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Background CHO (Chinese Hamster Ovary) cells are the cell line of choice for therapeutic protein production. Although the achieved volumetric titers have increased significantly over the past two decades, the establishment of wellproducing CHO cell lines is still difficult and not always successful [1]. Factors influencing productivity are the chosen host cell line, the genetic vectors, applied media, the cultivation strategy as well as the product itself. Several CHO host strains are available for recombinant protein production, however, they are often quite diverse in terms of growth rate, maximal achieved cell concentrations and specific productivities. Specific productivity is also related to the locus of integration of the transgenes due to positional effects caused by the chromatin environment. Previously it was described that Bacterial Artificial Chromosomes (BACs) carrying the Rosa26 locus are advantageous for the recombinant protein production in CHO cells, enhancing the specific productivity compared to plasmid derived recombinant CHO cells [2-4]. In this project we aim to identify factors influencing volumetric productivity using different CHO hosts, Rosa 26 BACs as genetic constructs and suitable cell culture media. First, different commonly used CHO host cell lines were analyzed in various cell culture media to identify which host strain performs best. Secondly, we generated a recombinant cell line, producing the highly glycosylated HIV envelope protein gp140 as an example for a difficult to express model protein. Gp140 expression was compared to an already existing gp140 cell line generated by a plasmid vector as expression system. Methods Cell culture: CHO-DUKX-B11 (ATCC-CRL-9096) and CHO-DG44 (life technologies) were serum-free cultivated in spinner flasks. CHO-K1 (ATCC-CCL-61) and CHO-S (life technologies) were serum-free cultivated in in shaker flasks. BAC Recombineering: E.coli carrying the Rosa 26 BAC (~220 kbp) were transformed with a plasmid coding for a recombinase. Consecutively, a plasmid carrying the gp140 (CN54) gene flanked by homologous regions to the BAC was used for the transformation of the recombinase positive E.coli cells. BAC positive colonies were selected and the BAC DNA was purified (NucleoBond Xtra BAC, Macherey Nagel). Transfection and selection: CHO-S host cells were transfected with linearized, lipid complexed (Lipofectin) CN54 Rosa26 BAC DNA. Recombinant clone selection was performed in 96-well plates using 0.5 mg/mL G418. BAC transfected CHO cells are able to express the transgene as well as a Neomycin resistance gene within the Rosa26 locus.

Powerful expression in Chinese Hamster Ovary cells using bacterial artificial chromosomes: parameters influencing productivity