Abstract
Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen—KLK7—the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.
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Acknowledgments
We greatly thank Luciano Puzer from the Universidade Federal do ABC, Sao Paulo, Brazil for his valuable scientific input and the biomaterials he made available for this research project. We also thank Daniela Gebauer, Nadine Konisch, and Anke Samuels for their excellent technical support during the experiments. This work was supported by the Helmholtz Protein Sample Production Facility (PSPF).
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König, J., Hust, M., van den Heuvel, J. (2021). Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System. In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_4
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_4
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