Fragmentation of a Monoclonal Antibody by Peroxotungstate.

Abstract

Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate. A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry. Exposure of a mAb to 1.07-1070ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH5-7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidativedisulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin. Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.