微生物来源的谷氨酰胺转氨酶 (mTG) 介导的单一定点修饰的PEG IFN-α2a

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

摘要:

PEG修饰被认为是改善重组蛋白药物特性的最有效手段，包括增加蛋白质药物在体内的血浆半衰期，降低免疫原性和抗原性。目前典型的PEG修饰手段为将PEG连接至蛋白质的游离氨基，包括赖氨酸和N-末端，但这种连接缺乏选择性，产物为混合物，活性及工艺稳定性差，难以控制。酶法PEG化修饰能有效克服上述缺点，其中谷氨酰胺转氨酶 (TGase) 可以作为PEG化定点修饰用酶。文中选择重组人干扰素α2a (IFN α2a) 进行酶法修饰反应，通过计算机模拟预测IFN α2a可以在第101位Gln特异性定点修饰。将IFN α2a与40 kDa的Y型PEG在微生物来源的谷氨酰胺转氨酶 (mTG) 催化下进行定点PEG化修饰。结果显示，mTG可以介导IFN α2a特异性位点Gln的单一定点PEG修饰，产生分子量为58 495.6 Da的PEG-Gln101-IFN α2a分子。圆二色谱结果显示，PEG-Gln101-IFN α2a与未修饰的IFN α2a具有相同的二级结构。SD大鼠药代结果显示，与IFN α2a相比，PEG-Gln101-IFN α2a能有效提高药代动力学参数，强于已上市PEGIFN α2a-PEGASYS?。

Abstract:

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.