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dc.contributor.authorMozziconacci, Olivier
dc.contributor.authorStobaugh, Jordan T.
dc.contributor.authorBommana, Rupesh
dc.contributor.authorWoods, Joshua
dc.contributor.authorFranklin, Edward
dc.contributor.authorJorgenson, James W.
dc.contributor.authorForrest, M. Laird
dc.contributor.authorSchöneich, Christian
dc.contributor.authorStobaugh, John F.
dc.date.accessioned2021-10-05T20:36:39Z
dc.date.available2021-10-05T20:36:39Z
dc.date.issued2017-07-04
dc.identifier.citationMozziconacci, O., Stobaugh, J. T., Bommana, R., Woods, J., Franklin, E., Jorgenson, J. W., … Stobaugh, J. F. (2017). Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-μm Particles. Chromatographia, 80(9), 1299–1318. doi:10.1007/s10337-017-3344-9en_US
dc.identifier.urihttp://hdl.handle.net/1808/31926
dc.description.abstractIn recent years, protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics, a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficulty to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently, we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kpsi range, accommodates the use of capillary columns in excess of 1 m packed with sub-2-µm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular, one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at a higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.en_US
dc.publisherSpringeren_US
dc.rights© 2017, Springer Natureen_US
dc.subjectPhotochemical mediated protein degradationen_US
dc.subjectXUPLCen_US
dc.subjectMeter capillary columnsen_US
dc.subjectSub-2 μm particlesen_US
dc.titleProfiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography–Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particlesen_US
dc.typeArticleen_US
kusw.kuauthorMozziconacci, Olivier
kusw.kuauthorBommana, Rupesh
kusw.kuauthorWoods, Joshua
kusw.kuauthorForrest, M. Laird
kusw.kuauthorSchöneich, Christian
kusw.kuauthorStobaugh, John F.
kusw.kudepartmentDepartment of Pharmaceutical Chemistryen_US
dc.identifier.doi10.1007/s10337-017-3344-9en_US
kusw.oaversionScholarly/refereed, author accepted manuscripten_US
kusw.oapolicyThis item meets KU Open Access policy criteria.en_US
dc.identifier.pmidPMC6346430en_US
dc.rights.accessrightsopenAccessen_US


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