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Novel in situ polymerized coatings for hydrophobic interaction chromatography media

Fexby, Sara LU ; Ihre, Henrik; Bülow, Leif LU and Van Alstine, James M. (2007) In Journal of Chromatography A 1161(1-2). p.234-241
Abstract
Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(D polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl... (More)
Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(D polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, (x-chymotrypsinogen A, myoglobin and (x-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70 mg protein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (Y-PYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media. (c) 2007 Published by Elsevier B.V. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
green fluorescent protein, hydrophobic interaction chromatography, polymer coating, sepharose
in
Journal of Chromatography A
volume
1161
issue
1-2
pages
234 - 241
publisher
Elsevier
external identifiers
  • wos:000248891500030
  • scopus:34447632993
ISSN
0021-9673
DOI
10.1016/j.chroma.2007.05.086
language
English
LU publication?
yes
id
8fc54303-9b88-4992-abe3-cf2882d05a2a (old id 688961)
date added to LUP
2007-12-11 09:47:39
date last changed
2017-01-01 07:16:42
@article{8fc54303-9b88-4992-abe3-cf2882d05a2a,
  abstract     = {Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(D polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, (x-chymotrypsinogen A, myoglobin and (x-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70 mg protein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (Y-PYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media. (c) 2007 Published by Elsevier B.V.},
  author       = {Fexby, Sara and Ihre, Henrik and Bülow, Leif and Van Alstine, James M.},
  issn         = {0021-9673},
  keyword      = {green fluorescent protein,hydrophobic interaction chromatography,polymer coating,sepharose},
  language     = {eng},
  number       = {1-2},
  pages        = {234--241},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Novel in situ polymerized coatings for hydrophobic interaction chromatography media},
  url          = {http://dx.doi.org/10.1016/j.chroma.2007.05.086},
  volume       = {1161},
  year         = {2007},
}