A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins

作者：Qian Jianing; El Khoury Graziella; Issa Hamzah; Al Qaoud Khaled; Shihab Penelope; Lowe Christopher R^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2012, 898: 15-23.

DOI：10.1016/j.jchromb.2012.03.043

• Synthetic affinity ligand
• Ugi multi-component reaction
• Mammalian immunoglobulin purification
• Camelid IgG

Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L. in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose (TM) resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C1111 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of similar to 65% purity, compared to similar to 62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1 M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg IgG ml(-1) resin and a dissociation constant (K-d) of 4.78 x 10(-6) M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.

• 出版日期2012-6-1