D N A synthesis and cell growth (Sammon, 1989) and delays cell death (Geaugey et al., 1989), but removal of toxic products during the growth cycle is a task much more difficult to achieve.

83

Conclusions (1) The r e g u l a t i o n of synthesis a n d secretion of I g G in h y b r i d o m a cell c u l t u r e a p p e a r s to b e t i m e - d e p e n d e n t . (2) Q A is increased when cells are arrested a n d m a i n t a i n e d in late G 1 / S p h a s e s a n d when v i a b i l i t y is decreased. (3) Passive release of a n t i b o d y f r o m n e c r o t i c a n d d y i n g cells d u r i n g the d e a t h p h a s e of b a t c h culture leads to a s u b s t a n t i a l increase in a n t i b o d y c o n c e n t r a t i o n in the m e d i u m . (4) T h e rates of p r o t e i n a n d D N A synthesis d u r i n g b a t c h c u l t u r e r e a c h their p e a k values d u r i n g the early e x p o n e n t i a l p h a s e a n d their m i n i m u m values at the d e a t h phase. (5) Loss of viability occurs d u r i n g the time of m i n i m u m D N A a n d p r o t e i n synthesis, cessation of cell division a n d decline in energy m e t a b o l i s m .

Acknowledgements W e gratefully a c k n o w l e d g e the help of A. M i l n e r a n d R. Bird for the F A C S analyses a n d S. R o o k e s for technical assistance.

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