title: Separation of protein charge variants by ultrafiltration.
abstract: The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine epsilon-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.
authors.affiliation: Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Mareia Frost
authors.initials: MF
authors.lastname: Ebersold
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Zydney
mesh_terms: D045424:Complex Mixtures / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification*; D004563:Electrochemistry / Q000379:methods*; D005240:Feasibility Studies; D009211:Myoglobin / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D055672:Static Electricity; D014462:Ultrafiltration / Q000379:methods*
chemical_list: D045424:Complex Mixtures; D009211:Myoglobin; D011506:Proteins