METAL AFFINITY CHROMATOGRAPHY WITH A CHITOSAN/CERAMIC MEMBRANE
Advancing the chemical engineering fundamentals
Membranes and Membrane Science - II (T2-8b)
Keywords: membrane, affinity, chromatography, chitosan, protein, purification
Affinity chromatography, mostly based on column configurations, is nowadays widely employed in protein purification. Unfortunately, due to high pressure drop, these systems are not appropriate for industrial applications where big volumes of non-clarified biological solutions have to be treated. In recent years, great interest has been paid to membranes as chromatographic media. This relatively new method combines the high-resolution separation of chromatography with the low pressure drop induced by the short-wide-bed of macroporous membrane systems, which generally involve organic supports [1, 2].
In this study, a hybrid chitosan/ceramic membrane is proposed with the aim of performing the purification of proteins by metal-affinity chromatography (histidine-tagged proteins mainly). Thanks to the attractive properties of chitosan, such as biocompatibility and non-toxicity, it was chosen to activate the ceramic support before the immobilization of the affinity ligand. Concerning the ceramic support, it will be in charge of conferring membranes the mechanical and chemical strength required for industrial applications [3, 4].
In the first stage of the study, a four step protocol was developed for membrane preparation: Chitosan is firstly coated onto a tubular alumina support (length 13cm; inner diameter 0.7cm; pore diameter 1.8μm) by cross-flow filtration of a chitosan solution. Then, the chitosan layer is cross-linked with epichlorohydrin. Iminodiacetic acid (IDA) is grafted on cross-linked chitosan. Finally the copper-ion is fixed by dead-end filtration with a 100ppm Cu2SO4 solution at a flow rate of 1ml/min. Tests show these membranes have a cooper adsorption capacity of 1.5 g of Cu2+/m2 of membrane.
Protein retention is tested with Bovine Serum Albumin (BSA) as a model protein. Experiments are carried out in dead-end filtration, with a 40ppm BSA solution at a flow rate of 0.25ml/min. Analyses show a BSA retention capacity of 0.33 g BSA/m2 of membrane; total capacity achieved in 230min. Protein retention is then followed by rinsing and elution steps.
This work is interesting in two aspects: in one hand, it proposes a new method to elaborate membrane media for protein purification and, in the other hand, it contributes in the study of this promising new membrane application process, nowadays not well known.
References
[1] C.Charcosset, Purification of Proteins by Membrane Chromatography, Journal of Chemical, Technology and Biotechnology 71(1998) 95-1107.
[2] V.Gaberc-Porekar, V.Menart. Perspectives of immobilized-metal affinity chromatography, Biochemical and biophysical methods 49(2001) 335-360.
[3] E.Klein, Affinity membranes: a 10-year review, Journal of Membrane Science 179(2000) 1-2.
[4] C.Wu, S-Y.Suen, Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes, Journal of Chromatography A 996(2003) 53–70.
Presented Wednesday 19, 15:00 to 15:20, in session Membranes and Membrane Science - II (T2-8b).
