Ex cell cd cho serum free medium

Light and heavy chain sequences of nivolumab were obtained from GenBank (MC034325) and long chain cDNAs were synthesized by Bioneer. To produce IgG1 and IgG4 nivolumab, light and heavy chains of nivolumab with IgG1 and IgG4 backbone were transfected into HEK cells (ATCC, American Type Culture Collection) to generate lentiviruses. The HEK cell media containing lentiviruses were treated in CHO WT and GD CHO cells. Nivolumab-producing cells grown to 80% confluency were refreshed with EX-CELL® CD CHO Serum-Free medium (Sigma) containing 1 mM sodium butyrate. Conditioned media containing antibody was obtained by further incubation for 14 days at 30 °C in 5% CO₂ incubator. The antibodies were collected and purified using a protein A column (Thermo Fisher Scientific). Buffer changes and sterilization were performed using an Amicon® Ultra-2 (UFC801024). The antibodies were analyzed using SDS-PAGE and coomassie blue staining, and their concentrations were quantified relative to the band intensities 0.5, 1.0, and 2.0 μg bovine serum albumin used as a standard.

HEK293T cells (ATCC, cat# CRL-3216) used in this study were cultured in Dulbecco’s Modified Eagle Medium, DMEM, supplemented with 10% FBS, 1% NEAA and 1% L-Glutamine at 37 °C and 5% CO₂. Genetically engineered CHO cells, B4GalT7 KO, Csgalnat1/2/Chsy1 KO, Extl3 KO, Ndst1/2 KO, Hs2st1 KO, Glce KO, Hs6st1-3 KO, NDST2 KI, HS3ST1 KI Csgalnact1/2/Chsy1 KO, HS3ST5 KI Csgalnact1/2/Chsy1 KO, CHST3 KI, CHST15 KI were maintained in suspension culture using an equal mix of EX-CELL® CD CHO Serum-Free Medium (#14360 C, Sigma-Aldrich) and BalanCD CHO Growth A medium (#91128, Fujifilm), supplemented by 2% GlutaMAX™ (#35050061, Gibco). These CHO cell lines were generated and are maintained in the Copenhagen Centre of Glycomics, the cell plasmids and gRNA constructs designed for each CHO cell are described in refs. ^{33 (link),34 (link)}. Protein expression was induced by transfection with plasmid DNA and polyethyleneimine as the transfection reagent.