Chapter 4
Validation of the Production of Antibodies in Different
Formats in the HEK 293 Transient Gene Expression System
Jens Ko ¨ nig, Michael Hust, and Joop van den Heuvel
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 oneor 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.
Key words Transient gene expression, Recombinant antibody formats, scFv, scFv-Fc, Fab, IgG,
Biolayer interferometry, Kallikrein-related protease 7
1 Introduction
1.1 Aim of the Study
The functional and structural analyses of mammalian protein and
protein complexes require simple, robust, and efficient protein
production systems. Virus-free transient gene expression in insect
cell lines and mammalian cell lines as well as the baculoviral expression vector system allows the purification of ample amounts of
high-quality protein [1, 2]. Furthermore, adequate biochemical
and biophysical methods are essential for the kinetic analysis of
the binding affinity. Biolayer interferometry is a simple method
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59
Validation of the Production of Antibodies in Different
Formats in the HEK 293 Transient Gene Expression System
Jens Ko ¨ nig, Michael Hust, and Joop van den Heuvel
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 oneor 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.
Key words Transient gene expression, Recombinant antibody formats, scFv, scFv-Fc, Fab, IgG,
Biolayer interferometry, Kallikrein-related protease 7
1 Introduction
1.1 Aim of the Study
The functional and structural analyses of mammalian protein and
protein complexes require simple, robust, and efficient protein
production systems. Virus-free transient gene expression in insect
cell lines and mammalian cell lines as well as the baculoviral expression vector system allows the purification of ample amounts of
high-quality protein [1, 2]. Furthermore, adequate biochemical
and biophysical methods are essential for the kinetic analysis of
the binding affinity. Biolayer interferometry is a simple method
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59