Analysis of Antibody siRNA Conjugate Using BioAccord System

Significance of the Topic

Therapeutic siRNA conjugates represent a novel class of targeted biologics that combine the gene-silencing capabilities of siRNA with the cell-specific binding of antibodies. Comprehensive molecular characterization is critical to ensure product identity, purity, and efficacy in both research and quality control settings.

Study Objectives and Overview

The primary goal of this application was to establish two complementary LC-MS workflows on the BioAccord System for the analysis of Antibody-siRNA Conjugates (ARC):

• Mass confirmation of the individual siRNA sense and antisense strands using ion-pair reversed-phase LC-MS
• Determination of the drug-to-antibody ratio (DAR) on the intact ARC via native SEC-MS

Methodology

A reversed-phase ion-pairing LC-MS method was optimized in negative ion mode to confirm the accurate mass of both siRNA strands. A non-denaturing SEC-MS protocol was developed to preserve the integrity of the double-stranded siRNA when determining DAR distribution.

Instrumentation Used

The analyses were performed on the BioAccord LC-MS System, comprising:

• ACQUITY UPLC I-Class PLUS
• Tunable UV Detector
• ACQUITY RDa Time-of-Flight Mass Detector
• Waters_connect compliance-ready informatics

Main Results and Discussion

The ion-pairing RPLC-MS assay produced deconvoluted masses for both sense and antisense strands that matched theoretical values within acceptable error margins. Native SEC-MS revealed distinct DAR1 and DAR2 species, as well as byproducts corresponding to single-stranded conjugates. Intact mass profiles confirmed proper mAb engineering and siRNA conjugation efficiency.

Benefits and Practical Applications

• Rapid and reliable confirmation of siRNA and ARC identity
• Quantitative DAR assessment to support batch release and stability studies
• Compliance-ready workflows facilitating transfer to QC laboratories
• Minimal method optimization required due to robust BioAccord performance

Future Trends and Applications

Further integration of automated sample preparation and high-throughput screening can expand applicability to diverse oligonucleotide-antibody constructs. Advances in native MS sensitivity and software algorithms may enable deeper profiling of conjugate heterogeneity, supporting next-generation therapeutic modalities.

Conclusion

The two LC-MS methods developed on the BioAccord System provide complementary, high-resolution data for the characterization of antibody-siRNA conjugates. These approaches streamline identity confirmation and DAR determination, underpinning robust analytical strategies for emerging biotherapeutics.

References

1. Shion H, Yu Y, Chen W. Enabling Routine and Reproducible Intact Mass Analysis When Data Integrity Matters. Waters Application Note 720006472EN. October 2020.
2. Shion H, Yu Y, Chen W. Analysis of Antibody Drug Conjugates by Native Mass Spectrometry on the BioAccord System. Waters Application Brief 720006570EN. May 2019.
3. Doneanu C, Fox J, Harry E, Knowles C, Yu Y, Fredette J, Chen W. Intact Mass Confirmation Analysis on the BioAccord LC-MS System for Extensively Modified Oligonucleotides. Waters Application Note 720007028EN. October 2020.