A Novel Workflow to Assess Serum Stability of Antibody–siRNA Conjugates

Abstract Antibody–siRNA conjugates (ARCs) are emerging as a promising approach for targeted delivery of small interfering RNA (siRNA) to specific cells outside the liver, harnessing the advantages of antibody-based delivery to overcome limitations inherent to siRNA molecules, including rapid degradation, limited tissue penetration, and poor specificity. To address the critical need for robust stability assessment of ARCs in biological matrices, we developed a novel in vitro serum stability assay featuring immunoaffinity purification that purified a tool molecule, ARC1, under neutral conditions from biological matrices to preserve the molecular integrity and prevent artificial degradation. The purified ARC1 was analyzed in three complementary liquid chromatography–high-resolution mass spectrometry (LC-HRMS) methods: native mass spectrometry, protein mass spectrometry, and oligonucleotide mass spectrometry. This assay enabled assessment of each individual ARC component, including the parental antibody, linker, sense strand, and antisense strand. The results demonstrated that the linker remained stable in serum for 4 days, while hydrolysis of the conjugated siRNA occurred in both sense and antisense strands. Various biotransformation products were identified by mapping the results from three LC-HRMS methods to visualize the degradation pathway of ARC1. This work represents the first in vitro serum stability analysis workflow of ARCs that can capture biotransformation of each ARC component using immunoaffinity purification. This method offers a valuable tool to proactively identify potential liabilities in ARC designs and inform ARC candidate selection, siRNA engineering, and strategic planning for bioanalytical, toxicity, and immunogenicity assessment during drug discovery and early development.