Growth and production kinetics of human × mouse and mouse hybridoma cells at reduced temperature and serum content

Journal of Biotechnology, 25 (1992) 319-331

319

© 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

BIOTEC00770

Growth and production kinetics of human × mouse and mouse hybridoma cells at reduced temperature and serum content Nicole Borth, R e n a t e H e i d e r , Ali Assadian and H e r m a n n Katinger Institute of Applied Microbiology, Unic'ersityof Agriculture, Vienna, Austria (Received 6 December 1991; revision accepted 2 February 1992)

Summary The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing Gl-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed. Cell cycle; Flow cytometry; Hybridomas; Production kinetics; RNA content

Introduction The hybridoma technology established by K6hler and Milstein in 1975 has in the meantime developed to the industrial scale. As long as monoclonal antibodies were Correspondence to." N. Borth, Institute of Applied Microbiology, University of Agriculture, Nussdorfer L~inde 1l, 1190 Vienna, Austria.

320 made by scientists for their personal research requirements on a lab scale the main demand on a production process was simplicity. On an industrial scale, however, the cost effectiveness and stability of a process turn out to be the main issue. Several attempts have been made to reduce the costs of hybridoma cultivation and of the ensuing purification processes. One strategy was the development of serum-free media containing various serum substitutes (Griffiths, 1986), another strategy was to establish high cell density growth systems, where cells are maintained in a nongrowing state (Comer et al., 1990; Iijima et al., 1988; Lazar et al., 1988; Nakamura et al., 1989; Ray et al., 1990; Reiter et al., 1990; Schumpp and Schlaeger, 1989; Tokashiki and Arai, 1989). The fact that in dense cell systems the external growth factor requirements are reduced makes these systems ideally suited for serum free media, which brings the two strategies described together (Lee and Palsson, 1990). The observation that prompted the development of these high density growth systems was that many hybridomas show inversely growth associated production kinetics. It is assumed that when cells do not actively grow more energy is available for antibody production. This behaviour was observed for many cell lines in chemostat culture (Miller et al., 1988), but also during stationary growth in batch systems (Merten, 1988). However, there have also been reports, that for certain cell lines specific production rates of monoclonal antibodies are growth rate-dependent (Low et al., 1987; Ray et al., 1990), and others that the amount of antibody produced is proportional to the integral of viable cells (Renard et al., 1988) and independent of growth rate (Goebel et al., 1990). It is generally accepted now, that antibody production is dependent on cell metabolism and that the increase in antibody titer during the stationary phase of a batch culture is due to enhanced productivity and only partly due to release of intracellularly stored antibody from dying and lysed cells (Merten et al., 1990). The development of flow cytometric methods has given an important stimulus for cell biological research in animal cell culture. Flow cytometric investigations on hybridomas have centered on the kinetics of surface and intraceUular immunoglobulin formation and distribution (A1-Rubeai et al., 1989, 1990; Heath et al., 1990; Meilhoc et al., 1989; Rupp et al., 1989; Sen et al., 1990). Several methods have been described that determine differences between the quiescent, proliferative and differentiated state. Hybridomas, like most transformed cells, can not reach a true quiescent (GQ) state. Indeed there are reports that they need to achieve a certain minimal rate of division to remain viable (AI-Rubeai et al., 1989; Birch, 1984). Nevertheless several investigations have shown that cellular parameters such as RNA content (Dalili and Ollis, 1990) or specific production rates (Dalili et al., 1990; Merten et al., 1990) change during the course of a batch culture though not to the same extent as they change when quiescent lymphocytes are stimulated. However, reports on different cell lines gave differing, partly contradictory results, so that one can not generalize such results to hold for all hybridomas. Most papers that have examined physiological changes in hybridomas at different growth states have done so under conditions of substrate limitation, either in batch or chemostat culture (Low et al., 1987; Miller et al., 1988). Flow cytometric

321 work done on cells arrested by immobilization was restricted to evaluation of changes in the concentration of intracellular immunoglobulin, which is believed to be directly related to the antibody production rate of cells (Altshuler et al., 1986). In the present investigation we have tried to maintain ceils at different growth rates by other means than substrate limitation. We have compared two cell lines, a human X mouse heterohybridoma and a mouse hybridoma, with respect to their growth kinetics and cellular activity and tried to explain some of the phenomena observed.

Material and Methods

Cell lines and media The human x mouse heterohybridoma 3D6-LC4 (Grunov et al., 1988) as well as the mouse hybridoma cell line P24 were grown in RPMI 1640 medium containing 2% fetal calf serum (Seromed, Germany). Both cell lines produce mAb against HIV-1. Total and viable cell count was obtained with a hemocytometer and the Trypan blue dye exclusion method. The amount of antibody produced was determined by Elisa, glucose concentrations were measured by HPLC (Weigang et al., 1989).

Flow cytometry Flow cytometric analyses were performed on a P A R T E C PASI1 flow cytometer with mercury arc lamp excitation.

