Evelyn Trummer

Transcriptional profiling of phenotypically different Epo‐Fc expressing CHO clones by cross‐species microarray analysis

Chinese hamster ovary (CHO) cells exhibit large variabilities regarding growth, recombinant protein production and post‐translational processing during cell line development and clone selection. To accelerate the development of stable high quality cell factories, new efficient strategies for cell screening and clone selection are required. In our work, we combined phenotypic characterisation of recombinant CHO clones during early cell line development with transcription profile analysis using cross‐species microarrays. The objective was to identify genes or gene patterns that correlate with clone specific alterations in terms of productivity, sialylation capacity and stress resistance. In all high producer clones transcriptional profiling revealed a common enrichment of gene ontology categories related to protein metabolism, transcription, nucleus and nucleolus, whereas no common genes were differentially regulated in clones showing higher sialylation capacities. Furthermore, we identified predictive stress‐related marker genes that were up‐regulated in one clone without showing the corresponding phenotype at an early stage of development. Thus, we successfully applied gene expression profiling to allocate transcriptomal differences to specific phenotypes that changed during cell line development. These promising results will further increase our efforts to develop CHO specific microarrays that deliver information about the suitability of a clone candidate for industrial production.

Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH

Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa–Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was co-localised to the target gene.

Analysis of Genetic Parameters in Order to Get More Information on High Producing Recombinant CHO Cell Lines

Recombinant mammalian cells for the production of glycoproteins and antibodies are mainly established by gene amplification techniques, using selection and screening methods based on specific productivity and growth rate. In the present study we will show the selection and screening process of a recombinant CHO cell line expressing a Fc fusion protein by analysis of gene copy numbers, mRNA-levels, specific productivity and integration locus.

CHARACTERISATION OF CHO SUBCLONES SHOWING PROFOUND CHANGES IN PERFORMANCE WHEN PROPAGATED IN DIFFERENT CULTIVATION SYSTEMS

Experiments were carried out with five serum-free adapted subclones of a recombinant CHO DHFR - line producing a monoclonal antibody. The bioreactor cultivation was performed in the multiple fermenter system Sixfors (Infors HT) in repeated batch mode. In the course of inoculum preparation cells were cultivated in T-flasks and spinner flasks (WV 0.125l and WV 0.5l). The General Cell Screening System (GCSS) was used as a tool for comparing growth characteristics of subclones derived from different cultivation systems in the course of inoculum preparation and bioreactor cultivation. It enables non-invasive determination of growth kinetics in 96 well plates [Steindl, 1990]. In addition, part of the inocula was used to perform batch cultivations in spinner flasks. The obtained results were compared with those found in the bioreactor. Cell concentrations were determined measuring the cell nuclei with a Coulter Counter after cell desintegration. Viability