title: Ion exchange chromatography of antibody fragments.
abstract: Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.
authors.affiliation: GE Healthcare, Bio-Sciences AB, SE-751 84 Uppsala, Sweden. Anders.Ljunglof@ge.com
authors.forename: Anders
authors.initials: A
authors.lastname: Ljunglöf
authors.forename: Karol M
authors.initials: KM
authors.lastname: Lacki
authors.forename: Jay
authors.initials: J
authors.lastname: Mueller
authors.forename: Chithkala
authors.initials: C
authors.lastname: Harinarayan
authors.forename: Robert
authors.initials: R
authors.lastname: van Reis
authors.forename: Robert
authors.initials: R
authors.lastname: Fahrner
authors.forename: James M
authors.initials: JM
authors.lastname: Van Alstine
mesh_terms: D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry*; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D000837:Anion Exchange Resins; D002411:Cation Exchange Resins; D007140:Immunoglobulin Fab Fragments