Cell cycle analysis. For cell cycle analysis cells were permeabilized in 0.1 M Tris-HC1 buffer containing 2 mM MgC12 and 0.1% Triton X-100. D N A was stained at a final concentration of 3 ~g m l - 1 DAP1 (Sigma) using a stock solution of 300 ~g m l - t . The stock solution was kept frozen at -20°C. R N A / D N A histograms. These were obtained using a modification of the method described by Darzynkiewicz et al. (1982). Briefly, cells were permeabilized in 0.15 M NaCI containing 0.02% Triton X-100 and stained with 13 I~M Acridine Orange (Molecular Probes, USA) in the presence of 1 mM EDTA. The R N A values presented were obtained by determining the channel number of the Gl-phase peak maximum. For each analysis a total of 20 000 ceils was measured. Variations in instrument setup and performance during the course of the experiments were corrected with fluorescent microbeads (Polysciences, USA). Growth rates. To determine growth rates in systems with low division rates or low viability, the Bromodeoxyuridine/HO33258 quenching method was used. Cells were incubated in the presence of 30 ~ M BrdU and 30 p~M Deoxycytidine. Viable

322

cells were separated by centrifugation on Ficoll (Seromed, FRG) and stained with 3 txg ml-1 Hoechst 33258 in Tris-HC1 buffer (see above).

Results To obtain cells at varying growth states two sets of culture conditions were chosen that reduce growth without limitation of substrates. Incubation of cells at temperatures below 37°C effectively reduces their division rate while maintaining very good viability. Figures 1 to 4 show the course of batch cultures at 30°C, 33°C and 37°C with respect to total and viable cell density, cell cycle distribution and RNA content. Already in this simple experiment very dissimilar behaviour of the two cell lines can be observed. While P24 accumulates in the Gl-phase of the cell cycle when grown at low temperatures (Fig. 4), little changes in the cell cycle distribution of 3D6-LC4 can be observed (Fig. 2). However, the RNA content of 3D6-LC4 is highest at high growth rates and decreases during the stationary phase of batch cultures. Figure 5 represents the R N A / D N A histograms measured of

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Fig. l. Batch cultures of the human × mouse hybridoma 3D6-LC4 at II, 30°C; + , 33°C; and *, 37°C. (A) Total - and viable . . . . . . cell density. (B) Antibody concentration in the culture supernatant. (C) Glucose consumption. (D) Lactate production.

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Fig. 2. Cell cycle distribution and RNA content of 3D6-LC4 during batch culture at (A) 30°C, (B) 33°C, and (C) 37°C. (D) RNA content. 3D6-LC4 cells cultured at 30°C and 37°C. As can be seen, all cell cycle phases are shifted towards lower R N A content with little difference between the relative reduction for all three phases. In contrast to these results, the R N A content of P24 remained unchanged throughout the experiment. As the R N A values were determined as the peak value of the Gl-fraction the slight increase during the exponential growth phase is presumably due to a higher percentage of cells in early S-phase. The position of the S-phase cells was constant for this cell line. Sureshkumar and Mutharasan (1991) reported that the amount of lactate produced per mol of glucose consumed changed at different temperatures. However, for the two cell lines used in this experiment, the ratio of lactate to glucose remained constant at 1.6 + 0.12 M lactate per M glucose for 3D6-LC4 cells and at 1.7 + 0.1 M lactate per M glucose for P24 at all growth rates (Figs. 1 and 3). The specific glucose consumption rates, however, varied according to growth rate (Fig. 6). A second set of experiments was performed using media with reduced serum content. The cell lines were routinely grown at serum concentrations of 2%. In serum-free, unsupplemented medium most cells will die within a week. At inter-

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Fig. 3. Batch cultures of the mouse hybridomaP24 at • 30°C, + 33°C and * 37°C. (A) Total ( ) and viable ( . . . . . . ) cell density. (B) Antibody concentration in the culture supernatant. (C) Glucose consumption. (D) Lactate production.

mediate serum concentrations, however, the ceils will reduce their growth rate and have an increased death rate. The two cell lines 3D6-LC4 and P24 were therefore washed in media containing 0.25%, 0.5%, 0.75%, 1% and 2% serum, respectively, and incubated for 4 days. Again, cell density, viability and R N A content of the cells were measured (Fig. 7). The Gl-phase fraction of LC4 cells again remained unchanged within a standard variation of 15%, while P24 reacted to reduced serum content by an increase in the Gl-fraction (data not shown). To further characterize the unusual behaviour of 3D6-LC4 two additional experiments were initiated. For a chemostat culture a 500 ml Spinner flask was adapted for continuous culture. Dilution rate was increased in four steps and maintained at each step for five complete changes of medium. The results obtained are presented in Fig. 8. It is well known that cells in suspension will grow to a characteristic cell density, above which growth rates will be strongly reduced. For the cell line 3D6-LC4 this characteristic cell density is in the range of 1-3 x 10 6 viable cells per ml. When cells are inoculated into dialysis tubing at densities above this value, the viable cell

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density will drop to 1-2 x 1 0 6 ml-~ within a week. Figure 9 presents the data for dialysis cultures inoculated at different densities and observed for 4 d. Figure 10 finally presents the specific antibody production rates during all these experiments plotted against growth rate. Again, very different behaviour of the two cell lines can be observed. While 3D6-LC4 shows a definite trend towards increased product'ivity at high growth rates, the specific production rates of P24 remain unchanged within a standard deviation of 30%, which presumably is due to the varying culture conditions. The RNA content of cells follows the same trend.

