title: A novel approach for oxidation analysis of therapeutic proteins.
abstract: Measuring and monitoring of protein oxidation modifications is important for biopharmaceutical process development and stability assessment during long-term storage. Currently available methods for biomolecules oxidation analysis use time-consuming peptide mapping analysis. Therefore, it is desirable to develop high-throughput methods for advanced process control of protein oxidation. Here, we present a novel approach by which oxidative protein modifications are monitored by an indirect potentiometric method. The method is based on adding an electron mediator, which enhances electron transfer (ET) between all redox species and the electrode surface. Specifically, the procedure involves measuring the sharp change in the open circuit potential (OCP) for the mediator system (redox couple) as a result of its interaction with the oxidized protein species in the solution. Application of Pt and Ag/AgCl microelectrodes allowed for a high-sensitivity protein oxidation analysis. We found that the Ru(NH3)6(2+/3+) redox couple is suitable for measuring the total oxidation of a wide range of therapeutic proteins between 1.1 and 13.6%. Accuracy determined by comparing with the known percentage oxidation of the reference standard showed that percentage oxidation determined for each sample was within ± 20% of the expected percentage oxidation determined by mass spectrometry.
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA. Electronic address: iva.turyan@biogen.com.
authors.forename: Iva
authors.initials: I
authors.lastname: Turyan
authors.affiliation: Rare Disease Research Unit (RDRU), Pfizer, Cambridge, MA 02142, USA.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Khatwani
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Sosic
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA.
authors.forename: Shiranthi
authors.initials: S
authors.lastname: Jayawickreme
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mandler
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D055664:Electrochemical Techniques; D013058:Mass Spectrometry; D008839:Microelectrodes; D009942:Organometallic Compounds / Q000737:chemistry; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012834:Silver / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D009942:Organometallic Compounds; D011506:Proteins; D012834:Silver
keywords: Indirect potentiometry; LC–MS; Oxidation analysis; Therapeutic proteins; Water-soluble raw materials composed of tween