A novel cytostatic process enhances the productivity of Chinese hamster ovary cells.

We have established a novel production process which allows up to fourfold higher production of a model secreted protein, the human secreted alkaline phosphatase (SEAP), in Chinese hamster ovary (CHO) cells. A cytostatic production phase is established in which cell proliferation is inhibited or completely abolished. Such a cytostatic production phase is established by overexpression of the tumor suppressor genes p21, p27, or p53175P (a p53 mutant showing specific loss of apoptotic function) under transcriptional control of a tetracycline-repressible promoter (P(hCMV*-1)).

Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity.

Overexpression of the cyclin-dependent kinase inhibitor p27 and exposure to low temperature (30 degrees C) represent two strategies to establish controlled proliferation processes for production of therapeutic proteins using Chinese hamster ovary (CHO) cells. Here we analyze the effect of growth inhibition on the quality of the human model glycoprotein SEAP (secreted alkaline phosphatase) for both strategies in monoclonal CHO-derived cell lines. Separation of purified SEAP samples using two-dimensional gel electrophoresis showed that production by proliferation-controlled CHO cultures did not alter the overall integrity of the product.

A novel autoregulated proliferation-controlled production process using recombinant CHO cells.

Controlled proliferation bioprocesses have shown great enhancement of heterologous protein production. This novel technology has been implemented here using a multicistronic expression unit encoding the product gene and a cytostatic cell-cycle-arresting gene (p27) under control of a single tetracycline-repressible (tet(off)) promoter. The strict genetic linkage of both genes allows the dissection of the production process into a nonproductive growth phase (dicistronic expression unit repressed) followed by a proliferation-inhibited production phase (dicistronic expression unit induced) when the cells have reached an optimal cell density.

Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells.

Proliferation of mammalian cells can be controlled by low cultivation temperature. However, depending on cell type and expression system, varying effects of a temperature shift on heterologous protein production have been reported. Here, we characterize growth behavior and productivity of the Chinese hamster ovary (CHO) cell line XM111-10 engineered to synthesize the model-product-secreted alkaline phosphatase (SEAP).

pTRIDENT, a novel vector family for tricistronic gene expression in mammalian cells.

We constructed tricistronic expression vectors for the simultaneous and coordinated expression of three independent genes in mammalian cells. One single promoter allows high level and, in some vectors, adjustable transcription of all three cistrons. Whereas the first cistron is translated in a cap-dependent manner, the subsequent ones utilize intercistronic regions of viral origin such as the internal ribosomal entry site of poliovirus or the cap-independent translation enhancer of encephalomyocarditis virus for enhanced translation.