Hitrap kappaselect column

PGZL1 HCs and LCs were cloned using Gibson Assembly Enzyme mix (NEB) into expression vectors with the appropriate IgG1, Igκ, or Igλ constant domains^{49 (link)}. Antibodies were expressed in FreeStyle 293F cells (Life Technologies Cat#R79007). Briefly, ~ 750 µg DNA (500 µg HC and 250 µg LC plasmid) were added to 25 ml Opti-MEM (Life Technologies, 31985-070), which was mixed with Opti-MEM containing 2250 µg polyethylene imine MAX (molecular weight 40,000 kDa; Polyscience, 24765-1). After incubation for 20 min at room temperature (RT), the transfection mix was added to 1 L cells at a density of ~ 1.2 × 10⁶ cells/ml in FreeStyle293 Expression Medium (Life Technologies, 12338018). The cells were incubated at 37 °C and 8% CO₂ for 6 days. After collecting the cells, the supernatant, containing IgG or Fab, was filtered and loaded into a protein A beads column (Thermo Scientific) or HiTrap KappaSelect column (GE Healthcare Life Sciences, 17545812). The column was washed with phosphate-buffered saline (PBS) and eluted with 0.2 M citric acid pH 3.0 or 0.1 M glycine pH 2.7. The fractions were concentrated and the buffer was changed to 20 mM sodium acetate pH 5.5. The Fab was loaded into a Mono S column and was eluted with a 0–60% linear gradient of 1 M sodium chloride and20 mM sodium acetate pH 5.5 buffer. The Fabs were concentrated and stored in 20 mM sodium acetate pH 5.5 at 4 °C.

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).

IgGs and Fabs were expressed following standard protocols as follows. Proteins were expressed in FreeStyle 293F cells (Thermo Fisher #R79007). About 25 mL Opti-MEM (Thermo Fisher #31985070) containing ∼750 μg DNA (500 μg heavy chain and 250 μg light chain plasmid) for Fab or ∼500 μg DNA (250 μg heavy chain and 250 μg light chain plasmid) for IgG was mixed with 25 mL Opti-MEM containing 2,250 μg polyethylene imine MAX (MW 40,000; Polyscience - 24765-1). After incubation for 20 min at RT, the transfection mix was added to 1L cells at a density of ∼1.2x106 cells/ml in FreeStyle 293 Expression Medium (Thermo Fisher - 12338018). The cells were incubated at 37°C and 8% CO2 for 6 days. After harvesting the cells, the supernatant, containing IgG or Fab, was filtered and loaded into a HiTrap protein A column (GE Life Sciences #17040301, for IgG) or HiTrap KappaSelect column (GE Life Sciences #17545812, for Fab). The column was washed with phosphate buffered saline and eluted with 0.1 M glycine pH 2.7. The fractions were concentrated, and the buffer was changed to 20 mM sodium acetate pH 5.5. The Fab was loaded into a Mono S column and was eluted with a 0 to 60% linear gradient of 1M sodium chloride in 20 mM sodium acetate pH 5.5 buffer. The Fabs were concentrated and stored in 20 mM sodium acetate pH 5.5, PBS or TBS at 4°C or at −80°C.