N-terminal Glutamate to Pyroglutamate Conversion in Vivo for Human IgG2 Antibodies
N-terminal Glutamate to Pyroglutamate Conversion in Vivo for Human IgG2 Antibodies
复制标题
DOI：
10.1074/jbc.m110.185041
复制
发表时间：
2011-04-01
影响因子：
4.8
通讯作者：
Flynn, Gregory C.
中科院分区：
文献类型：
--
作者：
Liu, Y. Diana;Goetze, Andrew M.;Flynn, Gregory C.
治疗性蛋白质含有大量的翻译后修饰，其中一些可能会影响其安全性或有效性。在这些变化之一中，焦谷氨酸可以在多肽链的N末端形成。重组单克隆抗体N端的谷氨酰胺和谷氨酸在体外都能自发环化为焦谷氨酸（pE）。谷氨酸转化为焦谷氨酸的速度比从谷氨酰胺转化为焦谷氨酸的速度慢，但在接近生理条件下已观察到。在这里，我们研究了人IgG 2 N-末端谷氨酸在体内转化为pE的程度。注射人体后，焦谷氨酸水平随时间推移而增加，多肽链之间的形成速率不同。在生理pH和温度条件下，相同抗体在体外重复了这些变化，表明在体内观察到的变化是由于化学转化而不是差异清除。抗体上轻链和重链之间转化率的差异通过使蛋白质变性来消除，揭示了结构元件影响pE形成率。通过从人血清中分离的内源性抗体中酶促释放pE，我们可以估计这种翻译后修饰的天然存在水平。总之，这些技术和结果可用于预测治疗性抗体的pE暴露量，并指导该属性的关键性评估。
Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.