Efficient Isolation of High Producing Mammalian Cells

The establishment of cell lines producing human antibodies for therapy of cancer or viral and bacterial infections is a very labour and cost-intensive process when using current methodology. The proposed project will establish and evaluate a technology that should reduce the expenditure required by a factor of at least 10 by using a methodology that allows the direct measurement of the specific production rate of single cells, so that they can be isolated using a flow cytometer and cell sorter. In addition to the specific production rate other cellular properties, which would be desirable for a production cell line will be incorporated into the screening process. Two such properties selected for this study are stability of gene expression and a change in the production kinetics to give high production rates during maintenance. The method will also be used as a very potent tool for the analysis of cellular physiology. As the specific production rate of single cells can be analysed, it will be possible to correlate this information to other cellular properties, such as cell cycle phase, intracellular product concentration or the concentration of helper proteins in the endoplasmic reticulum. Finally, the method will be modified to be usable for direct selection of hybridoma cells or lymphocytes. The modifications will also include the attempt to select for specifically binding hybridoma cells by replacing the second staining antibody by a fluorescence-labelled antigen. This should reduce the number of tests required significantly (f.i. for specificity), so that a desired cell line should be obtainable with far less cost and time.