High speed immuno-affinity chromatography on supports with gigapores and porous glass.
Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.
Schuste
M
M
Novartis Forschungsinstitut, Vienna, Austria.
Wasserbauer
E
E
Neubauer
A
A
Jungbauer
A
A
D002846:Chromatography, Affinity / Q000379:methods*; D005898:Glass; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*; D012709:Serum Albumin / Q000276:immunology / Q000302:isolation & purification*
D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C085706:FLAG peptide