Impact of bioprocessing on quality and yield of complex proteins

Background.

More and even most of newly developed biopharmaceuticals are produced in recombinant CHO cells, which are predetermined by the production clone in the master cell bank. Cellular variations of recombinant clones are often not tested in bioreactors since possible clones might be numerous and therefore lab-scale fermentation is too laborious. Development and characterisation of bioprocesses means to balance the possible quantitative output of a fermentation run to the product quality (Kelley et al., 2018). The result is defined firstly by the cellular biology, which is also called microscale environment and secondly by the process mode and –conditions, which define the macroscale environment (Radhakrishnan et al., 2018). In addition to process mode and conditions, the composition of production media and the chemical substances provided by media supplements significantly contribute to a successful process. Nonetheless, the exact chemically defined media formulations are still confidential and lack detailed description even in the respective datasheets. Despite all, critical substances necessary to propagate CHO cells and to promote protein expression are known and published but the development of cell culture media compositions is predominantly achieved via high throughput screening (HTS) and as such limited to big biotech companies.
Regarding macroscale strategies, we have evaluated down-scaling strategies to select process strategies in combination with media supplementation strategies (Reinhart et al., 2015). By applying experimental design, commonly known as design of experiment (DoE) (Mayrhofer and Kunert, 2019), we will be able to define process conditions, which balance the protein quality with the amount of secreted protein.

Aims and methods.

In the herein proposed PhD project, we will evaluate various unknown media compositions, individual media components and small molecules known as chemical chaperons (Kumar, 2009; Papp and Csermely, 2006) on their performance at different process conditions and operation modes. We will apply experimental design for the characterisation and assessment of numerous chemical chaperones. The definition of best performing parameters regarding product quality is based on biophysical properties of the protein. Biophysical properties might vary to a distinct degree and result in the micro-heterogeneity of the harvested protein. The acceptability of micro-heterogeneity differs from protein to protein and may result in adverse side reactions during human application and optimal functionality of distinct or accumulated isoforms. The prior mentioned strategy of media optimization will be applied to different process operation modes, including batch, fed-batch and continuous cultivation to investigate the consistency of cellular demands for growth and productivity of an individual production cell line. To characterize the behaviour of different host cell lines and biopharmaceutical proteins, we will include recombinant cell lines based on CHO-K1, CHO-DG44 and CHO-S host lineage (Reinhart et al., 2018a) expressing different highly complex proteins, including antibodies of IgA-, IgG- and IgM subclasses (Hennicke et al., 2019) or CD19/21-fusion variants. Finally, the influence of process operating conditions will be checked in the bioreactor with the possibility to control temperature, dissolved oxygen as well as pH.
Methods for protein characterization are already well established for IgG and partly for IgM but need to be defined in more detail for IgA and the CD19/CD21 chimeric protein. After identification and definition of optimal medium and process conditions for an individual clone harbouring the product of interest, we will characterize the cellular biology leading to optimized protein production (Reinhart et al., 2018b). This will be done in defined core facilities. Sampling will be planned accordingly and already collected during the evaluation run in lab scale fermentation under controlled conditions.

Collaborations.

The thesis will be supervised by Renate Kunert,
Véronique Chotteau, KTH Royal Institute of Technology (Stockholm, Sweden), will be the external supervisor – to be confirmed
Protein chemical and structural analysis will be done in collaboration with the group of Christian Obinger (VIBT, Boku)
Glycosylation analysis will be done in collaboration with the group of Prof. Friedrich Altmann (VIBT, Boku)
Modelling of continuous processes will be done in collaboration with the group of Prof. Alois Jungbauer (VIBT, Boku)

Hennicke, J., Reinhart, D., Altmann, F., and Kunert, R. (2019). Impact of temperature and pH on recombinant human IgM quality attributes and productivity. N Biotechnol 50, 20–26.
Kelley, B., Kiss, R., and Laird, M. (2018). A Different Perspective: How Much Innovation Is Really Needed for Monoclonal Antibody Production Using Mammalian Cell Technology? Adv. Biochem. Eng. Biotechnol. 165, 443–462.
Kumar, R. (2009). Role of naturally occurring osmolytes in protein folding and stability. Archives of Biochemistry and Biophysics 491, 1–6.
Mayrhofer, P., and Kunert, R. (2019). Screening of media supplements for high-performance perfusion cultures by Design of Experiment. 4th edition of Animal Cell Biotechnology - accepted manuscript
Papp, E., and Csermely, P. (2006). Chemical Chaperones: Mechanisms of Action and Potential Use. In Molecular Chaperones in Health and Disease, K. Starke, and M. Gaestel, eds. (Berlin, Heidelberg: Springer Berlin Heidelberg), pp. 405–416.
Radhakrishnan, D., Wells, E.A., and Robinson, A.S. (2018). Strategies to enhance productivity and modify product quality in therapeutic proteins. Current Opinion in Chemical Engineering 22, 81–88.
Reinhart, D., Damjanovic, L., Kaisermayer, C., and Kunert, R. (2015). Benchmarking of commercially available CHO cell culture media for antibody production. Appl. Microbiol. Biotechnol. 99, 4645–4657.
Reinhart, D., Damjanovic, L., Kaisermayer, C., Sommeregger, W., Gili, A., Gasselhuber, B., Castan, A., Mayrhofer, P., Grünwald-Gruber, C., and Kunert, R. (2018a). Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis. Biotechnol J e1700686.
Reinhart, D., Damjanovic, L., Castan, A., Ernst, W., and Kunert, R. (2018b). Differential gene expression of a feed-spiked super-producing CHO cell line. Journal of Biotechnology 285, 23–37.