Affinity Chromatographic Purification of Human Immunoglobulins by Hexamer Peptide Ligands

Antibodies are highly specific, naturally evolved molecules that recognize and eliminate pathogenic and disease antigens. The past 30 years of research have proven their potential as therapeutic agents to fight cancer, autoimmune diseases and infections. Today, antibody-based therapeutics account for one third of all new treatments in USA. The world's therapeutic monoclonal antibodies market exceeded ;A family of linear hexamer peptides, exhibiting the ability to recognize and bind immunoglobulin G (IgG) through its Fc portion, has been previously identified through a three-step screening of a synthetic solid phase combinatorial peptide library. As a family, all three peptide ligands HWRGWV, HYFKFD and HFRRHL have the ability to recognize both human IgA and IgM and have the potential for large-scale purification of hIgA and hIgM.;Hexamer peptide ligand HWRGWV demonstrates the strongest binding affinity to hIgM, followed by hIgA and hIgG, respectively. Relatively high recovery (> 90%) can be achieved for hIgG by using an elution pH larger than 4.0; however, high recovery (> 90%) can only be achieved for hIgA and hIgM by using elution pH lower than 4.0. N-terminal acetylation of the ligand would decrease the binding of hIgA and hIgM since electrostatic interactions play a role in hIgA and hIgM binding to the peptide. At low peptide density, salting-in salts such as MgCl2 and CaCl2 and non-electrolytes such as ethylene glycol, urea and arginine can be used as elution additives to facilitate hIgA and hIgM elution.;Hexamer peptide ligands demonstrate the ability to purify hIgA and hIgM from complex media, mammalian cell culture supernatants as well as Cohn fraction II/III. For antibody purification from cMEM with peptide ligand HWRGWV at four different peptide densities (0.04, 0.11, 0.22 and 0.55 mequiv./g), the improved recovery at higher peptide density due to increased binding affinity was compensated by the decrease in purity for all three antibodies. Over 80% recovery and 90% purity were achieved for hIgG and hIgA at peptide densities of 0.11 and 0.22 mequiv./g. For hIgM, 75.7% recovery and 86% purity were achieved at peptide density of 0.04 mequiv./g. For antibody purification from cMEM with other ligands HYFKFD and HFRRHL at peptide density of 0.11 mequiv./g, higher purities but lower recoveries were achieved with HYFKFD compared to HFRRHL, suggesting better binding specificity and possible lower dynamic binding capacity. About 95% recoveries and 90% purities were achieved for human IgA and secretory IgA purified from spiked Chinese hamster ovary cell culture supernatants using peptide ligand HWRGWV at peptide density of 0.11 mequiv./g. For purification of hIgM from spiked human B lymphocyte cell culture supernatants using peptide ligand HWRGWV at peptide density of 0.04 mequiv./g, the final recovery and purity of the antibody is feedstock dependent, but can reach levels of both recovery and purity as high as 95%. Because of the binding affinity difference, one-column purification of hIgA, hIgG and hIgM from Cohn fraction II/III was achieved. After pretreatment with caprylic acid precipitation or the combination of caprylic acid and polyethylene glycol precipitation, highly purified (> 95%) hIgG was obtained with hIgA enriched fraction and hIgM enriched fraction as by-products.;This work demonstrates hexamer peptide ligands, initially screened from a solid phase combinatorial peptide library for IgG purification; also have the potential for large-scale purification of human IgA and IgM.