Powerful expression of recombinant proteins

Background.

Mammalian cells and among these especially CHO (Chinese Hamster Ovary) cells (Puck et al, 1958) are nowadays widely used for recombinant protein production because of their ability of proper protein folding, assembly and post translational modifications (Wurm, 2004). Beside technological developments the generated recombinant cell lines are important for scientific purposes in terms of protein chemistry and serve as tools for cell biological investigations. Today, the achieved volumetric yields of recombinantly produced proteins are much higher than in the beginning of mammalian cell culture technology.

In terms of recombinant CHO cells the reported titers have increased more than 100-fold over the past two decades (Kim et al, 2012) but the establishment of well-producing cell lines remains difficult and is not always successful. The four main factors that are considered to be influential in productivity are the genetic construct, the host cell line, the cell culture medium as well as the applied fermentation strategy. Insufficient production yields are a major problem in biotechnological processes and can lead to unprofitable products. Therefore, improvement of mammalian protein production is a huge research field and higher yields are pursued by further investigations of expression vectors and host cell lines as well as media and fermentation strategy. Subsequently, the main parameters that influence the productivity have to be tuned accurately to define a powerful expression process. Based on these facts we want to optimize the whole process of cell line establishment and hopefully find a robust procedure to generate well-producing cell lines.

Aims and methods.

The efficient expression of recombinant proteins depends on the optimal combination of host cell line, genetic constructs and cultivation environment.

In this project we want to compare different CHO host cell lines and investigate the differences in their adaptation strategies. Moreover we will evaluate their robustness to metabolic by-products. The genetic construct will be a Bacterial Artificial Chromosome (BAC). The main advantageous factor of such a large BAC construct (~300 kbps) is the prevention of position and chromatin silencing effects. Beneficial expression with such BAC vectors was already proofed by different groups (Blaas et al, 2009 and 2012).

The model proteins will be IgG1 antibodies and the HIV-1 gp140 envelope protein to provide highly complex molecules with scientific data already available from the past.

Different media will be tested in batch and fed-batch experiments with the aim to establish a chemically defined medium suitable for the cultivation of high producing recombinant CHO cells.For fermentation strategies we will investigate fed-batch and perfusion methods. The challenge of this project will be the comprehensive design of experiments and statistically consolidated evaluation of data.

Puck, T. T., Cieciura, S. J. and Robinson, A. (1958) Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J Exp Med. 108, 945-956
Wurm, F. M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 22, 1393-1398
Kim, J. Y., Kim, Y. G. and Lee, G. M. (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol. 93, 917-930
Blaas, L., Musteanu, M., Eferl, R., Bauer, A. and Casanova, E. (2009) Bacterial artificial chromosomes improve recombinant protein production in mammalian cells. BMC Biotechnol. 9, 3
Blaas, L., Musteanu, M., Grabner, B., Eferl, R., Bauer, A. and Casanova, E. (2012) The use of bacterial artificial chromosomes for recombinant protein production in mammalian cell lines. Methods Mol Biol. 824, 581-593