Discussion The production kinetics of different hybridomas have been described by several authors. Many cell lines exhibited increased specific productivity at low growth rates. These results were obtained in perfusion systems (Searnans and Hu, 1990) as well as in chemostat cultures (Miller et al., 1988). The typical course of batch cultures as described by Merten (1988) also supports the theory that non proliferat-

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ing cells have more energy available for antibody production than actively growing cells. This has also been attributed to the fact that most hybridomas have their highest specific productivity during the Gl-phase of the cell cycle (Al-Rubeai et al., 1989; Suzuki and Ollis, 1989). As non dividing cells accumulate in this phase the specific production rate of a culture will increase. The main aim when establishing an optimal system should therefore be to reduce growth rates once a sufficient cell density has been obtained. This has been done using immobilization and high density techniques. However, very low growth rates are hard to maintain for hybridomas as these cells, like most transformed cell lines, can not reach a true

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quiescent state. It has been noted, that hybridomas need to achieve a certain minimal rate of division to remain viable (Birch, 1984; A1-Rubeai et al., 1990). Nevertheless, even hybridomas will usually be arrested in G1, when not dividing. In spite of these observations several reports have described growth associated production kinetics of hybridomas. Comer et al. (1990) e.g. have cultivated several hybridomas in dialysis tubing and have found increased as well as reduced specific productivity at low growth rates. Recently Bibila and Flickinger (1991) have published a model on antibody synthesis that explains these different types of kinetics as a consequence of the relationship of growth rates to antibody assembly rates.

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Fig. 10. Specific production rates and RNA content of 3D6-LC4 and P24 cells during the experiments described in dependence of growth rates: (A) specific production rates of 3D6-LC4. (B) R N A content of 3D6-LC4. (C) Specific production rates of P24. (D) RNA content of P24.

329 Regarding the results of batch and chemostat cultures one has to bear in mind that in both systems the growth reducing factor is substrate limitation which may in itself have a definite effect on productivity. It is known that different specific production rates can be obtained by limiting either glucose or glutamine (Dalili et al., 1990). It has also been observed that in situations of stress such as low pH, hyperosmolarity or substrate limitation hybridomas will produce more antibody on a per cell basis than under normal conditions (Miller et al., 1988). Considering these reports the behaviour of the cell line 3D6-LC4 can be explained. One aspect is the lack of cell cycle regulation observed for this line. While P24 exhibits a good correlation of the fraction of S-phase cells to growth rate, the cell cycle distribution of 3D6-LC4 cells remained rather constant. G1phase fraction varied from 45% to 90% for P24 cells, while it remained between 50% and 70% for 3D6-LC4 cells. Several attempts to synchronize 3D6-LC4 ceils (by hydroxyurea or excess thymidine blocks as well as by isoleucine deprivation) have failed. As 3D6-LC4 cells do not accumulate in Gl-phase under adverse growth conditions, but can be arrested in any phase of the cell cycle, the time that cells remain in this high-productivity phase will only be prolonged when cells cycle often. This can explain the increased production rates of 3D6-LC4 cells at higher growth rates. The strongest control on cell cycle distribution is obviously exerted by substrate limitation, as the highest fraction of Gl-phase can be observed during the stationary phase of batch cultures (irrespective of temperature and growth rate, Fig. 2) and at low dilution rates in chemostat culture (data not shown). These data seem to indicate that substrate limitation has a more pronounced influence on cell physiology of 3D6-LC4 cells than apparently density inhibition or serum reduction. This is also supported by the observation that mitochondrial membrane potential is dependent on glucose concentration in the culture medium (Borth et al., in preparation). In contradiction to the results obtained by Sureshkumar and Mutharasan (1991) the molar yield of lactate per mole of glucose remained constant with the cell lines used in this study. The specific glucose consumption rate of hybridomas has been reported to be a function of glucose availability in the medium (Borth et al., 1990). During growth at 33°C and 37°C this behaviour was again observed. At the higher temperature the absolute value of glucose consumed is increased due to the higher metabolic activity, but the trend remains the same.

Conclusion

According to the reports found in the literature and also to the results of this study it can be generally assumed that hybridoma cell lines show very differing kinetic behaviour with respect to cell specific antibody production rates. In the case of the cell line 3D6-LC4 the kinetics of production can be explained by the cell cycle kinetics of this line. As the production kinetics are an important parameter required to select the optimal process strategy, flow cytometric analysis of cell cycle kinetics and RNA content under varying conditions of growth

330 l i m i t a t i o n h a v e p r o v e n t o b e a u s e f u l t o o l in t h e c h a r a c t e r i z a t i o n cell l i n e b e h a v i o u r .

and prediction of

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