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. Further, oligosaccharide profiles were compared using HPEC-PAD analysis. No differences were detectable between SEAP profiles obtained from p27 growth-arrested and proliferating cultures. However, production at 30 degrees C led to a significant increase in the degree of sialylation, an effect that is generally considered beneficial for the in vivo efficacy of protein therapeutics. In the production context presented here, SEAP expression is controlled by the tetracycline- (tet) repressible gene regulation system. Here we show low temperature-induced upregulation of the tetracycline-dependent transactivator (tTA). This induction has been shown by Northern blot analysis to occur at the mRNA level and is independent of the promoters driving the transactivator. We also describe a novel bottleneck in productivity at low temperature found in p27 growth-arrested CHO cells cultivated at 30 degrees C.

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. Based on rapid degradation of the external repressible agents tetracycline (tet) and doxycycline (dox) in the cell culture medium, we developed a self-regulated process for transition from the growth phase to the production phase in a fashion that is dependent only on the starting cell population and the initial concentration of the tetracyclines. With this process, no change in medium is required to accomplish the transition from growth to production phase. The two-phase bioprocess achieved here by tet switch-controlled proliferation is reliable and allows a growth-arrested production phase of at least 7 days, during which cells remain in a well-defined, highly viable physiological state and show enhanced heterologous protein production. This Tet(SWITCH) process is readily adaptable to a variety of industrial processes designed for production of difficult-to-express protein pharmaceuticals.

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). Shift of cultivation temperature from 37 degrees C to 30 degrees C caused a growth arrest mainly in the G1 phase of the cell cycle concomitant with an up to 1.7-fold increase of specific productivity. A low temperature cultivation provided 3.4 times higher overall product yield compared to a standard cultivation at 37 degrees C. The cellular and molecular mechanisms underlying the effects of low temperature on growth and productivity of mammalian cells are poorly understood. Separation of total protein extracts by two-dimensional gel electrophoresis showed altered expression levels of CHO-K1 proteins after decrease in cultivation temperature to 30 degrees C. These changes in the proteome suggest that mammalian cells respond actively to low temperature by synthesizing specific cold-inducible proteins. In addition, we provide the first evidence that the cold response of mammalian cells includes changes in postranslational protein modifications. Two CHO proteins were found to be phosphorylated at tyrosine residues following downshift of cultivation temperature to 30 degrees C. Elucidating cellular events during cold exposure is necessary for further optimization of host-cell lines and expression systems and can provide new strategies for metabolic engineering.

Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27.

We constructed stable Chinese hamster ovary (CHO) cell lines which conditionally and coordinately express the model product gene secreted alkaline phosphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p53 mutant deficient in apoptotic but not cell-cycle arrest function. The use of dicistronic expression technology allowed the conditional expression of the model product gene and the cytostatic gene in a coordinated fashion from a single expression unit under the control of the tetracycline-responsive promoter PhCMV-1. Due to the presence of a cytostatic gene in the multicistronic expression unit, the growth behavior of the engineered CHO cell lines could be controlled by the addition or withdrawal of the exogenous agent tetracycline to or from the cell culture medium. Withdrawal of tetracycline resulted in sustained growth arrest of the stable cell lines for a prolonged period. The growth arrest of such cell lines was found to be accompanied by a 10-15-fold increase in their production of SEAP per cell. This controlled proliferation technology allows the design of a novel two-stage production process which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain growth-arrested and increase cell-specific production of a heterologous protein.

Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells.

