Photodegradation of peptides and proteins by near UV and visible light

Therapeutic proteins (TP) have been on the rise for the last 30 years for the treatment of diverse diseases, including cancer and autoimmune diseases. Monoclonal antibodies (mAbs) comprise about 50% of the TP approved by the FDA and are the major group of TP sold worldwide. Although the high specificity and good tolerance allow mAbs to be categorized as safe drugs, several side effects have arisen, including immunogenicity. It was demonstrated that immunogenicity is produced by aggregation and oxidation of mAbs. While near UV and visible light can lead to the oxidation of mAbs, no concise mechanism has been established for the photo-oxidation of mAbs. Here, we evaluated the formation of reactive species by carboxylic acid buffer-Fe3+ complexes, which can absorb light and cause the formation of various reactive species via the photo-Fenton reaction. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids. Specifically, for citrate buffer, the light-induced formation of H2O2, hydroxyl radicals (●OH), carbon dioxide radical anion (●CO2-), and formaldehyde was demonstrated. Several products were observed, including the formation of novel products on Tyr. The yields of oxidation products in acetate and succinate formulations were significantly lower. The photo-Fenton reaction in citrate buffer was evaluated in a model mAb (IgG2). We detected an increase in the formation of aggregates and fragments with increasing concentrations of added Fe3+. Light exposure resulted in the generation of more acidic products, evident from a shift of the protein pI detected by two-dimensions electrophoresis. Oxidation products of Tyr, His, Cys, and Trp were detected. The exposure to ambient light or light doses specified by the ICH guideline showed that mild doses of photo-irradiation can produce several oxidation products. We evaluated the photo-Fenton oxidation of peptides in the presence of diverse excipients, including amino acids (Gly, Arg, Lys, His and Met), sugars (mannitol, sucrose, and trehalose), EDTA, DTPA, and polysorbate 80. The antioxidant Met in citrate solutions leads to the generation of novel oxidation products. While Arg, Lys and His, mannitol, sucrose, and trehalose promoted the increase in yields of Met sulfoxide, the addition of EDTA or DTPA did not protect from the photo-oxidation. Polysorbate 80 was also susceptible to the oxidation by the photo-Fenton reaction, leading to the generation of organic peroxides. Altogether, the photo-Fenton reaction in pharmaceutical relevant conditions can produce reactive species that lead to aggregation, fragmentation, the formation of charge variants, and oxidation products in pharmaceutical proteins and peptides. The nature and yields of the oxidation products depended on the concentration of Fe3+ present in the solution, the concentration of buffer, and the excipients added.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2020

Author dc:creator

• Subelzu, Natalia J

Advisor dc:contributor.advisor

• Schoneich, Christian

• Pharmaceutical sciences
• Formulations
• Near UV light
• Peptides
• Photostability
• Proteins
• Visible light

Statement dc:rights

• Copyright held by the author.

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:17446

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/37289