Alkaline-stable peptide ligand affinity adsorbents for the purification of biomolecules.
A strategy of modification of resin surface chemistry is presented to produce hydrophilic peptide-based alkaline-stable affinity adsorbents for the purification of biopharmaceuticals from complex media. In this work, the peptide-based affinity adsorbent HWRGWV-Toyopearl resin for the purification of IgG is presented as an example. When prepared by direct peptide synthesis on the chromatographic matrix, the peptide-based resin showed lability under alkaline conditions. In fact, the regeneration with aqueous 0.1 M NaOH caused the leaching of 40% of the peptide ligand, resulting in a decrease of IgG yield from 85% to 23%. It was found that the ligand leaching was caused by the coupling of a significant amount of peptide by alkaline-labile ester bonds. A method was designed to prevent the formation of ester bonds and allow the synthesis of the ligand exclusively on alkaline-stable bonds. The method consists in activating the hydrophilic base resin, blocking the hydroxyl groups responsible for alkaline lability and performing the peptide synthesis exclusively via alkaline-stable amide bonds. Repeated cycles of IgG purification from a cell culture medium were performed, each followed by cleaning with aqueous NaOH (0.1 M, 0.5 M and 1 M). The IgG yield decreased from 91% to 85% after 200 purification cycles with 0.1 M NaOH. However, the IgG purity remained almost constant at around 95% based on SDS-PAGE analysis. The procedure presented is rapid, efficient and inexpensive and does not require any equipment other than the conventional instrumentation for peptide synthesis. The method also has a broad application since it is valid for any peptide ligand identified for the purification of a biopharmaceutical target.
Department of Chemical and Biomolecular Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Partner's Building I, Suite 3200, Box 7006, Raleigh, NC 27695-7565, USA.
Stefano
S
Menegatti
Amith D
AD
Naik
Patrick V
PV
Gurgel
Ruben G
RG
Carbonell
D000327:Adsorption; D000468:Alkalies / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding
D000468:Alkalies; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides