D5_AR, 10E8v4, VRC01, PGDM1400, and 10-1074 IgG1s were expressed and purified from Expi293F cells. Expression vectors for D5_AR were generated previously (20 (link)), expression vectors for VRC01 and 10E8v4 were sourced from the NIH HIV Reagent Program (see “NIH HIV Reagents”), and expression vectors for 10-1074 were gifted from Dr. Christopher Barnes (28 (link), 48 (link)). PGDM1400 heavy and light chain sequences were synthesized (Integrated DNA Technologies) and cloned into a mammalian expression vector under a CMV promoter using InFusion (Takara) and sequence verified.
Expi293F cells were cultured in 33% Expi293 Expression/66% FreeStyle Expression medium (Thermo Fisher Scientific) and grown in baffled polycarbonate shaking flasks (Triforest) at 37 °C and 8% CO2. Cells were grown to a density of ~3 × 106/mL and transiently transfected using FectoPro transfection reagent (Polyplus). For transfections, 0.5 μg total DNA (1:1 heavy chain to light chain plasmids) was added per mL final transfection volume to culture medium (1/10 volume of final transfection) followed by FectoPro at a concentration of 1.3 μL per mL final transfection volume and incubated at room temperature for 10 min. Transfection mixtures were added to cells, which were then supplemented withd -glucose (4 g/L final concentration) and 2-propylpentanoic (valproic) acid (3 mM final concentration). Cells were harvested 3 to 5 d after transfection via centrifugation at 18,000 × g for 15 min. Cell culture supernatants were filtered using a 0.22-μm filter prior to purification.
Filtered Expi cell culture supernatants were buffered with 1/10 volume 10× PBS and loaded onto a HiTrap MabSelect SuRe column (Cytiva) equilibrated in PBS (pH 7.4) using a Cytiva ÄKTA Pure system at a flow rate of 3.5 mL/min. The column was subsequently equilibrated with five column volumes PBS (pH 7.4) before elution with three column volumes 100 mM glycine (pH 2.8) into 1/10th volume of 1M Tris (pH 8.0). The column was washed with 0.5 M NaOH with a minimum contact time of 15 min between purifications of different antibodies. Elutions were concentrated using Amicon spin filters (molecular weight cut-off 10 kDa; Millipore Sigma) and were subsequently loaded onto a GE Superdex S200 increase 10/300 GL column preequilibrated in 1× PBS using a Cytiva ÄKTA Pure system. Protein-containing fractions were identified by A280 signal and/or SDS-PAGE, pooled, and stored at 4 °C or at −20 °C in 10% glycerol/1× PBS until use.
Expi293F cells were cultured in 33% Expi293 Expression/66% FreeStyle Expression medium (Thermo Fisher Scientific) and grown in baffled polycarbonate shaking flasks (Triforest) at 37 °C and 8% CO2. Cells were grown to a density of ~3 × 106/mL and transiently transfected using FectoPro transfection reagent (Polyplus). For transfections, 0.5 μg total DNA (1:1 heavy chain to light chain plasmids) was added per mL final transfection volume to culture medium (1/10 volume of final transfection) followed by FectoPro at a concentration of 1.3 μL per mL final transfection volume and incubated at room temperature for 10 min. Transfection mixtures were added to cells, which were then supplemented with
Filtered Expi cell culture supernatants were buffered with 1/10 volume 10× PBS and loaded onto a HiTrap MabSelect SuRe column (Cytiva) equilibrated in PBS (pH 7.4) using a Cytiva ÄKTA Pure system at a flow rate of 3.5 mL/min. The column was subsequently equilibrated with five column volumes PBS (pH 7.4) before elution with three column volumes 100 mM glycine (pH 2.8) into 1/10th volume of 1M Tris (pH 8.0). The column was washed with 0.5 M NaOH with a minimum contact time of 15 min between purifications of different antibodies. Elutions were concentrated using Amicon spin filters (molecular weight cut-off 10 kDa; Millipore Sigma) and were subsequently loaded onto a GE Superdex S200 increase 10/300 GL column preequilibrated in 1× PBS using a Cytiva ÄKTA Pure system. Protein-containing fractions were identified by A280 signal and/or SDS-PAGE, pooled, and stored at 4 °C or at −20 °C in 10% glycerol/1× PBS until use.