The eukaryotic cell cycle is regulated by a complex network of many proteins. Effective reprogramming of this complex regulatory apparatus to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production, can require coordinated manipulation of multiple genes. Previous efforts to establish inducible cell-cycle arrest of Chinese hamster ovary (CHO) cells by regulated expression of the cyclin-dependent kinase inhibitor (CDI) p21 failed. By tetracycline-regulated coexpression of p21 and the differentiation factor CCAAT/enhancer-binding protein alpha (which both stabilizes and induces p21), we have achieved effective cell-cycle arrest. Production of a model heterologous protein (secreted alkaline phosphatase; SEAP) has been increased 10-15 times, on a per cell basis, relative to an isogenic control cell line. Because activation of apoptosis response is a possible complication in a proliferation-arrested culture, the survival gene bcl-xL was coexpressed with another CDI, p27, found to enable CHO cell-cycle arrest predominantly in G1 phase. CHO cells stably transfected with a tricistronic construct containing the genes for these proteins and for SEAP showed 30-fold higher SEAP expression than controls.

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. Three multiple cloning sites with a total of up to 18 unique restriction sites allow sequential cloning of the genes of interest. The modular structure of this pBluescript(R)-based high copy number vector system allows straightforward movement of individual cistrons among members of the pTRIDENT family, and facilitates their combination with existing expression vectors.

Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells.

Regulated expression of a cloned gene often provides much higher final expression of the gene product. Also, regulated expression of an activity can enable optional metabolic engineering and simplify functional genomic research. We constructed di-, tri-, and quattrocistronic mammalian expression vectors which allow the simultaneous, coordinated, and adjustable expression of up to two product genes. A single, tetracycline-regulatable promoter, PhCMV*-1, drives high-level expression of a multicistronic expression unit, containing the product gene(s), the gene for tetracycline-responsive transactivator (tTA), and, in the case of pQuattro-tTA, also the neomycin resistance gene. This autoregulatory genetic configuration retains a very low basal transcription activity in the presence of tetracycline, thereby reducing or eliminating possible toxic effects of tTA expression. However, upon withdrawal of tetracycline, a positive feedback regulation loop is activated which leads to higher levels of tTA expression and consequently also to higher expression levels of all other cistrons encoded on the multicistronic expression unit. Since such multicistronic expression vectors combine all genetic elements necessary for high-level expression as well as regulation in a single multicistronic expression unit, they alleviate limitations of previously reported tetracycline-regulatable vector systems and allow straightforward, one-step genetic engineering of eucaryotic cells to give an adjustable phenotype under strict control of the external stimulus, here tetracycline. Because the expression vectors described here were used for the expression for several heterologous product genes such as the green fluorescent protein and the tumor suppressor gene p21 in several cell lines (CHO-K1, BHK-21, and HeLa), we expect these multicistronic, positive feedback regulation vectors to function in a wide variety of eucaryotic cells and to be useful for basic as well as for applied research applications. Other vectors based upon the same autoregulation and multicistronic expression concepts can be constructed using other regulator gene-regulated promoter elements.

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)). In order to minimize complications due to possible clonal variation of selected, stable cell lines, our investigations are based on transiently transfected subpopulations, that have become a useful tool in industrial R&D. These subpopulations have been selected by flow cytometry for the expression of genes encoded on a dicistronic expression vector. These vectors contain a dicistronic expression unit consisting of the genes encoding the green fluorescent protein (GFP) or SEAP, followed by one of the cytostatic genes p21, p27 or p53175P encoded by the second cistron. p21, p27 as well as p53175P block the cell cycle of CHO cells in the G1-phase for a prolonged period. However, these G1-arrested cells remain viable and proliferation proficient upon repression of expression of the cytostatic gene. All three of the cytostatic genes studied provided similar regulation of proliferation, and also similar enhancements in SEAP production, suggesting that higher productivity may be a general and intrinsic feature of G1-phase arrested CHO cells. Overall productivity is most likely enhanced because growth-arrested cells do not need to devote cellular resources to biomass production.

Two new bioactive diterpenes from Lepista sordida.

In a screening for inducers of the differentiation of human leukaemic cells, two new active diterpenoids were isolated from fermentations of the basidiomycete Lepista sordida. The structural elucidation by spectroscopic methods and the biological activities of both metabolites are described.