Separation is a critical process in the production and purification of bio-derived products. Among the existing separation techniques, affinity chromatography has attracted significant research attention owing to its high selectivity and resolution properties. These properties are specifically of great importance in protein separation applications. Selectivity depends on the binding interactions between the protein and a specific ligand on the chromatographic matrix. This results in effective elution of highly concentrated target molecules in high purity. Additionally, for the affinity chromatography-based purification systems, successful elution of the target molecules can only be achieved if the ligand attached to the matrix allows for the reversible and specific binding of target molecules.
Traditional affinity chromatography techniques are mainly based on gel bead packing. Unfortunately, developing such chromatography methods prove difficult due to three main factors: bead deformation, high-pressure drop across the column and packing comprehension, leading to poor reusability, low throughput and low flow rates. Significant efforts have been made to address these problems. Ceramic monoliths, commonly used to reduce pollutants in automotive catalytic converters, have been employed as supports in affinity chromatography. Unfortunately, ceramic monolith surfaces are chemically inert solids, making it difficult to attach spacer arms and ligands to the monolith. Moreover, previous solutions to this problem are based on coating the ceramic monolith with agarose, cyanate esters and L-asparagine, which pose serious safety and environmental concerns.
In light of the above challenges, Dr. Javier Sánchez Santiago, Professor Ramón Cerro and Professor Carmen Scholz from the University of Alabama in Huntsville developed a new robust affinity chromatography separation technique based on ceramic monolith coatings as a reliable support in the bioseparations. Specifically, this approach is environmentally friendly as the cyanate esters were eliminated and substituted with more robust stable support based on commercial poly(ethylene acrylic acid) copolymer. The coating consisted of this copolymer together with poly(L-lysine) as spacer arm and L-asparagine as ligand, forming a copolymer network. Their work is currently published in the research journal, Polymer International.
In their approach, L-asparaginase was used as the target molecule for the separation process. Poly(L-lysine), a standard biocompatible material, was prepared through ring-opening polymerization of ε-trifluoroacetyl- L-lysine N-carboxyanhydride, and was used as a spacer arm due to its excellent coupling properties. The construct was decorated using enzyme-specific L-asparagine groups, and the coating was prepared by attaching the poly(L-Lys) to the poly(ethylene acrylic acid) copolymer followed by attaching the L-asparagine without having to isolate the intermittent product. All the coupling reactions involved EDC/NHS catalysts. The practicality and feasibility of the presented approach were demonstrated through adsorption and elution experiments.
The authors observed reversible binding between the L-asparaginase and L-asparagine, followed by a release of L-asparaginase. Consequently, they recovered approximately 83% of active enzymes by elution with sodium chloride and D-asparagine solutions. Compared with traditional separation methods based on gel bead packings, the adsorption/elution process took less than one hour on the ceramic monolith system to achieve the same separation levels. Moreover, the coated monoliths could be reused and have a sufficing shelf-life as they are not subject to microbial degradation. Furthermore, the initial enzyme concentration influenced the degree of adsorption, and optimizing the experiment could result in better adsorption/elution rates.
In summary, the authors proposed and demonstrated the feasibility of affinity chromatography technique based on ceramic monoliths coated with a poly(amino acid)-based polymeric network for isolation of L-asparaginase. The developed polymeric construct met the key conditions: formed a thin and uniform film on the monolithic wall, got coated on the surface out of an aqueous solution, carried a covalently attached enzyme-specific ligand via the spacer arm, and was successfully used in the separation and purification of protein. The resulting separation process was superior to its traditional counterparts in terms of reduced separation time, reusability and efficiency. According the lead author Professor Carmen Scholz, the findings provide insights into the use of poly(amino acid) copolymers together with ceramic monoliths as a robust support for high-performance affinity chromatography based separation technique.
Santiago, J., Cerro, R., & Scholz, C. (2020). A robust affinity chromatography system based on ceramic monoliths coated with poly(amino acid)‐based polymeric constructs. Polymer International, 70(1), 41-50.