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Ion exchange chromatography of antibody fragments
Author(s) -
Ljunglöf Anders,
Lacki Karol M.,
Mueller Jay,
Harinarayan Chithkala,
van Reis Robert,
Fahrner Robert,
Van Alstine James M.
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21124
Subject(s) - chemistry , chromatography , sepharose , adsorption , agarose , confocal laser scanning microscopy , conductivity , ion chromatography , analytical chemistry (journal) , ion exchange , particle size , particle (ecology) , size exclusion chromatography , ion , phase (matter) , confocal , biophysics , biochemistry , organic chemistry , oceanography , geometry , mathematics , geology , biology , enzyme
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 Sepharose™ 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. Biotechnol. Bioeng. 2007;96: 515–524. © 2006 Wiley Periodicals, Inc.