Monoclonal antibodies constitute one of the most important and versatile groups of biological drugs. Affinity chromatography based on bacterial immunoglobulin-binding proteins (e.g. staphylococcal protein A and streptococcal protein G) is typically used for isolation and purification of therapeutic antibodies, but it has many shortcomings. The aim of this work was to construct and evaluate an affinity monolithic chromatographic column based on a peptide ligand for the purification and isolation of immunoglobulin G (IgG). Affinity columns with polymethacrylate monolith and cross-linked agarose stationary phases were prepared. A peptide ligand labeled with a C-terminal cysteine residue was coupled to the functionalized supports via tris(2-aminoethyl)amine and bromoacetate spacers. The IgG dynamic binding capacity of the chromatographic columns was analyzed using an HPLC system. IgG binding was confirmed for the peptide: agarose matrix, but not for the peptide: monolith matrix. Using the same coupling chemistry, we prepared affinity matrices functionalized with different model ligands, such as oligonucleotides (via amino and sulfhidryl groups) and protein A (via a cysteine residue), and compared them to those carrying the same ligands but immobilized in a different way. All the affinity matrices were functional judging from the determined dynamic binding capacity. We conclude that the hydrophobic nature of the peptide IgG ligand is not compatible with the relatively hydrophobic polymethacrylate monolithic support. We assume that due to hydrophobic interactions between the two, the peptide is not accessible for antibody binding.
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