Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave, Lawrence, Kansas, 66047, USA.
Department of Chemistry, University of Kansas, Lawrence, Kansas, 66045, USA.
Pharm Res. 2018 Sep 25;35(11):219. doi: 10.1007/s11095-018-2496-0.
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 1.07-1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5-7) 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 HO and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with HO caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide 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)SCy], followed by hydrolysis.
在预充式玻璃注射器中发现的钨和氧化钨浸出物被认为会引发蛋白质沉淀和聚集。在这里，我们测试了钨和氧化钨通过与过氧化氢反应（蛋白质配方中可能存在的一种杂质）来诱导蛋白质化学降解的可能性，从而产生过氧钨酸盐。
将单克隆抗体 (mAb) 与各种浓度的过氧钨酸盐孵育，并通过 SDS-PAGE 和质谱分析反应混合物。
将 mAb 暴露于 4°C 和 22°C（pH 5-7）温度下、基于钨含量为 1.07-1070 ppm 的过氧钨酸盐（pH 5-7）下，会诱导蛋白质片段化。片段化的程度随着温度升高、pH 值降低和过氧钨酸盐浓度升高而增加。mAb 片段被鉴定为含有不同重链 (H) 和轻链 (L) 的组合。当蛋白质暴露于 HO 和正钨酸盐时，即使在低至 5 ppm 的水平下，也会产生类似的 mAb 片段。此外，用于制造预充式玻璃注射器的钨针的提取物与 HO 结合，会导致 mAb 发生类似的片段化。通过质谱对片段的鉴定表明，通过重链和轻链之间的氧化二硫键断裂产生片段，这一点通过对产物形成的质谱数据得到证实。用胰岛素分别证实了氧化断裂的机制。
过氧钨酸盐引起 mAb 片段化的机制被提出是通过链间二硫键氧化形成亚砜 (CyS(═O)SCy) 和砜 [CyS(═O)SCy]，随后水解。
