Rupesh Bommana1, Olivier Mozziconacci1,2, Y John Wang3, Christian Schöneich4. 1. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA. 2. Analytical Sciences, Merck & Co., Inc., Kenilworth, New Jersey, USA. 3. Late Stage Pharmaceutical Development, Genentech, S. San Francisco, California, 94080, USA. 4. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA. schoneic@ku.edu.
Abstract
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.
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, humangrowth hormone (hGH), insulin and bovineserum 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.
Entities:
Keywords:
fluorogenic derivatization; human growth hormone; mass spectrometry; monoclonal antibody; protein oxidation
Authors: Igor A Kaltashov; Cedric E Bobst; Rinat R Abzalimov; Guanbo Wang; Burcu Baykal; Shunhai Wang Journal: Biotechnol Adv Date: 2011-05-17 Impact factor: 14.227
Authors: Ravuri S K Kishore; Sylvia Kiese; Stefan Fischer; Astrid Pappenberger; Ulla Grauschopf; Hanns-Christian Mahler Journal: Pharm Res Date: 2011-03-03 Impact factor: 4.200
Authors: Ninad Varkhede; Peters Björn-Hendrik; K Ryan Moulder; Philip Gao; Christian Schöneich; M Laird Forrest Journal: Anal Biochem Date: 2021-10-20 Impact factor: 3.365