Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies

Ada Hui; Xanthe M. Lam; Christopher Kuehl; Ulla Grauschopf; Y. John Wang

doi:10.5731/pdajpst.2015.01059

Abstract

When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H₂O₂) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H₂O₂, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H₂O₂ concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H₂O₂ was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H₂O₂. The consumption of H₂O₂ by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H₂O₂. However, mAb formulation excipients may affect the rate of H₂O₂ consumption. mAb formulations containing trehalose or sucrose had faster H₂O₂ consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H₂O₂ spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H₂O₂ level. Our kinetic modeling of the reaction between Fc-Met oxidation and H₂O₂ provides an outline to design a H₂O₂ spiking study to support the use of VPHP isolator for antibody drug product manufacture.

LAY ABSTRACT: Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H₂O₂) spiking studies may be performed to determine the sensitivity of monoclonal antibody (mAb) drug products to residual peroxide in the isolator. In this study, mAbs were spiked with H₂O₂; an increase in methionine (Met) oxidation of the mAbs with a decrease in H₂O₂ concentration was observed for various levels of spiked-in peroxide. The reaction between Met and H₂O₂ was 1:1, and its rate was dependent on mAb and H₂O₂ concentrations. Consumption of H₂O₂ by Met followed pseudo first-order kinetics; the rate was proportional to mAb concentration. Formulations containing trehalose or sucrose had faster consumption rates than formulations without the sugars, which could be due to excipient impurities. Based on H₂O₂ spiking study results, we can predict the amount of Met oxidation for a given mAb concentration and H₂O₂ level. Our modeling of the reaction between Fc-Met oxidation and H₂O₂ provides an outline to design a H₂O₂ spiking study that supports using VPHP isolators during manufacture of mAb products.

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