Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100.
An IgG1 monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.
Taipa
M A
MA
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisboa, Portugal. pcataipa@alfa.ist.utl.pt
Kaul
R H
RH
Mattiasson
B
B
Cabral
J M
JM
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D011232:Chemical Precipitation; D006825:Hybridomas; D011109:Polymethacrylic Acids; D013678:Technology, Pharmaceutical / Q000379:methods
D000911:Antibodies, Monoclonal; D011109:Polymethacrylic Acids; C038300:methylmethacrylate-methacrylic acid copolymer