Surfactant-aided size-exclusion chromatography for the purification of immunoglobulin G.
In the production of monoclonal antibodies, separate chains of the antibody are often present in the product mixture as well as other contaminating proteins. These fragments should be removed from the whole antibodies. This paper shows the purification of monoclonal immunoglobulin G (IgG) from its heavy chain contaminant. The heavy chain fragment is simulated experimentally using bovine serum albumin (BSA), which has approximately the same molecular weight. The purification is performed using traditional size-exclusion chromatography (SEC) and using surfactant-aided SEC (SASEC), testing two different surfactants (C(12)E(23) and Tween20) and two different gels (Sephacryl S200HR and Sephacryl S300 HR). Pulse experiments show that with SASEC both BSA and IgG are more distributed towards the solid phase than compared to using SEC. This effect is larger on IgG, the largest component than on BSA. As a consequence, azeotropes will be formed at a specific surfactant concentration. Above this concentration the selectivity is reversed and increased to values higher than obtained with conventional SEC. At 7.5% (w/w) of C(12)E(23), BSA actually elutes before IgG. These experiments further show that when using SASEC larger productivity, higher yields and lower solvent consumption can be achieved without loss of purity of IgG when compared to conventional SEC. Mathematical simulation of the separation of BSA and IgG using simulated moving bed (SMB) chromatography indicates a large increase in productivity when applying a surfactant gradient in SASEC SMB compared to conventional isocratic SEC-SMB. Furthermore, solvent consumption reductions with a factor 15 prove possible as well as concentrating the IgG by a factor 2.
Horneman
D A
DA
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Ottens
M
M
Keurentjes
J T F
JT
van der Wielen
L A M
LA
D002850:Chromatography, Gel / Q000379:methods*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008823:Micelles; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012997:Solvents / Q000737:chemistry
D007074:Immunoglobulin G; D008823:Micelles; D012997:Solvents; D012710:Serum Albumin, Bovine