Biomimetic Ligands for Immunoglobulin-M Purification

Abstract

Immunoglobulin-M (IgM) has been recognized as a diagnostic and therapeutic agent and a candidate for stem cell isolation. In order to exploit the full potential of IgM, large quantities in a highly pure and active form must be available at low costs for performing clinical trials and characterization studies. Most of the current practices for IgM purification rely on a multi-stage approach involving precipitation and conventional chromatographic techniques. Such an approach is costly, time consuming and frequently results in low IgM recoveries. An effective way to purify IgM is through affinity chromatography, which currently is limited by the availability of `suitable? capture agents. Peptide-based affinity chromatography represents a particularly promising approach because it combines the selectivity of a naturally-occurring biological receptor with the simplicity of a single small molecule. The focus of the present research was to develop biomimetic peptidic ligands which can mimic the binding activity of hpIgR, a naturally-occurring Fc (`universal?) receptor to IgM.

Synthetic peptides, pep12 and pep14, incorporating the complementary determining region 2 of Domain 1 of hpIgR were investigated for their interactions with hIgM and other hIgs. pep14 emerged as a unique ligand exhibiting specificity in its interaction to hIgM with negligible affinity for hIgG1, hIgA1, hIgE and BSA. Binding studies between modified peptides (pep13, pep5A and pep14A) and hIgM were performed that provided an insight into the binding mechanism of pep14 to hIgM. Various characterization studies including effect of pH, ionic strength, nature of binding buffer and ligand density on pep14-hIgM interaction, ligand binding region in hIgM, binding strength, static binding capacity and stability of pep14 to sanitizing agents, were performed to determine the suitability of pep14 as an affinity ligand for IgM purification. A unique method of introducing pre-concentration through the use of polyethylene glycol based linkers was devised. The method allowed significant increase in coupling efficiency when immobilizing hydrophobic peptidic ligands to hydrophilic chromatographic matrices.

The present work facilitates the development of a chromatographic methodology to purify IgM, especially hIgM, on a large scale at high purities and yields. The study demonstrated the uniqueness and competence of biomimetic ligands in isolating and purifying large proteins, opening up avenues of research to design and develop low molecular weight affinity ligands.