Development of a Microfluidic Capillary Electrophoresis-Mass Spectrometry Platform for the Characterization of Biotherapeutic Proteins Public Deposited

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  • March 21, 2019
Creator
  • Redman, Erin
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • This work describes the development of a mass spectrometry (MS) compatible microfluidic capillary electrophoresis (CE) analysis platform capable of characterizing large biomolecules. Initial efforts focused on developing a method for performing highly efficient CE separations of intact proteins with on-line MS analysis. Surface coating technology was optimized for the analysis of intact proteins. The ability to reproducibly generate uniform surface coatings for CE separations of biomolecules was paramount to achieving efficient separations of these large molecules. The effectiveness of the intact protein coating was demonstrated by analyzing whole blood lysate. The microfluidic CE-MS method proved to be a simple and rapid way to assess hemoglobin glycation and the results compared well with a commercially available technique used to measure glycated hemoglobin. Further application of this technology involved characterizing monoclonal antibody (mAb) based biotherapeutics at the intact level. The size and complexity of these molecules makes them difficult to analyze at the intact level. It was determined that maintaining some of the structural conformation of the mAbs was vital to achieving separation of intact variants. Thus, a background electrolyte was optimized to balance preserving antibody conformation and maximizing MS signal. In conjunction with the optimized surface chemistry, this resulted in the first MS compatible liquid phase separation of intact charge variants of mAbs. This technology was then adapted to perform other levels of antibody characterization. Both limited proteolysis and disulfide bond reduction were evaluated for a middle-up analysis of mAbs. Analysis of mAb fragments generated through disulfide bond reduction resulted in more reliable characterization of the molecules analyzed. Although the surface chemistry described in this work was developed for analyzing intact proteins, it has also proven effective for analyzing smaller molecules. The technology was used to perform bottom-up mapping experiments to generate in-depth information about biomolecules. It was determined that in many cases the resulting microfluidic CE-MS data was comparable to that achieved through LC-MS analysis, but with significantly shorter analysis times. The overall result of this work is a single microfluidic CE-MS analysis platform that is capable of rapidly performing multiple levels of protein characterization.
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Rights statement
  • In Copyright
Advisor
  • Ramsey, J. Michael
  • Walker, Glenn
  • Jorgenson, James
  • Taylor, Anne
  • Hicks, Leslie
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2016
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