For each antibody in the first set of 230 designs, nucleotide sequences encoding the designed heavy and light chain sequences were synthesized, cloned into an hIgG1 framework, and used to produce mAbs via transient transfection of HEK293 cells at ATUM (Newark, CA, USA).
For the second set of 204 designs, monoclonal antibody sequences were synthesized (Twist Bioscience), cloned into an IgG1 monocistronic expression vector54 (link) (designated as pVVC-mCisK_hG1), and expressed either at microscale in transiently transfected ExpiCHO cells55 (link) for screening, or at larger scale for down-stream assays. Sequences in this group of 204 designs all contain an additional arginine at the beginning of the light chain constant region with respect to sequences expressed in the first set. Larger-scale monoclonal antibody expression was performed by transfecting (30 ml per antibody) CHO cell cultures using the Gibco ExpiCHO Expression System and protocol for 125ml flasks (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). Purified monoclonal antibodies were buffer-exchanged into PBS and stored at 4 °C until use.
For the second set of 204 designs, monoclonal antibody sequences were synthesized (Twist Bioscience), cloned into an IgG1 monocistronic expression vector54 (link) (designated as pVVC-mCisK_hG1), and expressed either at microscale in transiently transfected ExpiCHO cells55 (link) for screening, or at larger scale for down-stream assays. Sequences in this group of 204 designs all contain an additional arginine at the beginning of the light chain constant region with respect to sequences expressed in the first set. Larger-scale monoclonal antibody expression was performed by transfecting (30 ml per antibody) CHO cell cultures using the Gibco ExpiCHO Expression System and protocol for 125ml flasks (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). Purified monoclonal antibodies were buffer-exchanged into PBS and stored at 4 °C until use.
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