A robust affinity chromatography system based on ceramic monoliths coated with poly(amino acid)-based polymeric constructs


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Polymer International


Traditional chromatographic separation systems are disadvantaged by low flow rates, a high pressure drop across the column, low capacity and poor reusability. Searching for more efficient separation systems we introduced the use of a ceramic monolith as robust support in bioseparations. A coating consisting of L-asparagine as ligand, poly(L-lysine) as spacer arm and a commercial poly(ethylene acrylic acid) film forming copolymer network (Michem 4983-40R) was developed as a coating for these ceramic monoliths. Poly(L-lysine) was synthesized by ring-opening polymerization of ε-trifluoroacetyl-L-lysine N-carboxyanhydride and coupled to a commercial film-forming poly(ethylene acrylic acid) network. This construct was then 'decorated' with L-asparagine via the terminal amino functional groups of poly(L-lysine) and coated onto the ceramic monolith to selectively bind L-asparagi-nase. Adsorption/elution experiments showed reversible binding between L-asparagine and L-asparaginase, and the subsequent release of L-asparaginase, and between 83% and 94% of the active enzyme was recovered by elution with D-asparagine and NaCl solutions. The functional activity of the eluted L-asparaginase was verified by a Nessler's assay. While traditional separation processes (adsorption and elution) using gel bead packings take many hours, the ceramic monolith system achieves the same of level of separation in about 1 h. This new system served as a proof of concept for its application in protein separation and purification. This work paves the way to a better understanding of the use of ceramic monoliths as stationary phase coated with a stable polymer construct for more robust and efficient supports in affinity chromatography.



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