(312a) One Step Purification of Human Immunoglobulins A, G and M by Small Peptide Affinity Chromatography

Authors: 
Gurgel, P. V., North Carolina State University
Liu, Z., North Carolina State University
Carbonell, R. G., Kenan Institute for Engineering, Technology & Science, NC State University


The small peptide ligand HWRGWV, exhibiting the ability to recognize and bind human immunoglobulin G (hIgG) through its Fc portion, has been previously identified through a three-step screening of a synthetic solid-phase peptide ligand library. Preliminary investigations suggest that the peptide ligand has high binding affinity for human immunoglobulin M (hIgM), and human immunoglobulin A (hIgA) as well as human immunoglobulin G (hIgG). Different elution additives were employed for the elution of bound antibodies at peptide densities of 0.04 and 0.55 mequiv/g. For electrolytes, at low peptide density, salting-in salts such as MgCl2 and CaCl2 facilitate the elution due to favorable interaction with the proteins resulting in higher recoveries. On the other hand, salting-out salts such as NaCl increase the specific binding between antibodies and the peptide ligand leading to lower recoveries. At high peptide density, the recovery decrease or remains low no matter which salt was used because of the increased non-specific hydrophobic interaction. For non-electrolytes, at low peptide density, ethylene glycol, urea and arginine facilitate the elution because of their ability to decrease hydrophobic interaction, hydrogen bonding and electrostatic interactions. At high peptide density, the additives almost had no effect on elution because the decreased interactions could not overcome the increased non-specific hydrophobic interaction. Based on the different elution conditions for human immunoglobulins A, G and M; one-step purification scheme for human immunoglobulins A, G and M mixture using the small peptide ligand HWRGWV is proposed in order to separate these three components from Cohn Fraction II + III Paste.

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