Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels.
The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 microm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)6-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques.
Dainiak
Maria B
MB
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, SE-22100 Lund, Sweden.
Kumar
Ashok
A
Plieva
Fatima M
FM
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D020106:Acrylamide / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning
D007128:Immunoglobulin Fragments; D020106:Acrylamide