Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer–polymer and polymer–salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the t...

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Bibliographic Details
Published in:	Journal of Chromatography A Vol. 1162; no. 1; pp. 103 - 113
Main Authors:	Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F., de Vries, J., Korporaal, R., Verhoef, H.J., Visser, T.J., Aires-Barros, M.R.
Format:	Journal Article Conference Proceeding
Language:	English
Published:	Amsterdam Elsevier B.V 24-08-2007 Elsevier
Subjects:	Affinity Analytical, structural and metabolic biochemistry Animals Aqueous two-phase systems Biological and medical sciences Chemical Fractionation - methods Chromatography, Affinity Cricetinae Dextrans Functionalised PEGs Fundamental and applied biological sciences. Psychology Glycoproteins Human antibodies Humans Immunoglobulin G - chemistry Immunoglobulin G - isolation & purification Models, Chemical Osmolar Concentration Partitioning Phase Transition Polyethylene Glycols - chemical synthesis Proteins Sensitivity and Specificity Synthesis Water Affinity Aqueous two-phase systems Synthesis Human antibodies Partitioning Functionalised PEGs Amino group End group Telechelic polymer IgG Partition coefficient Ethylene oxide polymer Separation method Aqueous medium Carboxyl group Functional group Human Purification Antibody Glycoprotein CHO cell line Biphasic system Cell line Triazine derivatives Isolation Lipophilicity Physicochemical properties
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Summary:	The partitioning of human immunoglobulin (IgG) in a polymer–polymer and polymer–salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG–COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150–COOH and 40% (w/w) PEG 3350–COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350–COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150–COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150–COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0021-9673
DOI:	10.1016/j.chroma.2007.03.067