title: Hydrophobic interaction chromatography for purification of monoPEGylated RNase A.
abstract: The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ~85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.
authors.affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey, Monterrey, NL 64849, Mexico.
authors.forename: Karla
authors.initials: K
authors.lastname: Mayolo-Deloisa
authors.forename: Ma Elena
authors.initials: ME
authors.lastname: Lienqueo
authors.forename: Barbara
authors.initials: B
authors.lastname: Andrews
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
authors.forename: Juan A
authors.initials: JA
authors.lastname: Asenjo
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D011092:Polyethylene Glycols / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D011092:Polyethylene Glycols; C020927:phenyl-sepharose; C052754:butylagarose; D012685:Sepharose; D012259:Ribonuclease, Pancreatic; D000645:Ammonium Sulfate