Glycosylated Anti-PD-L1 Antibody Production

The variable region for the glycosylated anti-PD-L1 variants is based on the sequence of atezolizumab (Genentech) (23 (link)). The antibody sequences of the variable heavy (VH) and light (VL) region were cloned into expression vectors containing sequences for the human constant domains of the IgG1 κ light chain and heavy chain (Glycotope), respectively. Both plasmids were co-transfected in two GlycoExpress cell lines (Glycotope) (28 (link)) characterized by normal and reduced core fucosylation followed by selection and gene amplification by increasing concentrations of methotrexate (Sigma-Aldrich, #M8407) and puromycine (Clontech, #631306). High producing cell clones isolated from semisolid matrix medium by the ClonePix system (Molecular Devices) were expanded and used for production of supernatants in spinner culture flasks or 2 l perfusion bioreactors. Antibodies were purified using protein A chromatography on MabSelect Sure (GE-Healthcare, #29049104) to a monomer content >98% and showed no obvious endotoxin-contamination as tested via LAL endotoxin assay (GenScript).
atezolizumab as a reference material was purchased from Genentech (PZN#11306050).