Application of nSMOL coupled with LC-MS bioanalysis for monitoring the Fc-fusion biopharmaceuticals Etanercept and Abatacept in human serum

Abstract

The principle of nano-surface and molecular-orientation limited (nSMOL) proteolysis has a unique characteristic Fab-selective proteolysis for antibody bioanalysis that is independent of a variety of monoclonal antibodies by the binding antibody Fc via Protein A/G in a pore with 100 nm diameter and modified trypsin immobilization on the surface of nanoparticles with 200 nm diameter. Since minimizing peptide complexity and protease contamination while maintaining antibody sequence specificity enables a rapid and broad development of optimized methods for liquid chromatography-mass spectrometry (LC-MS) bioanalysis, the application of regulatory LC-MS for monitoring antibody biopharmaceuticals is expected. nSMOL is theoretically anticipated to be applicable for representative Fc-fusion biopharmaceuticals, because Protein A/G-binding site Fc exists on the C-terminus, and its functional domain is available to orient and interact with the reaction solution. In this report, we describe the validated LC-MS bioanalysis for monitoring Ethanercept and Abatacept using nSMOL technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 μg/mL using the signature peptide VFCTK (aa.43-47), and that of Abatacept was from 0.391 to 100 μg/mL using the signature peptide MHVAQPAVVLASSR (aa.1-14). Both proteins fulfilled the guideline criteria for low-molecular-weight drug compounds. The results indicate that the clinical and therapeutic monitoring for antibody and Fc-fusion biopharmaceuticals are adequately applicable using nSMOL proteolysis coupled with LC-MS bioanalysis.

Keywords: Abatacept; Etanercept; LC‐MS; bioanalysis; clinical pharmacokinetics; nano‐surface and molecular‐orientation limited proteolysis; therapeutic drug monitoring.

Figure 1

Schematic view of nSMOL reaction principle

Figure 2

The ClustalW sequence alignment of (A) TNFR (TNR1B) and Etanercept (ETN), and (B) CTLA‐4 (CTLA4) and Abatacept (ABT). The black area shows identical amino acid residues. The red lines show the selected signature peptide of each Fc‐fusion protein. The blue arrow represents the position of the beginning of fused Fc domain

Figure 3

The 3‐D structure of the extracellular domain and signature peptide configuration of (A) TNFR (from Protein Data Bank ID 3ALQ), peptide VFCTK (aa.42‐46), and (B) CTLA‐4 (PDB ID 3OSK), peptide MHVAQPAVVLASSR (aa.1‐13). The red position shows the selected signature peptide. Green residues indicate cysteine, and the dashed lines show the site of intradisulfide bridge

Figure 4

The ClustalW alignment of reported N‐terminal Abatacept sequences. ClustalW alignment of the N‐terminal portion from DrugBank (ABT‐1), Review report from PMDA (ABT‐2), and KEGG drug (ABT‐3) is shown