24th European Society for Animal Cell Technology (ESACT) Meeting: C2P2: Cells, Culture, Patients, Products.

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P7 Identification of bottlenecks in antibody expression using targeted gene integration Patrick Mayrhofer, Alexander Mader, Bernhard Kratzer, David Reinhart, Willibald Steinfellner, Wolfgang Sommeregger, Renate Kunert Vienna Institute of BioTechnology (VIBT), Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, 1190, Austria; Institute of Immunology, Medical University Vienna, Vienna, 1090, Austria E-mail: [email protected] BMC Proceedings 2015, 9(Suppl 9):P7 Introduction: Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers [1]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products. Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure 1). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions. Materials and methods: All cell lines (CHO-K1, DUKX-B11) were grown under serumand protein-free conditions in chemically defined media in suspension using shaker flasks. Transfections were performed with the cationic 25kDa linear polymer polyethylenimine (PEI). Stable subclones were either generated by limited dilution subcloning or fluorescenceactivated cell sorting (FACS).Germline antibody sequences were identified by the IgBlast program. Variable antibody sequences were synthesized and cloned into vectors containing leader and constant antibody regions. For recombinase-mediated cassette exchange a CMV (for DUKX-B11) or CAGGS (for CHO-K1) promoter was placed 5’ upstream of the first flippase recognition target site to establish a promoter trap in the parental RMCE-cassette. A selection/reporter marker was placed in-between two heterospecific FRT sites harboring a gfp/thymidine-kinase/neomycin-phosphotransferase fusion protein. Antibody producing cells were developed after recombination with the reporter marker by negative selection with ganciclovir. Antibody product was purified by protein A chromatography. Results: The observed expression levels in cell cultures are influenced by many different parameters that should be controlled for a valid comparison of different antibody variable sequences. Besides different host cell lines and media formulations our group also has standardized fermentation process protocols and biophysical analytical tools for indepth investigations of possible correlation between cell culture performance, product quality attributes and expression levels. Recently, we also engineered a CHO DUKX-B11 host cell line suitable for recombinasemediated cassette exchange (RMCE) [2] to control the integration locus by targeted gene integration leading to equal gene copy numbers (GCN) and mRNA levels between different subclones or even between different antibody variants [3]. Although we could demonstrate a single RMCEintegration locus, facilitating the generation of isogenic cell lines, the overall productivity remains relatively low. Therefore a second generation RMCE host cell line was designed based on CHO-K1 using the CAGGS Figure 1(abstract P7) Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (A). Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (B). Respective germline antibody variants were constructed by combination of germline V-, Dand J-segments BMC Proceedings 2015, Volume 9 Suppl 9 http://www.biomedcentral.com/bmcproc/supplements/9/S9 Page 25 of 111