Mass Spectrometric Characterization of Antibody-RNA Conjugates using the Agilent 6545XT AdvanceBio LC/Q-TOF

Importance of Topic

Antibody-RNA conjugates combine the specificity of antibodies with oligonucleotide therapies, enabling delivery of RNA payloads to tissues beyond the liver and targeting previously undruggable pathways. Mass spectrometric characterization is critical to assess conjugate integrity, stoichiometry and stability, supporting therapeutic development and quality control.

Objectives and Study Overview

The study aimed to establish an LC/MS workflow for intact antibody-RNA conjugates under both denaturing and native conditions using the Agilent 6545XT AdvanceBio LC/Q-TOF. Key goals included:

• Development of separation methods to resolve unconjugated and mono-conjugated species (DAR=1)
• Evaluation of conjugate stability and dissociation in denaturing vs. native MS
• Implementation of relative quantitation and structural assignment strategies

Methodology and Instrumentation

A partially reduced monoclonal antibody was conjugated to activated RNA via thiol linkers, capped, and purified by ion-exchange chromatography to isolate DAR=1 species. Analytical conditions:

• Denaturing LC/MS: AdvanceBio PLRP-S column, organic/acidic solvents, SWARM autotune, m/z range up to 30,000
• Native LC/MS: SEC AdvanceBio column, 100 mM ammonium acetate buffer (pH 7), Bio-Rad Bio-Spin P-30 desalting
• Mass spectrometer: Agilent 6545XT AdvanceBio LC/Q-TOF with extended mass range

Main Results and Discussion

Under denaturing conditions, deconvoluted spectra revealed multiple dissociation products (light/heavy chains with or without RNA), indicating weak non-covalent interactions. Native SEC-MS preserved intact conjugates:

• Separated two chromatographic peaks corresponding to DAR=1 variants
• Deconvolution confirmed species carrying one RNA or cap modifications
• Native MS reduced fragment artifacts and delivered accurate mass assignments

Benefits and Practical Applications

This workflow enables precise characterization and relative quantitation of antibody-RNA conjugates, improving analytical support for:

• Therapeutic pipeline development and batch release testing
• Structural confirmation during bioconjugation optimization
• Comparative studies of linker chemistries and payload stability

Future Trends and Potential Applications

Advancements may include higher-resolution MS instruments, automated data analysis, and application to diverse conjugate formats. Integration with ion mobility and top-down fragmentation approaches will deepen structural insights and accelerate therapeutic design.

Conclusion

The Agilent 6545XT AdvanceBio LC/Q-TOF workflow under native MS conditions overcomes dissociation issues seen in denaturing analyses, providing robust, accurate mass characterization and quantitation of antibody-RNA conjugates. This method supports advanced biotherapeutic research and QC practices.

Reference

1. Crooke S. T. et al. RNA-targeted therapeutics. Cell Metab. 2018;27(4):714-739.
2. Cuellar T. L. et al. Nucleic Acids Res. 2015;43(2):1189-1203.
3. Sugo T. et al. J Control Release. 2016;237:1-13.