Antioxidants for Prevention of Methionine Oxidation in Recombinant Monoclonal Antibody HER2

Abstract

Recombinant humanized monoclonal antibody HER2, rhuMAb HER2, in liquid formulations undergoes oxidation when exposed to intense light and elevated temperatures (30 & 40 degrees C). Met-255 in the heavy chain of the Fc region of the antibody is the primary site of oxidation. Met-431 of the Fc fragment can also be oxidized under extreme conditions. The amount of oxidation was determined by cleaving the Fab and Fc fragments by papain digestion, and the oxidized Fc fragment was detected by hydrophobic interaction chromatography. Oxidation of rhuMAb HER2 was also formulation dependent. The presence of NaCl in the rhuMAb HER2 formulation caused an increase in oxidation at higher temperatures after contact with stainless steel containers or stainless steel components in the filling process. The corrosion of stainless steel by chloride ions at the low pH of the formulation buffer generated iron ions that catalyzed methionine oxidation in rhuMAb HER2. Temperature-induced oxidation of rhuMAb HER2 occurred by the formation of free radicals, and light-induced oxidation of rhuMAb HER2 occurred via single oxygen pathway. Antioxidants, such as methionine, sodium thiosulfate, catalase, or platinum, prevented Met oxidation in rhuMAb HER2, presumably as free radicals or oxygen scavengers. The minimum effective levels (molar ratios of protein to antioxidant) required to inhibit temperature-induced oxidation were 1:5 and 1:25 for methionine and thiosulfate, respectively. A thiosulfate adduct of rhuMAb HER2 was observed by cation-exchange chromatography. These studies demonstrate that stoichiometric amounts of methionine and thiosulfate are sufficient to eliminate temperature-induced oxidation of rhuMAb HER2 caused by free radicals that were generated by the presence of metal ion and peroxide impurities in the formulation.

Mass spectrometry analysis of photo-induced methionine oxidation of a recombinant human monoclonal antibody

“…This result supports the hypothesis by Lam et al [7], which suggested that a singlet oxygen formed from the reaction between molecular oxygen and photo-activated polysorbate-20 reacts with Met to form an intermediate, which reacts with a second Met leading to the formation of two Met sulfoxides. Polysorbate is commonly used in formulations.…”

“…The susceptibility of these Met residues was demonstrated by incubating the antibodies in liquid formulation either with [3,5,6] or without [6,7] the addition of a mild oxidizing reagent, tBHP. Lam et al [7] reported that exposure to high intensity fluorescence light also increased the level of Met oxidation.…”

“…The susceptibility of these Met residues was demonstrated by incubating the antibodies in liquid formulation either with [3,5,6] or without [6,7] the addition of a mild oxidizing reagent, tBHP. Lam et al [7] reported that exposure to high intensity fluorescence light also increased the level of Met oxidation. It was previously observed that Met256 and Met432 (Figure 1) of the antibody used in this study were randomly oxidized on the two HCs when the antibody was incubated with tBHP, which was not the case observed with the accelerated stability sample [6].…”

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

“…It was previously observed that protein oxidation can be effectively inhibited by antioxidants like Met. [60][61][62] Since the stability of some proteins has been shown to be affected when formulated with certain antioxidants, the antioxidant should be carefully selected for each protein. We conducted additional work to screen excipients that would improve the chemical stability of the otelixizumab product formulated with PS80.…”

“…Several different types of antioxidants (Met, Cys, Glut, MTG, and EDTA) were selected and tested based on the presence in marketed protein formulations and their mechanism of action. [60][61][62][63][64][65] Results from our stability studies demonstrate that incorporation of Met and EDTA as antioxidants in the PS80 formulation substantially improved the stability of this mAb and provided protection from oxidation without altering the protein structure and solution properties. These data also suggested that oxidation occurs not only through a peroxide-mediated pathway, but also through a metal-catalyzed reaction and highlighted the value of a combination approach toward ensuring a stable formulation.…”