Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.

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In this study several analytical techniques were applied to obtain information about the stability of expression, the yield, and the integrity of a monoclonal antibody (Mab) produced by hybridoma cell line RIV6 in a homogeneous continuous perfusion culture system. The total antibody as well as the isotype-specific antibody contents decreased continuously during the course of cultivation, while the viable cell concentration remained constant. The origin of the discrepancy between the Mab contents observed by two enzyme-linked immuno sorbent assay (ELISA) systems during the steady state was due to fragmentation of the IgG molecule, either cytoplasmic or in the culture fluid, as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The IgG-secreting cells as well as the fraction of cells containing a high cytoplasmic IgG content decreased continuously during cultivation. The isoelectric focusing (IEF) pattern showed the appearance of two additional bands after five days of cultivation. This work indicates that cell line RIV6 is unstable in the culture system used, with respect to cell properties and product formation.

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension batch culture. The relative cell size, cytoplasmic and membrane IgG content and the viability were monitored. Besides, the specificity of the cytoplasmic and membrane IgG was ascertained by means of a synthetic peptide containing the antigenic epitope recognized by the antibody. Cell size was found to increase during the exponential growth phase. The viability as determined by FC follows a similar pattern with the viability data obtained by the conventional trypan blue exclusion test. The relative cytoplasmic and membrane IgG contents were high during the exponential growth and low during stationary phase. Measurement of cell cycle distribution and the antibody content in the culture fluid, indicated that the major part of the cytoplasmic IgG is secreted by cells in the G1-phase. It is concluded that flow cytometry is a useful tool to characterize hybridoma cell lines in a suspension batch culture.

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