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Contenido archivado el 2024-04-16

Improved techniques for establishing a high expression production system forrecombinant proteins from animal cells


Construction or selection of mammalian cell lines which allow by genetic engineering stable and high expression of recombinant proteins.
A system was established which allows the identification and isolation of highly active chromosomal loci in production cell lines. Expression levels of a reporter gene which is integrated as a single copy into the chromosome of a large number of cell clones indicate the transcriptional activity of the integration loci. The isolation of flanking deoxyribonucleic acid (DNA) sequences of these highly transcribed reporter genes involve the next step. To achieve this inverse polymerase chain reaction (PCR) was established. Single copy chromosomal DNA fragments which are appropriate for targeting have been identified. Two cell lines secreting recombinant antibody were established by conventional transfection procedures and used for physiological studies in bioreactors. A new sampling system for aggregate flocks has been developed. Natural aggregation is a generic phenomenon. Aggregate size is only a function of the cell line and hydrodynamics of the system. The correlation of cell growth and productivity depends on the cell line.

The major result of the first year of this project was that all bottleneck technologies could be established. These included the methods of identifying transcriptionally active chromosomal loci in which the genes of interest have to be targeted, the establishment of an aggregated cell bioreactor system and the bioreaction on a fixed bed bioreaction system.
To obtain cell lines allowing the stable expression of recombinant proteins highly active chromosomal loci in several cell types (CHO, BHK, myeloma) which promote the transcription of any integrated transgene will be defined. These sites will be targeted with the gene of interest in appropriate vectors by means of homologous recombination. Physiological and physical properties in high density flocculated and immobilized culture systems will be tested in order to select cell clones which favour the intended expression.


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