N-terminus of Etanercept is Proteolytically Processed by Dipeptidyl Peptidase-4

• Published in: Pharmaceutical research 2022-10, Vol.39 (10), p.2541-2554
• Main Authors: Masui, Sho, Yonezawa, Atsushi, Yokoyama, Kotoko, Iwamoto, Noriko, Shimada, Takashi, Onishi, Akira, Onizawa, Hideo, Fujii, Takayuki, Murakami, Kosaku, Murata, Koichi, Tanaka, Masao, Nakagawa, Shunsaku, Hira, Daiki, Itohara, Kotaro, Imai, Satoshi, Nakagawa, Takayuki, Hayakari, Makoto, Matsuda, Shuichi, Morinobu, Akio, Terada, Tomohiro, Matsubara, Kazuo
• Format: Article
• Language: English
• Subjects: Arthritis; Biochemistry; Biological products; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Biotransformation; Enzyme-linked immunosorbent assay; Enzymes; Etanercept; Fc receptors; Fusion protein; Immunoglobulin G; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Medical colleges; Medical Law; Medical research; Medicine, Experimental; N-Terminus; Original Research Article; Peptidase; Pharmacokinetics; Pharmacology/Toxicology; Pharmacy; Proteolysis; Rheumatoid arthritis; Rheumatoid factor; Sitagliptin; Tumor necrosis factor; Tumor necrosis factor-TNF; Tumor necrosis factor-α
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-022-03371-2

Purpose Biologics 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 . Methods An 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. The in vitro N-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. Results In 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 ETN in vitro . Sitagliptin, a DPP-4 inhibitor, inhibited N-terminal truncation both in vivo and in 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 product in vivo and in vitro . Conclusions ETN 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.