Insight into single cell cloning in serum-free media

Chinese hamster ovary (CHO) cells have been used as host cells for the manufacturing of therapeutic recombinant proteins over the past decade. It is thought that the development of high performance cell lines, which satisfy both productivity and regulatory expectations, is one of the key success drivers to establish good manufacturing processes. The cell line for the clinical and commercial productions should be derived from a single progenitor or clone, and so the single cell cloning is an essential step during the cell line development. Recently serum-free media have been widely applied for this step. But under such conditions, the cloning efficiency varies significantly among the clones. This might be because the serum-free conditions can be stressful for the CHO cells exposed to such an unexpected cloning process. In this study, we performed re-cloning from two pre-cloned cell lines to evaluate the impact of serum-free cloning on the resulting cell line characteristics; various parameters such as cell growth, productivity, fed-batch culture performance, product quality and cell stability were evaluated. As a result, most of the clones showed exactly the same performance before and after the cloning process, but some clones did not. The detail of these results will be presented and also the proper evaluation to be needed during cell line development, especially after the single cell isolation, will be discusse

Efficient production of recombinant proteins in suspension CHO cells culture using the Tol2 transposon system coupled with cycloheximide resistance selection

Abstract DNA recombination techniques in mammalian cells has been applied to the production of therapeutic proteins for several decades. To be used for commercial production, established cell lines should stably express target proteins with high productivity and acceptable quality for human use. In the conventional transfection method, the screening process is laborious and time-consuming since superior cell lines had to be selected from an enormous number of transfected cell pools and clonal cell lines with a wide variety of transgene insertion locations. In this study, we demonstrated that the combination of a Tol2 transposon system and cell selection by cycloheximide resistance is an efficient method to express therapeutic proteins, such as human antibody in suspension culture of Chinese hamster ovary cells. The resulting stable cell lines showed constant productivity and cell growth over a long enough cultivation periods for recombinant protein production. We anticipate that this approach will prove widely applicable to protein production in research and development of pharmaceutical products