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Studies of Protein Stability by Hydrogen Exchange-Mass Spectrometry
Demalgiriya Gamage, Chamalee
Demalgiriya Gamage, Chamalee
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Abstract
With the rapid growth in using proteins as therapeutics, studying the stability of therapeutic proteins has become an essential part in the manufacturing of therapeutics to ensure the quality, safety, and efficacy of a drug product. Proteins can get exposed to various stresses and undergo various modifications during their production and formulation to a final drug product. These stress conditions and modifications of therapeutic proteins can lead them to degrade causing potential risks to the safety and efficacy of a drug. Evaluating the stability of therapeutic proteins to understand product liabilities is important both in discovery and development phases of a drug. It is also important to employ analytical tools that can reliably predict stability of proteins to reduce long development times. Hydrogen Exchange-Mass Spectrometry (HX-MS) is increasingly being used in pharmaceutical industry for studying protein-protein, protein-ligand, and protein-receptor interactions. However, there is much potential for using HX-MS in a variety of applications in manufacturing and development of biotherapeutics. In this dissertation work, we explored the potential of using HX-MS in studying deamidation and aggregation of proteins which are considered as major degradation pathways of therapeutic proteins. In chapter 2 and 3, we assessed the ability of using HX-MS to predict protein deamidation and understand structural dynamics of mAbs leading to interfacial mediated agitation-induced aggregation, respectively. In chapter 4, we generated a panel of model proteins that will be used to evaluate detection limits in HX-MS measurements in characterization of protein higher-order structures such as comparability studies of biosimilars.Estimating deamidation propensities of proteins is essential for predicting their long-term stabilities. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. In chapter 2, we assessed the potential of using HX-MS to predict deamidation propensities of proteins because HX-MS would offer a rapid method compared to accelerated and long-term stability studies used for deamidation predictions. Therefore, we investigated the correlation between hydrogen exchange kinetics and deamidation kinetics at deamidation sites of wild-type and mutant MBP. We observed a power law correlation between hydrogen exchange and deamidation half-lives at the NG sites of MBP. This correlation demonstrates that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins. Agitation-induced aggregation is a common issue that therapeutic protein formulations encounter during their manufacturing and transportation. Agitation increases exposure of proteins to the air-water interface and adsorption of proteins to the air-water interface induces aggregate formation. In chapter 3, we explored structural dynamics of three mAbs at the air-water interface using HX-MS to understand the structural changes of the mAbs that could lead to aggregate formation. Using three mAbs with variable propensities to form aggregates under agitation stress, we observed that all three mAbs underwent partial unfolding to variable. Among the three, the mAb which had the highest risk to aggregate unfolded extensively while showing cooperative unfolding in the Fab domain. The other two mAbs partially unfolded only in some regions of the molecules including some regions of the CDRs, but the unfolded regions were molecule specific. We also observe a protection against the partial unfolding of the molecules when the agitation stress was provided in the presence of polysorbate 20. This study provides direct evidence that local unfolding events resulting from interface exposure precede aggregate formation and may play a causal role in the process. HX-MS has proven to be a reliable method for the assessment of protein higher-order structures. However, the limit of detection in the HX-MS measurements in detecting subtle differences in higher-order structures has not been tested rigorously. With the aim of fulfilling this need, as the first step, we generated a panel of model proteins with subtle structural changes and altered function in chapter 4. Here we expressed and purified a set of mutants of VHH-F5 and a binding assay was optimized to identify the mutants with differences in relative binding affinities. The mutants with detuned binding affinities will be used to perform HX-MS experiments to identify their higher-order structural differences.
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Date
2022-05-31
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University of Kansas
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Keywords
Chemistry, Analytical chemistry, Biochemistry, Hydrogen Exchange, Mass Spectrometry, Protein Aggregation, Protein Deamidation, Protein Stability