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Project E5 - Process development/control, production of proteins and virus particles

PI
Project description

The IBPT works on platform technologies and methods for cell and particle-based therapy with the opportunity to optimize and control the material transport processes through modern imaging processes at a cellular level as well as accompany process analysis based on automated procedures.

In this context, engineering questions related to the  development of licensed nano and micro-particulate transport systems l for therapy, the development of licensed reaction systems for cell-based and nanoparticulate (virus particles) therapy, and the optimization and scale-up of cell cultivation systems (stem cells, insect cells) will be addressed.

e5

The Czermak group is working on the characterization and optimization of GMP-compliant processes for the expansion and harvesting of stem cells for therapy as well as on the characterization and optimization of processes for the production and purification of oncological measles viruses for cancer therapy. Moreover, questions about PAT and GMP are being addressed in process development for the production of medicines for new therapies (Advanced Therapy Medicinal Products, ATMP). The expertise of IBPT at THM is available in these fields of work. Along with the development of bioreaction systems, research and development for downstream processes of cell and virus-based therapeutic products as well as the transfer from processes to production are also being carried out.

Another focus of the work group is the recombinant production of proteins/peptides, in which the following developments and questions are addressed: i) knowledge-based selection of the appropriate expression system è successfully established expression systems that are also further developed as part of Golden Gate cloning: E. coli, Pichia pastoris, Kluyveromyces lactis, and the insect cells S2, Sf9/21. Furthermore, various animal cells are available (e.g. CHO, Vero); ii) process development in consideration of GMP and PAT guidelines and recommendations for upstream and downstream processes as well as application studies in various fields; iii) the integration of “single use” units into upstream and downstream processing; iv) know-how and process transfer as well as scale-up/scale-down studies. Along with optimizing the protein/peptide, e.g., via protein engineering, the development of a suitable expression system for standardized production and drug development are crucial for therapeutic application. In this way, for instance, the entire process chain (identification, characterization, expression, purification, formulation, impact studies) was successfully developed and carried out for the insect metalloproteinase inhibitor IMPI. This was achieved by developing an expression strain using Golden Gate cloning (E. coli Rosetta Gami 2 (DE3) PLysS plasmid GG49: T7 promoter, GST fusion protein, thrombin cleavage site, IMPI, T7terminator), which is able to maintain the peptide’s disulfide bonds and synthesize it in its soluble form (by integrating a fusion protein) in the cytoplasm, optimize fermentation, and develop a purification strategy. In order to start initial toxicity studies and animal experiments, the inhibitor was produced several times at a 20 l scale and purified, and the product was given to the Fraunhofer ITEM in Hannover, Germany. The established know-how to build a plasmid database based on the Golden Gate cloning principle can be made available to the consortium. This contains various sets of promoters, tags, fusion proteins, protease cleavage sites, and peptides of interest (POI) that have already been identified in the FhG. This plasmid database can be extended indefinitely if new interesting tags or fusion partners are identified, and it also allows fully tag-free production of POIs. Thus, the expression of gloverin by using insect cells (BVES, S2) and the production of the antifungal peptide AFP with E. coli and Pichia pastoris and the antimicrobial peptide BR021 with E. coli and insect cells (S2) were successfully carried out.

The characterization and optimization of a production process for a recombinant vaccine with the baculovirus expression system was successfully implemented using PAT (Process Analytical Technology).This depends on the exact observance of temperature and inoculation strategies as well as on the oxygen dependence and sensitivity of the Sf21 cells and the baculovirus - also during the infection. Additionally, there is the identification of an optimal inoculation titer and the establishment of online monitoring. This is used to establish robust bioprocesses that meet the requirements of GMP and PAT.

References E5: 1. Spohner & Czermak (2016) New Biotechnol 33:473-9 2. Spohner et al. (2016) J Biotechnol 222:104-16 3. Grein et al. (2016) Process Biochem 51:1109-19 4. Weiss et al. (2015) Eng Life Sci 15:425-36 5. Kuhn et al. (2015) E J Biotechnol 18:252-5 6. Grein et al. (2014) Meth Mol Biol 1104:459-91.