Application of <scp>nSMOL</scp> coupled with <scp>LC</scp>‐<scp>MS</scp> bioanalysis for monitoring the Fc‐fusion biopharmaceuticals Etanercept and Abatacept in human serum

<jats:title>Abstract</jats:title><jats:p>The principle of nano‐surface and molecular‐orientation limited (<jats:styled-content style="fixed-case">nSMOL</jats:styled-content>) 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 (<jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content>) bioanalysis, the application of regulatory <jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content> for monitoring antibody biopharmaceuticals is expected. <jats:styled-content style="fixed-case">nSMOL</jats:styled-content> 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 <jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content> bioanalysis for monitoring Ethanercept and Abatacept using <jats:styled-content style="fixed-case">nSMOL</jats:styled-content> technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 μg/mL using the signature peptide <jats:styled-content style="fixed-case">VFCTK</jats:styled-content> (aa.43‐47), and that of Abatacept was from 0.391 to 100 μg/mL using the signature peptide <jats:styled-content style="fixed-case">MHVAQPAVVLASSR</jats:styled-content> (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 <jats:styled-content style="fixed-case">nSMOL</jats:styled-content> proteolysis coupled with <jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content> bioanalysis.</jats:p>