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Novel Cys crosslinks and Trp side chain cleavages in proteins and peptides exposed to light
Bane, Jessica Mae
Bane, Jessica Mae
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Abstract
Protein pharmaceuticals, like monoclonal antibodies (mAbs), are sensitive to many degradation pathways, such as oxidation and photodegradation. Photostability testing provides essential information to characterize protein stability because mAbs are exposed to light during the production and storage process, which could lead to instability problems. Photostability testing has also become increasingly important to demonstrate likeness between biosimilars and original protein therapeutics. All amino acids are sensitive to oxidation, but only the aromatic amino acids and the disulfide bond are directly sensitive to light. The Trp residue is the most sensitive to light, and its photochemistry has been studied extensively, but the photochemistry involving both Trp and the disulfide bond in peptides and proteins has not been fully characterized, although the Trp residue is frequently located near disulfide bonds. To improve our understanding of the mechanisms of degradation and stability of protein pharmaceuticals, we have exposed disulfide-containing, Trp-containing, and disulfide and Trp-containing peptides, and an IgG1 molecule to light at λ = 254 nm and/or λmax = 305 nm. By mass spectrometry, chemical derivation, and NMR analysis, we observed a novel Cys crosslinked product after photoirradiation, tentatively identified as isothiazole-3(2H)-one. By mass spectrometry, we observed cleavage of the Trp side chain to Gly and/or Gly hydroperoxide in IgG1 at three separate Trp residues located in both the heavy and light chains, all of which were within close proximity to a disulfide bond. We also detected the cleavage of the Trp side chain to Gly and/or Gly hydroperoxide in two model peptides. In a third model peptide, we observed the cleavage and trapping of the Trp side chain by nearby Lys and Tyr residues. The Cys crosslink and Trp cleavage products highlight the damage light exposure can have by not only inducing a significant amino acid modification, but also leading to protein aggregation or amino acid hydroperoxides that have the potential to induce additional protein oxidation.
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Date
2015-05-31
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University of Kansas
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Keywords
Pharmaceutical sciences, Chemistry, Physical chemistry, Disulfide bonds, IgG1, Model peptides, Monoclonal Antibodies, Photochemistry, Tyrptophan