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Combined effects of potassium chloride and ethanol as mobile phase modulators on hydrophobic interaction and reversed-phase chromatography of three insulin variants

Johansson, Karolina LU ; Frederiksen, Søren S.; Degerman, Marcus; Breil, Martin P.; Mollerup, Jørgen M. and Nilsson, Bernt LU (2015) In Journal of Chromatography A 1381. p.64-73
Abstract (Swedish)
Abstract in Undetermined

The two main chromatographic modes based on hydrophobicity, hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC), are widely used for both analytical and preparative chromatography of proteins in the pharmaceutical industry. Despite the extensive application of these separation methods, and the vast amount of studies performed on HIC and RPC over the decades, the underlying phenomena remain elusive. As part of a systematic study of the influence of mobile phase modulators in hydrophobicity-based chromatography, we have investigated the effects of both KCl and ethanol on the retention of three insulin variants on two HIC adsorbents and two RPC adsorbents. The focus was... (More)
Abstract in Undetermined

The two main chromatographic modes based on hydrophobicity, hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC), are widely used for both analytical and preparative chromatography of proteins in the pharmaceutical industry. Despite the extensive application of these separation methods, and the vast amount of studies performed on HIC and RPC over the decades, the underlying phenomena remain elusive. As part of a systematic study of the influence of mobile phase modulators in hydrophobicity-based chromatography, we have investigated the effects of both KCl and ethanol on the retention of three insulin variants on two HIC adsorbents and two RPC adsorbents. The focus was on the linear adsorption range, separating the modulator effects from the capacity effects, but some complementary experiments at higher load were included to further investigate observed phenomena. The results show that the modulators have the same effect on the two RPC adsorbents in the linear range, indicating that the modulator concentration only affects the activity of the solute in the mobile phase, and not that of the solute-ligand complex, or that of the ligand. Unfortunately, the HIC adsorbents did not show the same behavior. However, the insulin variants displayed a strong tendency toward self-association on both HIC adsorbents; on one in particular. Since this causes peak fronting, the retention is affected, and this could probably explain the lack of congruity. This conclusion was supported by the results from the non-linear range experiments which were indicative of double-layer adsorption on the HIC adsorbents, while the RPC adsorbents gave the anticipated increased tailing at higher load. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Hydrophobic interaction chromatography, Reversed-phase chromatography, Insulin, Combined modulator effect, Salting-in effect, Salting-out effect
in
Journal of Chromatography A
volume
1381
pages
64 - 73
publisher
Elsevier
external identifiers
  • pmid:25595534
  • wos:000349590600008
  • scopus:84922782985
ISSN
0021-9673
DOI
10.1016/j.chroma.2014.12.081
language
English
LU publication?
yes
id
d107f00f-6928-4c4e-a98f-549affc0040e (old id 5035274)
date added to LUP
2015-01-30 08:47:47
date last changed
2017-09-24 03:54:49
@article{d107f00f-6928-4c4e-a98f-549affc0040e,
  abstract     = {<b>Abstract in Undetermined</b><br/><br>
The two main chromatographic modes based on hydrophobicity, hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC), are widely used for both analytical and preparative chromatography of proteins in the pharmaceutical industry. Despite the extensive application of these separation methods, and the vast amount of studies performed on HIC and RPC over the decades, the underlying phenomena remain elusive. As part of a systematic study of the influence of mobile phase modulators in hydrophobicity-based chromatography, we have investigated the effects of both KCl and ethanol on the retention of three insulin variants on two HIC adsorbents and two RPC adsorbents. The focus was on the linear adsorption range, separating the modulator effects from the capacity effects, but some complementary experiments at higher load were included to further investigate observed phenomena. The results show that the modulators have the same effect on the two RPC adsorbents in the linear range, indicating that the modulator concentration only affects the activity of the solute in the mobile phase, and not that of the solute-ligand complex, or that of the ligand. Unfortunately, the HIC adsorbents did not show the same behavior. However, the insulin variants displayed a strong tendency toward self-association on both HIC adsorbents; on one in particular. Since this causes peak fronting, the retention is affected, and this could probably explain the lack of congruity. This conclusion was supported by the results from the non-linear range experiments which were indicative of double-layer adsorption on the HIC adsorbents, while the RPC adsorbents gave the anticipated increased tailing at higher load.},
  author       = {Johansson, Karolina and Frederiksen, Søren S. and Degerman, Marcus and Breil, Martin P. and Mollerup, Jørgen M. and Nilsson, Bernt},
  issn         = {0021-9673},
  keyword      = {Hydrophobic interaction chromatography,Reversed-phase chromatography,Insulin,Combined modulator effect,Salting-in effect,Salting-out effect},
  language     = {eng},
  pages        = {64--73},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Combined effects of potassium chloride and ethanol as mobile phase modulators on hydrophobic interaction and reversed-phase chromatography of three insulin variants},
  url          = {http://dx.doi.org/10.1016/j.chroma.2014.12.081},
  volume       = {1381},
  year         = {2015},
}