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

Abstract

AbstractTraditional 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‐asparaginase. 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. © 2020 Society of Industrial Chemistry