Protein a resin

Purified recombinant CV3-25 IgG was mixed with LysC (NEB) at a ratio of 1 μg LysC per 10 mg of IgG and incubated at 37 °C for 18 h with nutation. The cleaved product was incubated with 1 mL of Protein A resin (GoldBio) per 10 mg of initial IgG and incubated at room temp for 1 hr to bind any uncleaved IgG and digested Fc. The purified Fab was further purifed by SEC using a HiLoad 16/600 Superdex 200 pg column.

The secreted antibodies were captured by Protein A resin (GoldBio) equilibrated with 0.1 M Tris pH 8.0, 500 mM NaCl, eluted with 0.1 M glycine pH 2.5, and 500 mM NaCl and neutralized with 1/10 volume of 1 M Tris pH 8.0. For the preparation of Fabs, the buffer of antibodies was exchanged using a Hitrap desalting column (Cytiva) equilibrated with the fresh digestion buffer (1× PBS with 0.02 M EDTA and 0.02 M l-cysteine, pH 7.0). Antibodies were digested with papain-immobilized resin (32 mg AB/mL settled resin; Thermo Fisher Scientific) in the digestion buffer overnight at 37°C. Fabs were collected from the flowthrough fractions after passing through the HiTrap rProtein A FF column equilibrated with 0.1 M Tris pH 7.5 and 0.5 M NaCl. Full-length antibodies and Fabs were analyzed using SDS-PAGE gel. The concentration of antibodies was estimated using an extinction coefficient of 1.4 (mg/mL).