Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications
Hana Hanaee-Ahvaz,
Monika Cserjan-Puschmann,
Florian Mayer,
Christopher Tauer,
Bernd Albrecht,
Paul G. Furtmüller,
Birgit Wiltschi,
Rainer Hahn,
Gerald Striedner
Affiliations
Hana Hanaee-Ahvaz
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Monika Cserjan-Puschmann
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria; Corresponding author.
Florian Mayer
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Christopher Tauer
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Bernd Albrecht
Biopharma Austria, Process Science, Boehringer Ingelheim Regional Center Vienna GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, A-1121, Vienna, Austria
Paul G. Furtmüller
University of Natural Resources and Life Sciences, Vienna, Department of Chemistry, Institute of Biochemistry, Muthgasse 18, 1190, Vienna, Austria
Birgit Wiltschi
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Rainer Hahn
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Gerald Striedner
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190, Vienna, Austria
Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like μ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications.
