Combined Approach to the Analysis of Recombinant Protein Drugs Using Hollow-Fiber Flow Field-Flow Fractionation, Mass Spectrometry, and Chemiluminescence Detection - Analytical Methods & Tools for Lyophilization

Combined Approach to the Analysis of Recombinant Protein Drugs Using Hollow-Fiber Flow Field-Flow Fractionation, Mass Spectrometry, and Chemiluminescence Detection
Received for review June 28, 2005. Accepted December 1, 2005.
Web Release Date: January 7, 2006
Aldo Roda,* Daniela Parisi, and Massimo Guardigli
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, Via Massarenti 9, 40138 Bologna, Italy
Andrea Zattoni and Pierluigi Reschiglian
Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi 2, 40126 Bologna, Italy
Anal. Chem.
ACS Publications
Copyright © 2006 American Chemical Society
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.
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