An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization

• Published in: Pharmaceutical research Vol. 34; no. 7; pp. 1428 - 1443
• Main Authors: Bommana, Rupesh, Mozziconacci, Olivier, John Wang, Y., Schöneich, Christian
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.07.2017; Springer; Springer Nature B.V
• Subjects: Albumin; Amino acids; Analysis; Animals; Benzoxazoles - chemistry; Biochemistry; Biodegradation; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Bovine serum albumin; Calibration; Catalysis; Cattle; Chemical reactions; Drug Stability; Fluorescence; Formulations; Growth hormones; High resolution; Homeopathy; Human Growth Hormone - chemistry; Humans; Immunoglobulin G; Immunoglobulin G - chemistry; Insulin; Insulin - chemistry; Investigations; Mass spectrometry; Mass spectroscopy; Materia medica and therapeutics; Medical Law; Methionine; Methionine - analogs & derivatives; Methionine - chemistry; Methods; Monoclonal antibodies; Oxidation; Oxidation-Reduction; Oxidation-reduction reaction; Peroxides - chemistry; Peroxyl radicals; Pharmacology/Toxicology; Pharmacy; Phenols; Phenylalanine - chemistry; Proteins; Proteolysis; Research Paper; Serum Albumin, Bovine - chemistry; Somatotropin; Sulfoxides; Therapeutics; Tyrosine; Tyrosine - chemistry; fluorogenic derivatization; mass spectrometry; monoclonal antibody; human growth hormone; protein oxidation
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-017-2159-6

Purpose The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Methods Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. Results The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Conclusions Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.