title: Principles and applications of steric exclusion chromatography.
abstract: We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9×10(12) plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60 mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30 min to 2 h depending on the product concentration in the feed stream.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Lee
authors.forename: Hui Theng
authors.initials: HT
authors.lastname: Gan
authors.forename: Sarah Maria Abdul
authors.initials: SM
authors.lastname: Latiff
authors.forename: Cindy
authors.initials: C
authors.lastname: Chuah
authors.forename: Wan Yee
authors.initials: WY
authors.lastname: Lee
authors.forename: Yuan-Sheng
authors.initials: YS
authors.lastname: Yang
authors.forename: Bernard
authors.initials: B
authors.lastname: Loo
authors.forename: Say Kong
authors.initials: SK
authors.lastname: Ng
authors.forename: Pete
authors.initials: P
authors.lastname: Gagnon
mesh_terms: D000818:Animals; D017104:Bacteriophage M13 / Q000302:isolation & purification; D002478:Cells, Cultured; D002850:Chromatography, Gel / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000821:virology; D057927:Hydrophobic and Hydrophilic Interactions; D007075:Immunoglobulin M / Q000302:isolation & purification; D008322:Mammals; D008968:Molecular Conformation; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D011109:Polymethacrylic Acids
chemical_list: D007075:Immunoglobulin M; D011109:Polymethacrylic Acids; C030613:polymethacrylic acid; D011092:Polyethylene Glycols