Roda Aldo, Parisi Daniela, Guardigli Massimo, Zattoni Andrea, Reschiglian Pierluigi
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Anal Chem. 2006 Feb 15;78(4):1085-92. doi: 10.1021/ac0511492.
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.
大规模重折叠过程生产的重组蛋白药物中存在的杂质不仅会影响产品安全性，还会与表达的蛋白质相互作用。为了将杂质谱与蛋白质药物的构象和功能联系起来，应优先选择不会降解样品成分的分析方法。在这项工作中，通过将中空纤维流动场-流分级分离与基质辅助激光解吸-电离飞行时间质谱（MALDI/TOFMS）以及化学发光酶活性测定相结合，对在酿酒酵母中表达的来自黄曲霉的尿酸氧化酶（尿酸酶）药物和在大肠杆菌中表达的来自球形假丝酵母的试剂级尿酸酶进行了分析。通过常规方法如配备电喷雾电离四极杆-TOF MS的反相高效液相色谱以及结合SDS-PAGE和二维SDS-PAGE的MALDI/TOFMS对样品杂质进行初步检测和鉴定。结果表明，从不同微生物获得的重组尿酸酶样品具有不同的杂质和不同的酶活性，并且溶液中存在不同的尿酸酶寡聚体。
