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

Significance of the Topic

Small interfering RNAs are emerging as powerful gene-silencing therapeutics, but their delivery into cells remains a major challenge. Monoclonal antibodies offer a targeted vehicle for siRNA transport, forming covalent mAb-siRNA conjugates. Detailed characterization of these conjugates under non-denaturing conditions is essential for drug development and quality control.

Study Overview and Objectives

This work presents a native LC/MS strategy to analyze intact mAb-siRNA conjugates. The main goal was to establish a workflow that preserves noncovalent interactions and enables accurate mass determination and quantitation of antibody-oligonucleotide species without denaturation.

Methodology

The workflow comprises:

• Selective reduction of antibody disulfide bonds and coupling to activated siRNA via SMCC linker
• Anion-exchange purification to isolate DAR1 species
• Buffer exchange into volatile ammonium acetate for native analysis
• Comparison of traditional reversed-phase denaturing LC/MS and size-exclusion native LC/MS

Used Instrumentation

• Agilent 1290 Infinity II LC system with high-speed pump, multisampler and column thermostat
• AdvanceBio SEC columns (4.6×30 mm and 4.6×300 mm, 200 Å, 1.9 µm)
• Agilent 6545XT AdvanceBio LC/Q-TOF with SWARM autotune for large molecules
• Bio-Rad Bio-Spin P-30 cartridge for desalting and buffer exchange

Key Results and Discussion

Denaturing LC/MS on a PLRP-S column showed extensive dissociation of conjugates into light and heavy chains and degraded species. Native SEC LC/MS preserved the intact mAb-siRNA conjugate, revealing two principal DAR1 forms carrying one or three capping moieties. High-accuracy mass measurements (<5 ppm) confirmed species assignments and demonstrated the method’s ability to resolve closely related conjugates.

Benefits and Practical Applications

• Maintains native conformation and noncovalent linkages during analysis
• Provides precise mass determination for complex bioconjugates
• Enables rapid separation and quantitation of drug-loading variants
• Facilitates quality control in biopharmaceutical manufacturing

Future Trends and Applications

Advancements may include integration of native MS/MS for localization of conjugation sites, automation of sample preparation, expansion to multi-antibody or multi-payload constructs, and coupling with ion-mobility separations to further dissect structural heterogeneity.

Conclusion

This native LC/MS workflow offers a robust approach for comprehensive characterization of mAb-siRNA conjugates. By preserving native structures and delivering high-accuracy mass data, it supports the development, optimization and quality assurance of novel antibody-based RNA therapeutics.

References

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5. Sensitive Native Mass Spectrometry of Macromolecules Using Standard Flow LC/MS Application Note 5994-1739EN Agilent Technologies 2020
6. Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF Application Note 5991-7813EN Agilent Technologies 2017