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

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.

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.

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.

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.