Cross–Species Comparison of Expression Systems: Genome–Wide Analysis of Recombinant Protein Expression in CHO Cells

Background.

Recombinant protein production has been accomplished in various eukaryotic hosts ranging from microbes such as yeasts and filamentous fungi to higher eukaryotic expression systems including insect, plant and particularly mammalian cell lines. Each system provides specific benefits (e.g. high specific growth rate of yeasts and high secretory capacity of mammalian cells) but also exhibits specific drawbacks (e.g. inefficient protein secretion of yeasts and weak growth characteristics of mammalian cells).

In the past, a lot of valuable information on different molecular, biochemical and physiological properties has been obtained from different hosts. However, this information has been compiled in a variety of non–comparable ways, following different questions, applying various methods and incomparable experimental designs, thus providing very little information for generalisation. At present, a comparative European study of different microbial hosts (yeast, filamentous fungi and bacteria) is being performed evaluating the efficiency of secretion, folding and solubility at different critical environmental conditions (pH, temperature, osmolarity and oxidative stress) and analysing both the transcriptomic and proteomic level with the aim to identify main bottlenecks or major pacemakers of efficient protein production. In the course of this study, the transcriptional reaction of the yeast Pichia pastoris to protein production stress was analysed (Gasser B. et al., 2007).

Mammalian cell factories, in particular Chinese Hamster Ovary (CHO) cells, have been intensively investigated in the Austrian Center of Biopharmaceutical Technology (ACBT) over the last years. Transcriptomic analyses of mammalian cell lines revealed a clear correlation between transcriptional expression profiles and cell specific phenotypes in terms of recombinant protein expression, growth and even stress resistance (Trummer E. et al. 2008). Understanding the regulatory network of the cell will therefore break new ground for specifically affecting those cellular pathways that still represent major bottlenecks for high–level protein production, in particular product secretion and viable cell growth (Müller D. et al., 2008).

In order to enhance our efforts in eukaryotic host optimisation, a concerted project on analysing major eukaryotic production hosts(CHO cells and Pichia pastoris) has been initiated using state–of–the–art technologies and advanced imaging methods in combination with advanced data analysis. This will enable the identification of both common patterns of cellular reactions involved in the expression of complex proteins and specific features of a certain expression system. This novel approach will guide the mutual exploitation of beneficial knowledge about secretory components, cell growth and metabolic pathways to engineer expression systems with superior capabilities for expressing high–quality recombinant proteins.

Aims and methods.

This PhD project is part of the Austrian Centre of Biotechnology (ACIB) project "Cross-Species Comparison of Expression Systems". The general concept of the overall project is based on a comparative genome-wide analysis of different eukaryotic host species with different capacities for protein expression with a main focus on functional, structural and regulatory processes involved in expression of recombinant proteins. In order to identify organism–wide conserved mechanisms and pathways that influence growth or protein production, different yeast and CHO hosts will be analysed.

As a first step, CHO cell lines will be established that produce two different proteins (human serum albumin and a single–chain Fv–Fc fusion antibody) with high and low productivity, respectively. The selected proteins exhibit different properties (a monomeric and a larger homodimeric protein) in order to challenge the host system in different ways. Dihydrofolate reductase deficient (dhfr–) CHO cells will be used for transfection and the working clones will be selected at different states of post–transfectional gene amplification. These cells will then be cultivated under defined conditions in order to quantitatively analyse their pattern of transcription and protein abundance in response to different levels of heterologous protein production and secretion. Therefore, state–of–the–art transcriptomics and proteomics technologies will be applied. Microarray analysis will be conducted according to methods developed in our consortium for CHO cells (Ernst W. et al. 2006, Trummer E. et al. 2008). For differential proteomic expression profiling, two–dimensional difference gel electrophoresis (2D–DIGE) will be applied (Kumar N. et al. 2008, Meleady P. et al. 2008, Joon C. Y. et al. 2008).

The results obtained from transcriptome and proteome analyses of the recombinant CHO cell lines will be integrated in a common database together with comparable data derived from Pichia pastoris. This database will be the basis for the genome–wide cross –species comparison as well as for subsequent analysis and modelling. From these data we expect to identify cellular components that are crucial for high growth rates or efficient protein production and secretion. Furthermore, genes and pathways that are correlating to the specific strengths of the individual production systems will be identified (e.g. growth and cell density in yeast or secretion capacity of CHO cells). These components can then be functionally validated for their effect on cellular physiology using gene silencing or overexpression. Thus, novel engineering targets can be identified in order to increase productivity, process robustness, etc. of the individual production systems.

Ernst, W., Trummer, E., Mead, J., Bessant, C., Strelec, H., Katinger, H., Hesse, F. (2006). Evaluation of a genomics platform for cross–species transcriptome analysis of recombinant CHO cells. Biotechnology Journal, 1(6), 639-650.
Gasser, B., Sauer, M., Maurer, M., Stadlmayr, G., Mattanovich, D. (2007). Transcriptomics–based identification of novel factors enhancing heterologous protein secretion in yeasts. Applied and Environmental Microbiology, 73(20), 6499-6507.
Joon, C. Y., Gatti, M. D. L., Philp, R. J., Yap, M., Hu, W. S. (2008). Genomic and proteomic exploration of CHO and hybridoma cells under sodium butyrate treatment. Biotechnology and Bioengineering, 99(5), 1186-1204.
Kumar, N., Gammell, P., Meleady, P., Henry, M., Clynes, M. (2008). Differential protein expression following low temperature culture of suspension CHO–K1 cells. BMC Biotechnology, 8, art. no. 42
Meleady, P., Henry, M., Gammell, P., Doolan, P., Sinacore, M., Melville, M., et al. (2008). Proteomic profiling of CHO cells with enhanced rhBMP–2 productivity following co–expression of PACEsol. Proteomics, 8(13), 2611-2624.
Müller, D., Katinger, H., Grillari, J. (2008). MicroRNAs as targets for engineering of CHO cell factories. Trends in Biotechnology. 26(7), 359-365.
Trummer, E., Ernst, W., Hesse, F., Schriebl, K., Lattenmayer, C., Kunert, R., Vorauer-Uhl, K., Müller, D. (2008). Transcriptional profiling of phenotypically different epo–fc expressing CHO clones by cross–species microarray analysis. Biotechnology Journal, 3(7), 924-937.