Institute of Chemical Engineering
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EIS4Fabs

This project is granted by FFG and is done in cooperation with Alfred Gruber GmbH.

Within the project EIS4Fabs a biosensor for the detection of antibody fragments should be developed to monitor the production process of these high valuable molecules.

Motivation

Antibody fragments (Fabs) are highly valuable molecules with a broad application in medicine (e.g. Abciximab, Ranibizumab, Certolizumab [1, 2]). Similar to antibodies (Figure 1) they have an antigen binding site. They are, much smaller and do not require post-translational glycosylation for biological activity allowing for cheap production processes using microbials like Escherichia coli.

 

Figure 1 Antibody and antibody fragment (Fab).

This reduction in production costs and time makes them extremely promising biopharmaceutical molecules. After expression in E. coli, the Fabs are translocated into the periplasm, enabling the formation of disulfide bridges in an oxidizing environment. However, the recombinant protein expression causes stress and as consequence cell-leakiness, the loss of cell integrity. The valuable Fab-product will be released uncontrollably and lost into the fermentation broth [3]. Today, no real time analytics is available to detect this loss. A similar challenge in Fab production processes is their purification, where once more only time delayed tools like ELISA are available. These offline ELISA-assays, which currently are state of the art, make fast and efficient process development as well as in-process-control impossible.

Goal

We will develop a novel biosensor based on electrochemical impedance spectroscopy (EIS) [4, 5] to 1) monitor Fab production processes in bioreactors in real- time (we will focus on cell leakiness), and 2) quantify the Fab in the different unit operations during product purification. Therefore, we will produce protein L, an immunoglobulin binding molecule, which will be the biorecognition element that binds to the antibody fragments. The produced protein L will be immobilized on the gold surface (Figure 2). Afterwards the gold chip with the covalently bound protein L will be used to detect antibody fragments. The bound antibody fragments then cause a change in the impedance signal. This will enable us to detect even small amounts of Fabs highly specific and very sensitive in the upstream and downstream process.

 

Figure 2 Schematic sketch of the EIS sensor. Protein L will be covalently bound to a gold surface. The immobilized protein L will selectively bind antibody fragments. This change of the protein L – Fab complex will cause a change in die impedimetric signal.

 

Further reading/links/cooperation partners

https://www.keg-gruber.at/de/

https://www.ffg.at

Open positions

MSc Thesis: Impedimetric biosensors for process control

BSc/MSc students and interns are always welcome!

Contact

Assoc. Prof. Dr. Oliver Spadiut

DI Dr. Sabine Kubicek

DI Stefan Kittler

Julian Ebner

References

1. Fernandes, J.C., Therapeutic application of antibody fragments in autoimmune diseases: current state and prospects. (1878-5832 (Electronic)).

2. Kholodenko, R.V., et al., Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and Limitations. (1875-533X (Electronic)).

3. Wurm, D.J., et al., How to trigger periplasmic release in recombinant Escherichia coli: A comparative analysis. (1618-0240 (Print)).

4. Slouka, C., et al., A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations. LID - 1900. (1424-8220 (Electronic)).

5. Slouka, C., et al., Low-frequency electrochemical impedance spectroscopy as a monitoring tool for yeast growth in industrial brewing processes. Chemosensors, 2017. 5(3): p. 24.