N-terminus of Etanercept is Proteolytically Processed by Dipeptidyl Peptidase-4
N-terminus of Etanercept is Proteolytically Processed by Dipeptidyl Peptidase-4
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DOI：
10.1007/s11095-022-03371-2
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发表时间：
2022-08-19
影响因子：
3.7
通讯作者：
Matsubara，Kazuo
中科院分区：
文献类型：
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作者：
Masui，Sho;Yonezawa，Atsushi;Matsubara，Kazuo
目的生物制品具有结构异质性，在体内可发生生物转化。依那西普（ETN）是一种融合蛋白，由可溶性肿瘤坏死因子（TNF）受体和人免疫球蛋白G1的Fc部分组成。ETN的N-末端具有被二肽基肽酶-4（DPP-4）切割的推定序列。本研究的目的是调查ETN在人类和小鼠的生物转化，并评估其对功能properties.MethodsAn分析方法，采用液相色谱-质谱（LC-MS/MS）建立。在类风湿性关节炎患者或接受ETN的小鼠的血清中评估ETN的N-末端异质性。用重组DPP-4进行了体外N端截短的研究。用酶联免疫吸附测定法（ELISA）研究了ETN与TNF-α或TNF-β的结合亲和力。结果在制剂中，约90%的ETN具有完整的N-末端，而在类风湿关节炎患者和小鼠血清中，ETN的N-末端截短形式最为丰富。重组人DPP-4在体外从ETN的N-末端切割两个氨基酸。DPP-4抑制剂西格列汀在体内和体外均能抑制N-末端截短。然而，N端截短不影响ETN与TNF-α或TNF-β的结合能力和药代动力学。结论ETN在体内发生N端截短，DPP-4通过N端蛋白水解作用切割外源性ETN。基于MS的测定的应用将检测治疗性蛋白质的新型生物转化。
PurposeBiologics are structurally heterogeneous and can undergo biotransformation in the body. Etanercept (ETN) is a fusion protein composed of a soluble tumor necrosis factor (TNF) receptor and the Fc portion of human immunoglobulin G1. The N-terminus of ETN has a putative sequence cleaved by dipeptidyl peptidase-4 (DPP-4). The purpose of this study was to investigate the biotransformation of ETN in humans and mice and evaluate its effects on functional properties.MethodsAn analytical method using liquid chromatography-mass spectrometry (LC–MS/MS) was established. The N-terminal heterogeneity of ETN was assessed in the serum of patients with rheumatoid arthritis or mice receiving ETN. Thein vitroN-terminal truncation was explored using recombinant DPP-4. The binding affinity to TNF-α or TNF-β was investigated using an in-house enzyme-linked immunosorbent assay.ResultsIn the formulations, about 90% of ETN had an intact N-terminus, while the N-terminal truncated form was most abundant in the serum of the patients with rheumatoid arthritis and mice. Recombinant human DPP-4 cleaved two amino acids from the N-terminus of ETNin vitro. Sitagliptin, a DPP-4 inhibitor, inhibited N-terminal truncation bothin vivoandin vitro. However, N-terminal truncation did not affect the binding ability to TNF-α or TNF-β and the pharmacokinetics of ETN. ETN biosimilars exhibited similar characteristics to the reference productin vivoandin vitro.ConclusionsETN undergoes N-terminal truncation in the body, and DPP-4 cleaves exogenous ETN via N-terminal proteolysis. The application of an MS-based assay will detect novel biotransformation of therapeutic proteins.