Influence of amino acid sequence on growth and secretion of recombinant CHO cell lines

Background.

Chinese hamster ovary (CHO) cells are extensively used for the production of monoclonal antibodies and other biopharmaceutical proteins in large-scale quantities up to 20.000 liters in a single production vessel (De Jesus and Wurm, 2011). Volumetric antibody titers of 1-5 g/L (Wurm et al., 2004) were achieved by optimizing each single step during clone development and production phase. Systematic investigations of the bottleneck in antibody expression have led to seminal improvements in gene delivery methods, selection and cultivation strategies. As an example, it was shown that the gene transcription level depends on the integration site of the recombinant gene of interest within the host genome, a phenomenon known as the "position effect". Innovative attempts were made to provide the cell with a gene of interest in a transcriptionally active chromatin region by using bacterial artificial chromosomes (BAC) (Blaas et al., 2009) or targeted integration by recombinase mediated cassette exchange (RMCE) (Schlake and Bode, 1994).

Besides the integration locus of the gene of interest also each other step during antibody processing may be rate limiting involving mechanisms in mRNA degradation, translation efficiency, posttranslational processing and secretion. Recently it was shown that two similar single-chain antibodies that differ only in the variable region led to significantly different specific productivities independent of the used expression vector (Mader et al., 2012) indicating that the antibody product itself has important influence on the secretion rate. Minor differences in the amino acid sequence of the antibody may lead to altered protein processing in the endoplasmic reticulum.

Aims and methods.

Two stable cell lines expressing 3D6scFc or 2F5scFc as model proteins established by stable integration of plasmids or BAC vectors were previously described by Mader et al. (2012) demonstrating that the 3D6scFc expressing cell lines were better producers compared to the cell lines expressing 2F5scFc, irrespective of the used vector. Further work will therefore include characterization of the product influence on growth characteristics and secretion rates of the already established cell lines based on plasmids, BAC or RMCE systems.

The anti-idiotypic antibody Ab2/3H6 mimicking the epitope of the anti-HIV-1 antibody 2F5 is used as another model protein and was developed at the Department of Biotechnology (Kunert et al., 2002). Different humanized Ab2/3H6 antibody variants with different amino acid substitutions will be obtained from antibody humanization approaches assisted by molecular dynamics (MD) simulations based on a previously study published by de Ruiter et al. (2011) in cooperation with the group of Chris Oostenbrink. Using mammalian cell culture techniques the antibody variants will be expressed from transient and stable cell lines comparing different transfection methods, transfection parameters, host cell lines (e.g. CHO-K1 or HEK 293F) and different antibody formats, i.e. expressed as scFc or complete IgG antibodies. The secretion rate and growth behavior of each antibody producing cell line will be examined. The conformation and thermodynamic data of the antibody will be analyzed by e.g. ECD or DSC and the binding properties of the monoclonal antibodies will be assayed by ELISA, Western-blot or label-free bio-layer interferometry (BLI) after isolation and purification from the culture supernatant.

The overall aim of this project is to get a better understanding about the influence of the product itself on the expression level by identification of rate-limiting steps during protein expression and investigation of the underlying mechanisms.

Mader, A., Prewein, B., Zboray, K., Casanova, E., Kunert, R. (2012) Exploration of BAC versus plasmid expression vectors in recombinant CHO cells. Appl Microbiol Biotechnol 1-6 doi:10.1007/s00253-012-4498-x
De Jesus, M. and Wurm, F. M. Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors. (2011) European Journal of Pharmaceutics and Biopharmaceutics 78, 184-188
De Ruiter, A., Mader, A., Kunert, R. Oostenbrink, C. (2011) Molecular Simulations to Rationalize Humanized Ab2/3H6 Activity. Aust. J. Chem. 64, 900-909
Blaas, L., Musteanu, M., Eferl, R., Bauer, A., Casanova, E. (2009) Bacterial artificial chromosomes improve recombinant protein production in mammalian cells. BMC Biotechnol. 9, 3
Wurm, F. M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotech 22, 1393-1398
Kunert, R. E., Weik, R., Ferko, B., Stiegler, G., Katinger, H.(2002) Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5. AIDS 16, 667-668
Schlake, T. and Bode, J. (1994) Use of Mutated FLP Recognition Target (FRT) Sites for the Exchange of Expression Cassettes at Defined Chromosomal Loci. Biochemistry 33, 12746-12751