Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the 'omic' technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.

A combined gene and protein expression profiling was performed to gain a deeper insight into the intracellular response of the antibody-producing GS-NS0 cell line in continuous perfusion culture. Growth rate, production rate, metabolic activity and viability declined with increasing cell density, dilution rate and time. Transcriptome and proteome analyses of cells at three different densities revealed 53 genes and 47 proteins as having significantly altered expression levels at HCD (high cell density). The results showed an increased up-regulation of genes/proteins involved in cellular energy production with increasing cell density. Furthermore, the intensified process triggered a cellular response to external stress stimuli, revealed by an overexpression of the genes/proteins implicated in cell-cycle arrest [e.g. Rb1 (retinoblastoma 1 gene) and Cdkn1b (cyclin-dependent kinase inhibitor 1B gene)] and in the induction of pro-apoptotic genes/proteins [e.g. Tnfrsf (tumour necrosis factor receptor superfamily gene), Nfkappa bia (gene coding for nuclear factor-kappaB inhibitor), HSP60 (heat-shock protein of molecular mass 60 kDa) and heterogeneous nuclear ribonucleoprotein K]. Interestingly, we observed a down-regulation of the transcription factor interferon regulatory factor 4 involved in the unfolded-protein-response process and protein disulfide-isomerase family members responsible for protein folding and assembly. Additionally, subunits of proteasome complex were highly expressed at HCD. Microarray, real-time quantitative reverse-transcription PCR and Western-blot analyses demonstrated a consistent trend of decrease in IgG heavy-chain level with increasing cell density. HSP60, which inhibits apoptosis by complexing with pro-apoptotic proteins such as Bax and Bak, was repressed at HCD. Overall, the data suggested that the balance among several factors involved in energy metabolism might be essential for fine-tuning the cell choice between survival and apoptosis, leaning towards the side of apoptosis at HCD. The results provide significant information for cell-engineering strategies and solutions to problems that prevail in HCD culture.

We investigated the transcriptional response of NS0 cells undergoing controlled nutrient growth change in continuous chemostat culture using mouse microarrays. A 50% reduction in growth rate resulted in detectable alterations in the expression of 29 genes in NS0 cells. Notably, expression of genes in three major biological processes, namely transcriptional, translational, and protein processing functions, were modified. To further elucidate the advantage of the chemostat environment for establishment of "omic" data sets, an expression profile of the over-expressed gene bcl-2 in NS0 cells was probed. Functional analysis revealed that the underlying altered molecular mechanism was particularly associated with G1 cell cycle progression, protein synthesis, and apoptosis. Importantly, these findings agreed with the physical function of the cells. Despite an increase in survival rate, bcl-2 over-expression resulted in a decrease of specific productivity, glucose consumption, oxygen uptake rate and intracellular protein content, indicating a lower energy generating metabolism. Further, a prolongation of G1 cell cycle phase was evident on lowering the growth rate. Overall, the application of microarray analysis to chemostat-grown cultures offers an excellent combination for the interpretation of transcriptomic profiles to elucidate the molecular mechanisms during nutrient growth change and bcl-2 over-expression.

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