Sho Masui1,2, Atsushi Yonezawa3,4, Kotoko Yokoyama5, Noriko Iwamoto5, Takashi Shimada5, Akira Onishi6, Hideo Onizawa6, Takayuki Fujii6,7, Kosaku Murakami8,9, Koichi Murata6,7, Masao Tanaka6, Shunsaku Nakagawa1, Daiki Hira1, Kotaro Itohara1, Satoshi Imai1, Takayuki Nakagawa1, Makoto Hayakari1, Shuichi Matsuda7, Akio Morinobu6,8, Tomohiro Terada1, Kazuo Matsubara1. 1. Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. 2. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. 3. Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. ayone@kuhp.kyoto-u.ac.jp. 4. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. ayone@kuhp.kyoto-u.ac.jp. 5. Shimadzu Corporation, Kyoto, Japan. 6. Department of Advanced Medicine for Rheumatic Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 7. Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 8. Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 9. Center for Cancer Immunotherapy and Immunobiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Abstract
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.
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.
Authors: Simone Schadt; Simon Hauri; Filipe Lopes; Martin R Edelmann; Roland F Staack; Roberto Villaseñor; Hubert Kettenberger; Adrian B Roth; Franz Schuler; Wolfgang F Richter; Christoph Funk Journal: Drug Metab Dispos Date: 2019-12 Impact factor: 3.922
Authors: L W Moreland; S W Baumgartner; M H Schiff; E A Tindall; R M Fleischmann; A L Weaver; R E Ettlinger; S Cohen; W J Koopman; K Mohler; M B Widmer; C M Blosch Journal: N Engl J Med Date: 1997-07-17 Impact factor: 91.245