Cellular limitations of poor protein secretion in mammalian cells

Background.

It is often claimed that highly complex proteins are difficult to express resulting in low titres accompanied by sometimes poor protein quality. This is most often emphasized by a considerable amount of micro-heterogeneity in terms of posttranslational modifications, formation of aggregates or degradation products accumulating intracellularly or after secretion to the culture supernatant (Alves and Dobrowsky, 2017).

Accountable reasons are intrinsic factors described by the genotype of the expression cell line in combination with extrinsic factors like medium composition, process management and product resting time in the bioreactor. Both intrinsic and extrinsic factors finally contribute to the resulting cellular phenotype defining the quantity and quality of protein expression. The improvement of extrinsic factors was realized in the last two decades by chemically defined media compositions and feed solutions for fed batch strategies and cell retention systems for continuous fermentation processes. In terms of intrinsic factors different up- or down-regulation engineering approaches were applied to prolong the batch process by reducing apoptosis or diminish the stress-related drop of productivity (Kunert and Reinhart, 2016). However, not many engineered host cell lines have found their way into large-scale technological application. This fact is proved by the negligible number of newly registered mammalian production cell lines with new or genetically modified hosts in contrast to traditional cell lines.

Despite most of the research and development strategies conducted with monoclonal antibodies, it is rarely indicated that not all mAbs have a comparable expression potential. However, it is commonly accepted that minor mutations or singular alterations in the amino acid primary sequence might change the expressibility and developability (Jarasch et al., 2015) of a highly similar protein dramatically (Stoops et al., 2012). 

Aims and methods.

Even though it is commonly accepted that stress factors induce endoplasmic reticulum associated protein degradation or even apoptosis, scientists most often try to cure the symptoms by overexpression or down-regulation of distinct regulator proteins (Fischer et al., 2015).

In this thesis we will study the effect of the primary amino acid sequence of individual mAbs on the general performance of the recombinant cell line, which was observed in different projects (Schwaigerlehner et al., 2018; Chromikova et al., 2015; Reinhart et al., 2014).

Therefore we have set up a systematic framework by establishing recombinant CHO cell lines expressing a panel of eight defined mAbs (PhD thesis Schwaigerlehner). We engineered a CHO-K1 and a CHO-DUKXB11 host cell line by integrating a cassette for homologous recombination of two antibody chains including a promoter trap for assured isogenic expression only from this chromosomal locus (Mayrhofer et al., 2014). The mAbs’ cDNAs are integrated in this isogenic environment to exclude all intrinsic factors except the transgenic sequence of the mAb. The cell lines have already been developed and the results from the cell cultivation as well as stability of the mAbs are under manuscript preparation.

Next we are going to investigate the differences of cellular biology of the individual cell lines. Therefore, we will cultivate the cell lines under controlled process conditions applying identical seeding densities and identical media. Such experiments are crucial for all conclusions which are drawn afterwards. The quality as well as the purity of the proteins under study will be ensured by various biophysical and biochemical methods (e.g. SEC-MALS, DSC, CD). Moreover, the product will be analysed for charge variants (CEX-HPLC) and glycan profile (LC-ESI-MS). In terms of cell biology we will evaluate the intracellular distribution of the expressed antibody and various described stress factors immunochemically. Distinct samples will be chosen for proteomics analyses to give a detailed view of all changes which are directly influencing cellular behaviour and compare the results with previous experiments of scFv-Fc expressing clones (Sommeregger et al., 2016). Cellular physiology will be characterized by metabolic flux analysis and related to differences of cell lines. After finalizing the experiments in batch modus, we will also evaluate different cell lines in continuous process mode as we have recently shown that different cell lines might be preferably suitable for distinct process conditions (Reinhart et al., 2018).

Collaborations within this thesis include ALTMANN, OBINGER and OOSTENBRINK.

Alves, C.S. and Dobrowsky, T.M. (2017) Strategies and Considerations for Improving Expression of "Difficult to Express" Proteins in CHO Cells. Methods Mol. Biol. 1603, 1-23. doi: 10.1007/978-1-4939-6972-2_1
Chromikova, V., Mader, A., Hofbauer, S., Gobl, C., Madl, T., Gach, J.S., Bauernfried, S., Furtmuller, P.G., Forthal, D.N., Mach, L., Obinger, C., Kunert, R. (2015) Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM. Biochim. Biophys. Acta 1854, 1536-1544. doi: 10.1016/j.bbapap.2015.02.018
Fischer, S., Handrick, R., Otte, K. (2015) The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnol. Adv. 33, 1878-1896. doi: 10.1016/ j.biotechadv.2015.10.015
Jarasch, A., Koll, H., Regula, J.T., Bader, M., Papadimitriou, A., Kettenberger, H. (2015) Developability assessment during the selection of novel therapeutic antibodies. J. Pharmac. Sci. 104, 1885-1898. doi: org/10.1002/jps.24430
Kunert, R. and Reinhart, D. (2016) Advances in recombinant antibody manufacturing. Appl. Microbiol. Biotechn. 100, 3451-3461. doi: 10.1007/s00253-016-7388-9
Mayrhofer, P., Kratzer, B., Sommeregger, W., Steinfellner, W., Reinhart, D., Mader, A., Turan, S., Qiao, J., Bode, J., Kunert, R. (2014) Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells. Appl. Microbiol. Biotechnol. 98, 9723-9733. doi: 10.1007/s00253-014-6011-1
Reinhart, D., Sommeregger, W., Debreczeny, M., Gludovacz, E., Kunert, R. (2014) In search of expression bottlenecks in recombinant CHO cell lines-a case study. Appl. Microbiol. Biotechnol. 98, 5959-5965. doi: 10.1007/s00253-014-5584-z
Reinhart, D., Damjanovic, L., Kaisermayer, C., Sommeregger, W., Gili, A., Gasselhuber, B., Castan, A., Mayrhofer, P., Grünwald-Gruber, C., Kunert, R. (2018) Bioprocessing of recombinant CHO-K1, CHO-DG44 and CHO-S: CHO expression hosts favor either mAb production or biomass synthesis. Biotechnol. J., doi: 10.1002/biot.201700686
Schwaigerlehner, L., Pechlaner, M., Mayrhofer, P., Oostenbrink, C., Kunert R. (2018) Lessons learned from merging wet lab experiments with molecular simulation to improve mAb humanization. Protein Eng. Des. Sel., doi: 10.1093/protein/gzy009
Sommeregger, W., Mayrhofer, P., Steinfellner, W., Reinhart, D., Henry, M., Clynes, M., Meleady, P., Kunert, R. (2016) Proteomic differences in recombinant CHO cells producing two similar antibody fragments. Biotechnol. Bioeng. 113, 190-112. doi: 10.1002/bit.25957
Stoops, J., Byrd, S., Hasegawa, H. (2012) Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis. Biochim. Biophys. Acta 1823, 1643-1657. doi: 10.1016/ j.bbamcr.2012.06.015