Advanced

A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.

Jespersen, Gry LU ; Matthiesen, Finn; Pedersen, Anja Kallesøe; Andersen, Henrik Sune; Kirsebom, Harald LU and Nielsen, Anders Lærke (2014) In Journal of Biotechnology 173. p.76-85
Abstract
Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the... (More)
Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biotechnology
volume
173
pages
76 - 85
publisher
Elsevier
external identifiers
  • pmid:24445170
  • wos:000331207300013
  • scopus:84893344748
ISSN
1873-4863
DOI
10.1016/j.jbiotec.2013.12.015
language
English
LU publication?
yes
id
6dd75fcb-3fb5-4e69-adea-16e482452570 (old id 4291025)
date added to LUP
2014-02-24 12:17:48
date last changed
2017-01-01 03:57:17
@article{6dd75fcb-3fb5-4e69-adea-16e482452570,
  abstract     = {Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.},
  author       = {Jespersen, Gry and Matthiesen, Finn and Pedersen, Anja Kallesøe and Andersen, Henrik Sune and Kirsebom, Harald and Nielsen, Anders Lærke},
  issn         = {1873-4863},
  language     = {eng},
  pages        = {76--85},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.},
  url          = {http://dx.doi.org/10.1016/j.jbiotec.2013.12.015},
  volume       = {173},
  year         = {2014},
}