Monitoring of Peroxide in Gamma Irradiated EVA Multilayer Film Using Methionine Probe

Abstract

In this study, the oxidation of methionine is used as a proxy to model the gamma radiation-induced changes in single-use bags; these changes lead to the formation of acids, radicals, and hydroperoxides. The mechanisms of formation of these reactive species and of methionine oxidation are discussed. With the help of reaction kinetics, the optimal conditions for the use of these single-use bags minimizing the impact of radical chemistry are highlighted. Biopharmaceutical bags gamma irradiated from 0 kGy to 260 kGy and aged from 0 to 36 months were filled with a methionine solution to follow the oxidation of the methionine. The methionine sulfoxide was measured with HPLC after different storage times (0, 3, 10, 14, 17, and 21 days). Three main results were analyzed through a design of experiments: the oxidative induction time, the methionine sulfoxide formation rate, and the maximum methionine sulfoxide concentration detected. A key aspect of the study is that it highlights that methionine is oxidized not necessarily directly by hydro(gen) peroxide but throughperacid, and likely peracetic acid. The answers to the design of experiments were considered to obtain the desirability domain for the optimization of the conditions of use for the single-use bags limiting the oxidation of methionine as well as the release of reactive species thereof.

Investigations at the Product, Macromolecular, and Molecular Level of the Physical and Chemical Properties of a γ-Irradiated Multilayer EVA/EVOH/EVA Film: Comprehensive Analysis and Mechanistic Insights

“…Hence, the perfect conditions for suppressing oxidation issues would be: oxidation induction time of more than 10 days, oxidation rate close to or less than 3 mM.day −1 , and level of oxidation lower than 3 mM. Then, it is possible to define a desirability function that provides a response surface as displayed in Figure 15 [ 42 ]. Hence, for 50 kGy, the maximum of desirability (50%) is reached for long ageing (30 months).…”

“…By contrast, amino acids such as cysteine or methionine are often used to probe the presence of oxidant in biological solutions. Therefore, we adapted a procedure based on the oxidation of methionine to investigate the presence of oxidants such as peroxide, peracids, and hydrogen peroxide [ 42 ]. Importantly, it is well known that hydrogen peroxide reacts with acetic acid to generate peracid in situ ( Scheme 3 ) [ 42 , 43 ] affording the methionine sulfoxide derivative ( Scheme 4 ).…”

“…Therefore, we adapted a procedure based on the oxidation of methionine to investigate the presence of oxidants such as peroxide, peracids, and hydrogen peroxide [ 42 ]. Importantly, it is well known that hydrogen peroxide reacts with acetic acid to generate peracid in situ ( Scheme 3 ) [ 42 , 43 ] affording the methionine sulfoxide derivative ( Scheme 4 ). Thus, with oxidation models using methionine, and taking into account a lengthy experience (up to 21 days for a one-month aged bag, Figure 7 ), we showed that methionine is oxidized to a significant extent.…”

“…Therefore, HPLC titration is used to detect and monitor only the presence of oxidant species. As mentioned in the analysis at the product level section, methionine oxidation depends a lot on dose, ageing, and solution storage time [ 42 ]. Moreover, with a titration curve it is possible to determine three parameters, the maximal concentration of oxidant, the rate of oxidation, and the retardation time, useful to understand the efficiency of the oxidation process as a function of irradiation dose, ageing, and storage time.…”

“…LOQ for limit of quantification and LOD for limit of detection. Reproduced from [ 42 ] with the permission of Polymers.…”