Amgen Inc., Process and Product Development, Analytical and Formulation Sciences Group, Thousand Oaks, California, 91320, USA.
Pharm Res. 2010 Mar;27(3):447-56. doi: 10.1007/s11095-009-0032-y. Epub 2010 Feb 2.
L-histidine, a commonly used buffer for protein formulations, has the potential to oxidize and form multiple byproducts. Previous studies were performed using metal catalyzed oxidation with Fe(2+) or Cu(2+). We re-examined the oxidation of L-histidine under conditions more appropriate to protein formulations.
Solutions of free L-histidine, protected from light, were initially reacted with tert-butylhydroperoxide and the products analyzed by UV absorption spectroscopy, reversed phase HPLC and mass spectrometric analysis and NMR. Experimental parameters investigated were oxidizing agent, pH, temperature, metal ion and metal chelator content.
The initial reaction produced a number of known products, along with an unknown product that was identified as 4(5)-imidazolecarboxaldehyde. The reaction was highly pH and oxidizing-agent specific. The product was not observed at pH 5.0 or below, while there was a dramatic increase for reactions carried out at pH 6.0 or above. Addition of FeSO(4) to the reaction dramatically increased the amount of 4(5)-imidazolecarboxaldehyde produced, while addition of the metal chelators EDTA or DTPA completely inhibited the reaction.
The presence of oxidants and trace concentrations of metal ions in high purity L-histidine solutions results in the formation of 4(5)-imidazolecarboxaldehyde which has the potential to covalently modify proteins.
L-组氨酸是一种常用于蛋白质制剂的缓冲剂，它有氧化并形成多种副产物的潜力。以前的研究是使用 Fe(2+)或 Cu(2+)金属催化氧化进行的。我们在更适合蛋白质制剂的条件下重新检查了 L-组氨酸的氧化。
将游离 L-组氨酸溶液避光，先用叔丁基过氧化氢反应，然后通过紫外吸收光谱、反相高效液相色谱和质谱分析及 NMR 分析产物。考察的实验参数有氧化剂、pH 值、温度、金属离子和金属螯合剂含量。
初始反应产生了一些已知的产物，以及一种未知的产物，被鉴定为 4(5)-咪唑甲醛。该反应具有高度的 pH 值和氧化剂特异性。在 pH 值为 5.0 或以下时，未观察到产物，而在 pH 值为 6.0 或更高时，产物的生成量显著增加。向反应中添加 FeSO4 会大大增加 4(5)-咪唑甲醛的生成量，而添加金属螯合剂 EDTA 或 DTPA 则完全抑制了反应。
在高纯度 L-组氨酸溶液中存在氧化剂和痕量金属离子会导致 4(5)-咪唑甲醛的形成，而 4(5)-咪唑甲醛有可能使蛋白质发生共价修饰。
