(122e) A Search for Affinity Ligands for Igm Purification

Current findings of immunoglobulin-M (IgM) as a candidate for cancer treatment and isolation of stem cells have spurred the interests of researchers for its isolation and purification. IgMs have also found application in immuno-precipitation, for immuno-histochemical staining, immunological identification of proteins and detection of cell secretions. Conventional approaches for purification of IgMs have been based on a combination of different fractionation techniques, such as precipitation, gel permeation chromatography, ion-exchange chromatography and electrophoresis. Besides being non-specific in nature, a multiple number of steps are involved making these techniques unsuitable for large scale production. Affinity chromatography, a technique based on the principle of molecular recognition, deserves particular attention as it allows for several fold purification of a highly dilute solution in a single step often with a high recovery. Despite its potential, the use of affinity chromatography for purification of IgMs has been limited by the availability of suitable ligands. Natural IgM binding proteins, for example the human secretory component, complementary protein C1q, mannose binding protein, are not directly amenable for use in affinity chromatography as they suffer from a number of disadvantages including high cost of production, their biological origin (they have to be isolated from human and animal fluids), the requirement for accurate analytical tests to ensure the absence of toxic contaminants and the poor stability to cleaning and sanitizing agents. The objective of this research was to develop low molecular weight synthetic ligands that can circumvent the drawbacks of natural binding ligands by imparting resistance to chemical and biochemical degradation, are facile with low cost of production, and are amenable to in situ sterilization.

Our approach to this was based on the ?structural template approach', one that requires the knowledge of the protein structure and knowledge of the interaction between the protein and its natural ligand. Studies conducted in the past have shown that the human secretory component (SC) binds specifically and non-covalently to IgM. The studies further suggest that 1) binding between IgM and SC depends preferentially on a strong interaction with domain 1 of SC and 2) domain 1 of SC consists of three CDR (complimentary determining region) -like loops and the region containing the CDR2-like loop is most important in the pentameric IgM-binding process. Based on these observations, a 14 amino acids (a.a.) long peptide containing the CDR2 like loop and a protein representing domain 1 of SC (114 a.a.) were chosen as probable affinity ligands and investigated for their suitability for IgM purification.

Surface Plasmon resonance (SPR) biosensor assays were carried out on a CM5 chip to obtain the strength of binding between the synthesized ligands and the analyte molecules: IgM, IgG and albumin. The results obtained by SPR studies ascertained our approach to the ligand design. Adsorption studies were performed on immobilized silica beads to determine the static binding capacity of IgM as well as the competitive adsorption isotherms in the presence of IgG and albumin. In these studies, a polyethylene glycol (PEG) based linker was used as a spacer arm between the silica beads and the ligands. The hydrophilic PEG linker reduced the non-specific binding of proteins to the silica surface and enhanced the binding of IgM to the ligands by reducing the steric hindrance. In addition, the stability of these ligands when exposed to cleaning and sanitizing agents was also validated.