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dc.contributor.advisorSchöneich, Christian
dc.contributor.authorPrajapati, Indira
dc.date.accessioned2021-04-25T20:36:08Z
dc.date.available2021-04-25T20:36:08Z
dc.date.issued2020-08-31
dc.date.submitted2020
dc.identifier.otherhttp://dissertations.umi.com/ku:17403
dc.identifier.urihttp://hdl.handle.net/1808/31621
dc.description.abstractProtein biotherapeutics, especially monoclonal antibodies (mAbs), have been on the rise due to their high efficacy, potency, and low toxicity. They are mainly used for the treatment of cancer, autoimmune diseases, infectious diseases, and organ transplantation. While mAbs are well-tolerated by patients and have fewer adverse effects, their stability can be challenged by physical and chemical degradation. Hence, they are formulated with buffers and other excipients such as polysorbate 80 (PS80), which help to maintain the pH of the formulation and prevent aggregation, respectively. However, when the drug product is exposed to light, heat, or contains metal impurities, they are likely to undergo degradation. Some of the reactive amino acids of the proteins such as Trp, Tyr, His, Met, and Cys residues are more prone to photooxidation. Thus, we have investigated the effect of both UV and visible light on peptides, and proteins and further looked into the role of protein- and buffer-derived radicals on degradation of PS80. We explored the role of methionine sulfur cation, generated by photosensitization of 4-carboxybenzophenone in the Met-Xn-His-containing peptides (n = 0 - 2), a common sequence present in the biotherapeutics such as mAb and human parathyroid hormone. Here, we report on the formation of novel photo-oxidation products and cross-links between Met oxidation product(s) and a neighboring histidine residue. Mechanisms for the formation of these products will be proposed. Specifically, the formation of cross-links is hypothesized to involve photo-oxidation of Met to an aspartate semialdehyde, followed by reaction with the imidazole side chain of His, and elimination of water. When a full mAbZ (mAb obtained from AstraZeneca) was exposed to visible light, discoloration of the solution was observed. The chromophoric product responsible for the color change was identified using a model Trp-containing compound, N-acetyl-L-tryptophan amide (NATA). The product was identified as NATA-33, a conjugated product formed after loss of 33 Da from NATA, by mass spectrometry and NMR. The mAbZ formulation contains polysorbate 80 (PS80) that has unsaturated fatty acids such as oleic acid and linoleic acid. Hence, we address the question of how protein-derived radicals may affect the composition of PS80. Isobaric products of PS80 were identified by means of mass spectrometry, suggesting cis/trans isomerization of unsaturated fatty acids of PS80. This mechanism was confirmed by the analysis of isolated fatty acids, demonstrating, e.g., the conversion of oleic acid to elaidic acid. Another commonly used excipient in mAb biotherapeutics is citrate buffer. In the presence of metal impurities such as iron, citrate buffer and iron can generate citrate-derived degradants such as carbon dioxide radical anion after exposure to UV-A light. Such radical anion can donate an electron to a disulfide bond and form a thiyl radical, which can also induce cis/trans isomerization of unsaturated fatty acids of PS80. Therefore, photo-induced radicals and radical ions may lead to cross-linking of amino acids, color change in concentrated mAbs, and cis/trans isomerization of PS80.
dc.format.extent216 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsCopyright held by the author.
dc.subjectPharmaceutical sciences
dc.subjectcross-links
dc.subjectimmunoglobins
dc.subjectphotooxidation
dc.subjectphotosensitizers
dc.subjectradical ions
dc.subjectsurfactants
dc.titleGeneration and effect of photo-induced radicals on cross-linking, color change, and isomerization in formulations of peptides and proteins
dc.typeDissertation
dc.contributor.cmtememberStobaugh, John F.
dc.contributor.cmtememberSiahaan, Teruna
dc.contributor.cmtememberTolbert, Thomas
dc.contributor.cmtememberDhar, Prajnaparamita
dc.thesis.degreeDisciplinePharmaceutical Chemistry
dc.thesis.degreeLevelPh.D.
dc.identifier.orcid


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