Detector Considerations in Developing of Performance Qualification Criteria for HPLC Systems Used for Peptide Mapping

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Thomas E. Wheat, Beth Gillece-Castro, Ziling Lu, Uwe D. Neue, and Jeffrey R. Mazzeo [Waters]
WCBP, January 24-27, 2006
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Peptide mapping is a particularly important tool for the characterization of therapeutic proteins. When this technique is used in a validated analytical method, it is necessary to characterize the system and to apply appropriate qualification procedures. A performance qualification test based on peptide analysis is required for predicting system performance with real samples. Such qualification protocols, however, only apply to a single instrument configuration. It is common practice, however, to use different detectors for specific purposes at different stages of development. We have, therefore, established performance standards for reversed-phase HPLC in conjunction with both UV and MS detection for systems dedicated to running peptide maps. The qualified optical detectors include tunable UV and photodiode array. The mass spectrometers include single quadrupole, oa-Tof, and tandem quadrupole-oa-Tof. The criteria and specifications include retention time reproducibility and chromatographic selectivity. Mass accuracy was included for MS configurations. Since injector reproducibility and injector linearity can only be judged in conjunction with the selected detector, separate specification criteria were defined were each of the five detectors. The combinations of optical and mass detectors were also tested. This qualification method is based the separation of a well-characterized peptide analyte mix on a tested HPLC column specific for peptide analysis. The consistent separation chemistry and test materials ensure that the test may be efficiently adapted to the selected detector. The developed procedure could also be used as a routine system suitability test

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