Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

<h4>Background</h4>Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by S...

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Autores principales:	Marion Giffard, Natalie Ferté, François Ragot, Mohamed El Hajji, Bertrand Castro, Françoise Bonneté
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f1f31aa56b2f4905b134e421e9bf51c2
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spelling	oai:doaj.org-article:f1f31aa56b2f4905b134e421e9bf51c22021-11-18T06:54:09ZUrate oxidase purification by salting-in crystallization: towards an alternative to chromatography.1932-620310.1371/journal.pone.0019013https://doaj.org/article/f1f31aa56b2f4905b134e421e9bf51c22011-05-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21589929/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions.<h4>Methodology/principal findings</h4>Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible.<h4>Conclusions</h4>The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.Marion GiffardNatalie FertéFrançois RagotMohamed El HajjiBertrand CastroFrançoise BonnetéPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 5, p e19013 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marion Giffard Natalie Ferté François Ragot Mohamed El Hajji Bertrand Castro Françoise Bonneté Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
description	<h4>Background</h4>Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions.<h4>Methodology/principal findings</h4>Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible.<h4>Conclusions</h4>The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.
format	article
author	Marion Giffard Natalie Ferté François Ragot Mohamed El Hajji Bertrand Castro Françoise Bonneté
author_facet	Marion Giffard Natalie Ferté François Ragot Mohamed El Hajji Bertrand Castro Françoise Bonneté
author_sort	Marion Giffard
title	Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
title_short	Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
title_full	Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
title_fullStr	Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
title_full_unstemmed	Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
title_sort	urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/f1f31aa56b2f4905b134e421e9bf51c2
work_keys_str_mv	AT mariongiffard urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography AT natalieferte urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography AT francoisragot urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography AT mohamedelhajji urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography AT bertrandcastro urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography AT francoisebonnete urateoxidasepurificationbysaltingincrystallizationtowardsanalternativetochromatography
_version_	1718424265881026560

Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

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