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Author ORCID Identifier



Open Access Dissertation

Document Type


Degree Name

Doctor of Philosophy (PhD)

Degree Program


Year Degree Awarded


Month Degree Awarded


First Advisor

Igor A. Kaltashov

Second Advisor

Richard W. Vachet

Third Advisor

Paul L. Dubin

Fourth Advisor

Stephen J. Eyles

Subject Categories

Analytical Chemistry


Biotherapeutics, an emerging class of medicines containing biopolymers (e.g., proteins, peptide, and polysaccharides) have been developed for a variety of indications including cancer, autoimmune, genetic, and blood diseases. Among these biopolymers protein therapeutics have been the rapidly growing segment in the pharmaceutical industry. This trend combined with the complexity of proteins has necessitated the development of powerful and robust analytical methods to study their structure and interactions with physiological partners. Mass spectrometry (MS) has become an indispensable tool to analyze various attributes of protein drugs such as profiling of intact mass, amino acid sequencing, and post translational modifications (PTMs). In this work novel analytical methods have been developed by combining non-denaturing chromatographic separations (such as size exclusion chromatography and ion exchange chromatography) with native ESI MS. In addition to providing information of mass and non-covalent assemblies, native ESI MS has an advantage of probing conformational integrity of proteins by analyzing ionic charge state distributions in the mass spectrum. Size exclusion chromatography with online native ESI MS detection method developed in this work allowed characterization of higher order structure of proteins and probe aggregation propensity. Using the same SEC/native ESI MS workflow a method to find kinetics and equilibrium binding constant of transient protein interactions have also been developed. Combination of ion exchange chromatography (IXC) with native ESI MS and MS/MS detection was developed and shown to be effective in characterization of positional isomers of protein conjugates and PTMs of a biotherapeutic recombinant human interferon beta 1a. To characterize highly heterogeneous PEGylated glycoprotein (conjugated with a 20kDa PEG) analytical methods using IXC/native ESI MS combined with limited charge reduction and collisional activation were developed.