Degradation of therapeutic proteins: Screening methods and identification of epimerized amino acids and local conformational changes in light exposed proteins
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Issue Date
2017-12-31Author
Bommana, Rupesh
Publisher
University of Kansas
Format
234 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Pharmaceutical Chemistry
Rights
Copyright held by the author.
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Show full item recordAbstract
Protein-based pharmaceuticals are a fast growing class of therapeutics, which are widely used in the treatment of various diseases such as cancers and autoimmune diseases. Proteins are susceptible to multiple degradation pathways including oxidation, deamidation and photodegradation. Over the past decade, the pharmaceutical industry has become increasingly cognizant of the sensitivity of proteins towards light. The exposure of proteins to light during their development and patient administration is inevitable, and may result in protein degradation. In the recent past there have been multiple reports indicating formation of aggregates, discoloration, oxidation, covalent crosslinking and fragmentation on exposure of protein pharmaceuticals to light. These degradation pathways could enhance immunogenicity or cause inactivation. Therefore, to increase the stability of these proteins, an understanding of their degradation is necessary. The research covered in this dissertation focused on two major degradation mechanisms for an IgG monoclonal antibody (mAb): chemical and physical degradation by photo irradiation. First, this dissertation explored the effects of certain pharmaceutical excipients on the extent and site-specificity of epimerization in UV irradiated mAb formulations. Amino acid analysis of UV irradiated mAb revealed formation of D-Ala, D-Glu, and D-Val in mAb formulations. Secondly, the underlying mechanism behind the increased aggregation propensity of a photo irradiated IgG1 mAb was investigated using hydrogen deuterium exchange mass spectrometry and biophysical characterization. Specific correlations were established between changes in dynamics of distinct segments in the CH2 domain of the IgG1 mAb and decreased thermal stability as well as increase in aggregation propensity of the IgG1 mAb on light exposure. This dissertation also describes development of a rapid screening methodology to identify protein oxidation by monitoring Tyr and Phe oxidation using fluorogenic derivatization. The Fluorogenic derivatization technique was adapted to a 96-well plate, in which several protein formulations can be screened in a short time. In addition, this dissertation also outlines the capability of an extreme ultra pressure liquid chromatography system in investigating complex chemical degradation problems.
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