Immobilisation of proteins onto CIM® – developing the most efficient affinity chromatographic monolith
Differently pre-activated CIM monoliths enable immobilisation of a range of proteins, peptides, nucleotides and other affinity ligands. The first parameters to be defined for a new immobilisation are the activation chemistry of the matrix and the linkage protocol. Due to the varying physico-chemical characteristics of different ligands, however, a platform process cannot be achieved.
In the following case study, covalent binding of Recombinant Prokaryotic Lectins (RPLs) onto CIM chromatographic monoliths is used as an example to describe the optimisation of an immobilisation protocol. The affinity of lectins for specific glycans can be exploited for the separation and isolation of glycosylated biomolecules and their glycoforms based on the glycan expression on the biomolecules. Affinity chromatography overcomes the limitations of traditional separation techniques based on size, charge distribution, or hydrophobicity/hydrophilicity, which are often unsuitable for the separation of molecules with closely related structure.
Galactose-specific lectin (RPL-Gal1) was immobilized onto CIMmic columns. Two different types of pre-activated resin chemistries were used, namely aldehyde-based (AE) and carbonylimidazole-based (CDI) monoliths. The composition of the immobilisation buffer and the protocol for deactivation of remaining active groups were varied to produce a total of 7 different affinity columns which were tested chromatographically as outlined below.