Partitioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems

Ana Margarida Nunes da Mata Pires de Azevedo; Ana Gabriela Gonçalves Neves; Paula Alexandra Albuquerque de Jesus; Isabel Filipa Prates Acciaioli Hilário; Ângela Miguel Miranda Oliveira; M.R. Aires-Barros

Key information

Authors:

Ana Margarida Nunes da Mata Pires de Azevedo (Ana Margarida Nunes da Mata Pires de Azevedo); Ana Gabriela Gonçalves Neves (Ana Gabriela Gonçalves Neves Gomes); Paula Alexandra Albuquerque de Jesus (Paula Alexandra Albuquerque de Jesus Rosa); Isabel Filipa Prates Acciaioli Hilário (Isabel Filipa Prates Acciaioli Hilário Ferreira); Ângela Miguel Miranda Oliveira (Ângela Miguel Miranda Oliveira Pisco); M.R. Aires-Barros (Maria Raquel Murias dos Santos Aires Barros)

Published in

2 de fevereiro de 2009

Abstract

Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15\% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a hybridoma cell culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a hybridoma cell culture supernatant in a serum-containing media was achieved using 15\% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8\% (w/w) PEG 3350, 8\% (w/w) citrate and 15\% (w/w) NaCl at pH 6, IgG was recovered with a 99\% yield, 44\% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5\%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99\%, with a HPLC purity of 76\% and with a IgG/protein ratio of 0.96. (C) 2007 Elsevier B.V. All rights reserved.