IMMUNOGLOBULIN PURIFICATION BY AFFINITY CHROMATOGRAPHY USING PROTEIN A MIMETIC LIGANDS PREPARED BY COMBINATORIAL CHEMICAL SYNTHESIS

Abstract

Affinity chromatography using protein A from Staphylococcus aureus as the ligand has been widely used for the isolation of immunoglobulin G (IgG) from various species. Since ligand leakage from the affinity support can occur, time consuming analytical controls are required to detect the presence of contaminants associated with the isolated IgG prior to its use for therapeutic purpose in humans. Besides, protein A is an expensive bacterial product, whose isolation involves complex and labor intensive procedures. Combinatorial chemistry enables the synthesis of a wide variety of ligands within a short period of time. Therefore, chemically defined, stable and inexpensive ligands, which can mimic the action of protein A, have been developed to isolate immunoglobulins. Two different types of ligands, synthesized following the techniques of combinatorial chemistry, have been used to isolate immunoglobulins. One of them is a synthetic peptide (TG 19318) comprising four identical tripeptide chains linked to a central polylysine core. Immobilized TG 19318 has been used to isolate polyclonal and monoclonal antibodies of different classes (IgG, IgM, IgA, IgE) from different sources (serum, ascities and cell supernatants) and species. The ligand has a binding capacity that can reach upto 25 mg IgG/mL of the support. It is stable when treated with sanitation agents such as ethanol and 0.1 M sodium hydro xide. Computer-aided molecular design and combinatorial chemistry have been used to develop an IgG binding affinity ligand (22/8), which consists of two organic aromatic amines (3-aminophenol and 4-amino-1-napthol) linked to a scaffold of cyanuric chloride (triazine). Ligand 22/8 displayed wider specificity than protein A, as it isolated IgG from a number of species, the order of adsorption being human>chicken>cow>rabbit>pig>horse>rat>goat>sheep>mouse. It showed an apparent binding capacity of 51.9 mg IgG/g moist gel and can isolate human IgG from plasma in 60% yield with a purity of 92%. The ligand is stable, as it withstood incubation in 1M NaOH for a week without loss of binding capacity for IgG. These findings suggest that synthetic affinity ligands, which are inexpensive, stable and specific can facilitate the purification of immunoglobulins in a cost-effective manner.