title: Use of ion-exchange chromatography and hydrophobic interaction chromatography in the preparation and recovery of polyethylene glycol-linked proteins.
abstract: Cation- and anion-exchange chromatography can be used to purify a polyethylene glycol-linked protein dimer (PEG dimer) made with M, 20 000 PEG bis-vinylsulfone, even when there are no net charge differences between the components that are being separated. The retention time on ion-exchange generally is inversely proportional to the PEG:protein ratio (on a mass basis). One of the biggest challenges in developing the process for making this PEG dimer was the quality of the PEG linker. Reversed-phase HPLC can be used to determine both size heterogeneity and the degree of end-group activation of Mr 20 000 PEG bis-vinylsulfone. In addition, we have found that hydrophobic interaction chromatography can be used make more size homogeneous preparations of Mr 20000 PEG bis-vinylsulfone, which significantly increased the recovery of the PEG dimer.
authors.affiliation: Amgen Colorado Process Development, Boulder 80301, USA. jseely@amgen.com
authors.forename: J E
authors.initials: JE
authors.lastname: Seely
authors.forename: C W
authors.initials: CW
authors.lastname: Richey
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D019281:Dimerization; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols