Expansion and Purification of BDBV223 Antibody

Human hybridoma cells secreting BDBV223 were expanded in post-fusion medium^{24 (link)}. Briefly, cells were fused with HMMA2.5 myeloma cells, and then hybridomas producing BDBV223 were cloned by two rounds of limiting dilution and by single-cell fluorescence-activated cell sorting. Once cloned, hyridoma cells were expanded in post-fusion medium (ClonaCell-HY Medium E, STEMCELL Technologies #03805) until 50% confluent in 75-cm² flasks (Corning #430641). For production of BDBV223, cells from one 75-cm² flask were collected with a cell scraper and expanded to four 225-cm² flasks (Corning #431082) in serum-free medium (Hybridoma-SFM, Gibco #12045–076). After 21 days, the supernate was clarified by centrifugation and sterile filtered using a 0.2-μm pore size filter device. HiTrap Protein G or HiTrap MabSelectSure columns (GE Healthcare Life Sciences #17040501 and #11003494 respectively) were used to purify BDBV223 from filtered supernate. Fab fragments were generated from purified IgG through digestion with 4% papain for 6 h followed by purification over HiTrap KappaSelect column (GE Healthcare). The BDBV 620–635 peptide was generated synthetically (GeneScript).