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

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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.

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Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ANOVA:

Analysis of variance

Ccps:

Counts per second

Δ1AA:

Cleavage of 1 amino acid

Δ2AA:

Cleavage of 2 amino acids

DPP-4:

Dipeptidyl peptidase-4

ELISA:

Enzyme-linked immunosorbent assay

ETN:

Etanercept

FL:

Full length

HRP:

Horseradish peroxidase

IgG1:

Immunoglobulin G1

IS:

Internal standard

KD :

Dissociation constant

LC–MS/MS:

Liquid chromatography-mass spectrometry

MRM:

Multiple reaction monitoring

nSMOL:

Nano-surface and molecular-orientation limited

RA:

Rheumatoid arthritis

rh:

Recombinant human

SD:

Standard deviation

TNF:

Tumor necrosis factor

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Funding

This study was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid for Scientific Research (B 19H03389), by the Japan Agency for Medical Research and Development (AMED) under Grant Number 21mk0101152h0503 and by the Nakatomi Foundation to A.Y.

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Authors and Affiliations

  1. Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan

    Sho Masui, Atsushi Yonezawa, Shunsaku Nakagawa, Daiki Hira, Kotaro Itohara, Satoshi Imai, Takayuki Nakagawa, Makoto Hayakari, Tomohiro Terada & Kazuo Matsubara

  2. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Sho Masui & Atsushi Yonezawa

  3. Shimadzu Corporation, Kyoto, Japan

    Kotoko Yokoyama, Noriko Iwamoto & Takashi Shimada

  4. Department of Advanced Medicine for Rheumatic Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Akira Onishi, Hideo Onizawa, Takayuki Fujii, Koichi Murata, Masao Tanaka & Akio Morinobu

  5. Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Takayuki Fujii, Koichi Murata & Shuichi Matsuda

  6. Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Kosaku Murakami & Akio Morinobu

  7. Center for Cancer Immunotherapy and Immunobiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Kosaku Murakami

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  1. Sho Masui
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Contributions

All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work, and have given their approval for this version to be published. S.Masui, A.Y., T.S., and K.Matsubara contributed to the study conception and design, and preparing a draft of the manuscript. S.Masui, K.Y., N.I., T.S., S.N., D.H., K.I., S.I., T.N., and M.H. performed the experiments and analyzed the data. A.O., H.O., T.F., K.Murakami, K.Murata, M.T., S.Matsuda, and A.M. contributed to the acquisition of patients’ data. S.Matsuda, A.M., T.T., and K.Matsubara supervised the study. All authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to Atsushi Yonezawa.

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Conflict of Interest

The Department of Advanced Medicine for Rheumatic Diseases is supported by two local governments (Nagahama City, Shiga, and Toyooka City, Hyogo, Japan) and five pharmaceutical companies (Mitsubishi Tanabe Pharma Co.; Chugai Pharmaceutical Co. Ltd.; UCB Japan Co. Ltd.; Asahi Kasei Pharma Corp.; and AYUMI Pharmaceutical Co.). The KURAMA cohort study is supported by a grant from Daiichi Sankyo Co. Ltd. A.Y. has received a research grant from Shimadzu Corporation and AYUMI Pharmaceutical Co. A.O. reports grants from Pfizer Inc., Bristol-Myers Squibb., Advantest, personal fees from Asahi Kasei Pharma Corp., Chugai Pharmaceutical Co. Ltd., Eli Lilly Japan K.K, Ono Pharmaceutical Co., Mitsubishi Tanabe Pharma Co., Eisai Co. Ltd., Abbvie Inc., Takeda Pharmaceutical Co. Ltd., and Daiichi Sankyo Co. Ltd. T.F. received speaking fees from Eisai Co. Ltd., Asahi Kasei Pharma Corp., Abbvie Inc., and Janssen Pharmaceutical K.K. K.Murata received a speaking fee and/or consulting fees from Eisai Co. Ltd., Chugai Pharmaceutical Co. Ltd.; Asahi Kasei Pharma Corp., Bristol-Myers Squibb, Mitsubishi Tanabe Pharma Co., Janssen Pharmaceutical K.K. and Daiichi Sankyo Co. Ltd. M.T. has received research grants and/or speaker fees from Abbvie Inc., Asahi Kasei Pharma Corp., Astellas Pharma Inc., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Eisai Co. Ltd., Eli Lilly Japan K.K., Janssen Pharmaceutical K.K., Kyowa Kirin Co. Ltd., Pfizer Inc., Taisho Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Teijin Pharma, Ltd., UCB Japan Co. Ltd. S. Matsuda received speaker fees from Pfizer Inc. A.M. has received honorarium from AbbVie G.K., Chugai Pharmaceutical Co. Ltd., Eli Lilly Japan K.K., Eisai Co. Ltd., Pfizer Inc., Bristol-Myers Squibb., Mitsubishi Tanabe Pharma Co., Astellas Pharma Inc., and Gilead Sciences Japan., and has received research grants from AbbVie G.K., Asahi Kasei Pharma Corp., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co. and Eisai Co. Ltd.

Consent to Participate

Written informed consent to participate in this study was obtained from all patients.

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Not applicable.

Ethical Approval

Written informed consent to participate in this cohort study was obtained from all patients. All patients’ data were de-identified and analyzed anonymously. The present study adhered to the principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (R0357). All animal studies were conducted in accordance with the guidelines for animal experiments at Kyoto University. The protocols were approved by the Animal Research Committee of the Graduate School of Medicine, Kyoto University (Permission No. MedKyo21113).

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Masui, S., Yonezawa, A., Yokoyama, K. et al. N-terminus of Etanercept is Proteolytically Processed by Dipeptidyl Peptidase-4. Pharm Res 39, 2541–2554 (2022). https://doi.org/10.1007/s11095-022-03371-2

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