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Supermacroporous cryogel matrix for integrated protein isolation - Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

Kumar, Ashok LU ; Bansal, V ; Andersson, Jonatan LU ; Roychoudhury, PK and Mattiasson, Bo LU (2006) In Journal of Chromatography A 1103(1). p.35-42
Abstract
A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the... (More)
A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column. (c) 2005 Elsevier B.V. All rights reserved. (Less)
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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
gel filtration, supermacroporous cryogels, process integration, animal cell culture, IMAC purification, urokinase
in
Journal of Chromatography A
volume
1103
issue
1
pages
35 - 42
publisher
Elsevier
external identifiers
  • wos:000234692100006
  • pmid:16368104
  • scopus:29844445608
ISSN
0021-9673
DOI
10.1016/j.chroma.2005.08.094
language
English
LU publication?
yes
id
d2d2822e-3271-4ab1-92e6-450ea913a882 (old id 419796)
date added to LUP
2016-04-01 15:25:01
date last changed
2021-08-25 01:06:27
@article{d2d2822e-3271-4ab1-92e6-450ea913a882,
  abstract     = {A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column. (c) 2005 Elsevier B.V. All rights reserved.},
  author       = {Kumar, Ashok and Bansal, V and Andersson, Jonatan and Roychoudhury, PK and Mattiasson, Bo},
  issn         = {0021-9673},
  language     = {eng},
  number       = {1},
  pages        = {35--42},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Supermacroporous cryogel matrix for integrated protein isolation - Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line},
  url          = {http://dx.doi.org/10.1016/j.chroma.2005.08.094},
  doi          = {10.1016/j.chroma.2005.08.094},
  volume       = {1103},
  year         = {2006},
}