Chemically defined medium f17

Unique scFv sequences isolated by antibody-phage display in MTPs or the complete output of the third panning rounds (all scFv encoding DNA fragments) were subcloned into pCSE2.7-hIgG1-Fc-XP using NcoI/NotI (New England Biolabs, Frankfurt, Germany) for mammalian production as scFv-Fc, an IgG-like antibody format. The vector pCSE2.7-hIgG1-Fc-XP is derived from the vector pCSE2.6-hIG1-Fc-XP^{61 (link)} with an additional AscI restriction site in the cloning site for improved NcoI/NotI cloning. The best neutralizing antibodies were converted into the human IgG1 format by subcloning of VH in the vector pCSEH1c (heavy chain) and VL in the vector pCSL3l/pCSL3k (light chain lambda/kappa)^{37 (link)}. EXPI293F (Thermo Fisher Scientific) cells were transfected with both vectors in parallel. For production, the transfected EXPI293F cells were cultured in chemically defined medium F17 (Thermo Fisher Scientific) supplemented with 0.1% pluronic F68 (PAN-Biotech, Aidenbach, Germany) and 7.5 mM L-glutamine (Merck). A subsequent protein A purification was performed as described previously^{61 (link)}.

For IgG production, selected antibodies were converted into the human IgG1 format by subcloning of VH in the vector pCSEH1c (heavy chain) and VL in the vector pCSL3l/pCSL3k (light chain lambda/kappa)^{76 (link)}, adapted for Golden Gate Assembly procedure with Esp3I restriction enzyme (New England Biolabs). Expi293F (Thermo Fisher Scientific) cells were transfected with 12.5 µg of both vectors in parallel in a 1:1 ratio. For production, the transfected Expi293F cells were cultured in chemically defined medium F17 (Thermo Fisher Scientific) supplemented with 0.1% pluronic F68 (PAN-Biotech) and 7.5 mM L-glutamine (Merck) for 7 days. A subsequent protein A purification was performed as described above.