Humanized Antibody Production and Purification

The variable domains of the humanized Apo-1 antibody (EP2920210B1), the humanized 2H7 antibody (EP2920210B1), the 4G7 antibody (GenBank no.: AJ555479 and AJ555622), and the MOPC-21 antibody (GenBank no.: AAD15290.1 and AAA39002.1) were codon-optimized using the GeneArt GeneOptimizer tool for the transfection of CHO cells (Thermo Fisher Scientific). V_H, V_L, and scFv sequences were synthesized de novo at GeneArt (Thermo Fisher Scientific). As previously described, the variable domains were inserted into a human IgGγ1sc backbone, which is designed to abolish FcR-binding and complement fixation [24 (link)]. IgGsc molecules were produced in the ExpiCHO^TM Expression System (Thermo Fisher Scientific) according to the manufacturer’s instructions and then purified by HiTrap^TM MabSelect^TM SuRe columns (Cytiva, Freiburg, Germany), before being subjected to preparative and analytical size exclusion chromatography (SEC) using HiLoad^TM 16/600 Superdex 200 pg and Superdex^TM 200 Increase 10/300 GL columns (Cytiva), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described [25 (link)]. The generation and purification of Fabsc molecules were described by Nalivaiko and colleagues [23 (link)].