The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.

A. Roda, D.Parisi, M. Guardigli, A.Zattoni, P. Reschiglian (2006). Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry and chemiluminescence detection. ANALYTICAL CHEMISTRY, 78, 1085-1092 [10.1021/ac0511492].

Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry and chemiluminescence detection

RODA, ALDO;PARISI, DANIELA;GUARDIGLI, MASSIMO;ZATTONI, ANDREA;RESCHIGLIAN, PIERLUIGI
2006

Abstract

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.
2006
A. Roda, D.Parisi, M. Guardigli, A.Zattoni, P. Reschiglian (2006). Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry and chemiluminescence detection. ANALYTICAL CHEMISTRY, 78, 1085-1092 [10.1021/ac0511492].
A. Roda; D.Parisi; M. Guardigli; A.Zattoni; P. Reschiglian
File in questo prodotto:
Eventuali allegati, non sono esposti

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/24697
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 0
  • Scopus 30
  • ???jsp.display-item.citation.isi??? 27
social impact