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Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels

Dainiak, Maria LU ; Kumar, Ashok LU ; Plieva, Fatima LU ; Galaev, Igor LU and Mattiasson, Bo LU (2004) In Journal of Chromatography A 1045(1-2). p.93-98
Abstract
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 mum) 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... (More)
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 mum) 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. (C) 2004 Published by Elsevier B.V. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cryogels, Antibodies, Proteins, Dimethylacrylamide
in
Journal of Chromatography A
volume
1045
issue
1-2
pages
93 - 98
publisher
Elsevier
external identifiers
  • wos:000223212000011
  • pmid:15378883
  • scopus:3843130508
ISSN
0021-9673
DOI
10.1016/j.chroma.2004.06.029
language
English
LU publication?
yes
id
af7e72c3-bdea-402e-a55e-9d0f39713ee2 (old id 140600)
date added to LUP
2007-07-02 09:35:46
date last changed
2017-11-19 04:05:22
@article{af7e72c3-bdea-402e-a55e-9d0f39713ee2,
  abstract     = {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 mum) 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. (C) 2004 Published by Elsevier B.V.},
  author       = {Dainiak, Maria and Kumar, Ashok and Plieva, Fatima and Galaev, Igor and Mattiasson, Bo},
  issn         = {0021-9673},
  keyword      = {Cryogels,Antibodies,Proteins,Dimethylacrylamide},
  language     = {eng},
  number       = {1-2},
  pages        = {93--98},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels},
  url          = {http://dx.doi.org/10.1016/j.chroma.2004.06.029},
  volume       = {1045},
  year         = {2004},
}