Thesis Lia Bersin v5 07DEC21

Reason: Some work contained in the dissertation is being prepared for journal publication.





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Pyroglutamate Formation and Hydrogen Deuterium Exchange in Lyophilized Therapeutic Proteins

posted on 09.12.2021, 01:09 by Lia Margaret BersinLia Margaret Bersin

Therapeutic proteins are vital to global health, yet they are challenging to develop due to their large size and complexity. Much about their behavior is still to be understood. The research presented in this dissertation explores chemical reactions in peptides and proteins in the solid-state in order to understand formulation and matrix properties that affect reactions in solid-state therapeutic proteins. More specifically, the work applies two reactions to study reactivity: pyroglutamate (pGlu) formation and solid-state hydrogen-deuterium exchange (ssHDX). pGlu is a chemical degradant found in peptides and proteins with either glutamate (Glu) or glutamine (Gln) at the N-terminal that can occur non-enzymatically during storage. N-terminal Glu and Gln are prevalent in monoclonal antibodies (mAbs), a growing class of biologics, thus understanding this chemical modification is relevant to the development of therapeutic proteins. ssHDX with mass spectrometric analysis (ssHDX-MS) is used as an analytic tool to provide high resolution information on protein structure, stability, and matrix interactions in solid-state peptides and proteins. In this reaction deuterium donors compete for hydrogen bonding sites, which allows interrogation of the protein hydrogen bond network. While well studied when applied to the solution state, the mechanism of exchange in the solid state is not fully understood. The research and findings can be divided into three sections described below.

The first section explores the mechanism of pGlu formation in the solution and solid states using a model peptide. pGlu formation and parent peptide loss were monitored by high performance liquid chromatography under accelerated storage conditions in lyophilize solid and solution formulations with vary ‘pH’ levels. ‘pH’ dependence in the solid state differed markedly from that in solution. Moreover, at the ‘pH’ where mAbs are often formulated the rate of pGlu formation is the solid state was greater than in solution.

The second section aims to develop formulations that inhibit pGlu formation and to identify formulation characteristic and matrix properties that are indicative of pGlu formation. Again, a model peptide was used to monitor pGlu formation in lyophilized solid and solution formulations with varied ‘pH’ and excipients stored under accelerated conditions. The results indicate that pGlu can be inhibited by low molecular weight hydrogen bonding excipients and low moisture content.

The final section aims to identify solid-state properties that affect ssHDX-MS. The effects of temperature, relative humidity (RH), and mobility on ssHDX were probed using formulations of lyophilized mAb plasticized with varying levels of glycerol. The results indicate that ssHDX kinetic parameters were influences by RH and temperature, but not glycerol level. There was a clear linear correlation with molecular mobility (T-Tg (glass transition temperature)). A first-order kinetic model was proposed that suggests a linear dependence of deuterium incorporation kinetic parameters on the product of RH and temperature, which provides a better correlation than T-Tg.

The outcomes of this dissertation provide insight into solid-state reaction behavior in peptides and proteins. They have implications for the rational design of stable formulations of therapeutic proteins by expanding our understanding of a relevant chemical instability, pGlu formation, and of a high-resolution analytical technique, ssHDX-MS, that can be used to probe solid-state proteins.


Degree Type

Doctor of Philosophy


Industrial and Physical Pharmacy

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Elizabeth M. Topp

Additional Committee Member 2

Sajal M. Patel

Additional Committee Member 3

Lynne S. Taylor

Additional Committee Member 4

Qi Tony Zhou