Growth and death of animal cells in bioreactors

<br/>Animal-cell cultivation is becoming increasingly important especially for the area of hunian- health products. The products range from vaccines to therapeutic proteins and the cells themselves. The therapeutic application of proteins puts high demands upon their quality with respect to purity and structure. For example, a correct folding and glycosylation is of importance for the activity, the <em>in vivo</em> clearance rate and the possible immunogenicity of the protein, and can often only be obtained by production in animal cells. An important class of proteins produced by animal cells is formed by monoclonal antibodies. Monoclonal antibodies are produced by hybridoma cells and have the capacity to bind very specifically to a particular molecular structure (epitope), a quality that makes them suitable for application in <em>in vivo</em> and <em>in vitro</em> diagnostics, in separation technology and for the <em>in vivo</em> targeting of drugs.<p>The occurrence of substantial cell death and the presence of cell debris is a major problem in animal-cell cultivation. It interferes with the attainment of high volumetric productivities and with a proper functioning of the process. In addition, it may affect the quality of the product and cause problems in down-stream processing. Cell death may follow two different pathways, being apoptosis and necrosis, which have very distinct physiological and morphological features. Necrosis is a passive process generally caused by sudden high levels of environmental stress, whereas apoptosis is an active, genetically controlled process induced by mild stress conditions or specific signals from the environment.<p>After the introduction in Chapter 1, the application of a general framework for the construction of segregated models is discussed in Chapter 2 with respect to the behaviour of animal-cell populations. For the construction of segregated models, the physiological state of an animal cell must be specified, which is discussed in this chapter with special attention for the experimental verification of the models. Finally, a number of age-structured, segregated models, which are of importance for animal-cell cultivation are reviewed in this chapter.<p>The required amounts of animal-cell products are expected to be in the order of kilograms or even tonnes on a yearly basis. In order to produce these amounts, scale-up is necessary, which is most easily done in conventional reactor systems like the stirred-tank, bubble-column, and air-lift reactor. A main problem in the scale-up of these reactors is the supply of sufficient oxygen to the culture, which often requires sparging. Hydrodynamic forces associated with sparging cause cell death. In Chapters 3, 4, and 5 the specific death rate of hybridoma cells in bubble-column and air-lift reactors is studied with the hypothetical-killing-volume theory as a central theme. The hypothetical killing volume is a hypothetical volume associated with an air bubble during its lifetime in the reactor in which all cells are killed. The first-order death-rate constant in bubble- column and air-lift reactors can then be derived to be the product of this hypothetical killing volume and the number of bubbles introduced into the reactor per unit time and per unit reactor volume. The specific death rate of the hybridoma cells in the bubble-column and air-lift reactors is shown to be proportional to the gas flow rate and the reciprocal reactor height. Furthermore, in bubble columns the specific death rate is shown to be proportional to the square of the reciprocal reactor diameter. These results are in accordance with the hypothetical-killing-volume theory. The main cause of cell death is found to be bubble breakup at the surface, although detrimental effects at the sparger cannot be excluded. In Chapter 6 the specific death rate of Vero cells immobilized on microcarriers is shown to be proportional to the gas flow rate. Since the height of the reactor is not varied, it cannot be excluded that in this case also the rising of the bubbles or the associated liquid flow cause cell damage.<p>A common method to reduce the detrimental effects of air bubbles is the use of protective additives. In this thesis it is shown that the addition of two such protectants, Pluronic F68 (Chapter 3) and serum (Chapter 4), respectively, reduces the amount of cell death as a consequence of sparging. Furthermore, as demonstrated in Chapter 4, the protective effect of serum has a fast-acting, physical, and a slow-acting, physiological component. In Chapter 5 the effect of the specific growth rate on the specific death rate of cells due to sparging is studied in air-lift loop reactors. Cells with varying specific growth rates are obtained from steady-state continuous cultures run at different dilution rates. Remarkably, the specific death rate of the cells due to sparging decreased as their specific growth rate decreased. Furthermore, in Chapter 6 it is shown that the specific death rate of Vero cells is reduced by immobilisation of the cells inside porous carriers.<p>Below a critical dilution rate in continuous culture as well as towards the end of batch cultures, the specific death rate of hybridoma cells increases rapidly. In this case, the cells mainly die through apoptosis as a consequence of substrate depletion and the accumulation of toxic products. In Chapter 7 an age-structured model is developed to describe the rate of apoptosis as a function of the dilution rate in continuous culture. In this model a critical specific growth rate is introduced below which the cells start becoming apoptotic. In addition to the specific deathand growth rate, the average cell volume of the viable cells and the specific consumption and production rates for glucose, glutamine, lactate and ammonia are calculated. The model can reasonably well describe a set of literature data, with respect to the specific growth- and death rate and the concentrations of viable cells, dead cells, glucose, glutamine, lactate, and ammonia. In Chapter 8 the model is extended with equations concerning two hypotheses for the production of monoclonal antibody being:<br/> (1) Passive release of antibody from dead cells.<br/> (2) Increased productivity by the apoptotic cells.<br/>Both hypotheses can describe the increase in productivity at decreasing dilution rates as observed in Chapter 5. Furthermore, the distribution of cells over the different phases of the cell cycle is calculated and the equations for the average cell volume are rewritten in terms of forward scatter as measured by flow cytometry. Model predictions concerning these variables are compared to results obtained in Chapter 5. The G <sub><font size="-2">1</font></sub> - and G <sub><font size="-2">2</font></sub> /M-phase fractions are not predicted correctly, which may be caused by a cell-cycle-phase specificity of apoptosis. In addition, a good prediction of all cell-cycle fractions can be obtained if it is assumed that the duration of the G <sub><font size="-2">2</font></sub> /M phase is not constant, but increases as the specific growth rate decreases from the maximum to its critical value. Furthermore, the calculated fraction of apoptotic cells is shown to be proportional to the forward scatter.<p>Cell death associated with sparging may be minimised by:<br/> (1) Maximizing the amount of oxygen transferred per bubble introduced in the reactor.<br/> (2) The addition of a shear-protective agent like Pluronic F68 or serum.<br/> (3) The immobilisation of cells inside porous microcarriers.<br/>Point one is most easily done by increasing the reactor height and the oxygen tension in the air bubbles. With respect to the second point, it should be mentioned that the additive must be removed from the final product and can cause problems in down-stream processing. This may lead to an increase in the product cost. Finally, in the case of immobilisation of cells inside carriers, transport limitations may occur, which may in turn induce apoptosis. Cell death through apoptosis as caused by low levels of shear, substrate limitation and the presence of toxic products may be reduced through a careful process design and genetic manipulation of the cells