Polarity- and Sequence-Dependent Ionization of Therapeutic Antibody-siRNA Conjugates: Enabling Intact Multi-attribute...

Area of Science:

• Bioconjugation Chemistry
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Antibody-siRNA conjugates (ARCs) are advanced therapeutics for targeted delivery of small interfering ribonucleic acids (siRNAs).
• Characterizing complex ARCs and monitoring their critical quality attributes (CQAs) presents significant analytical challenges during development and manufacturing.
• Robust analytical methods are essential for ensuring the safety, efficacy, and consistency of ARC-based therapeutics.

Purpose of the Study:

• To develop and qualify a novel analytical method for comprehensive characterization of ARCs.
• To establish a reliable approach for monitoring CQAs of ARCs in a Good Manufacturing Practice (GMP) environment.
• To gain fundamental insights into the ionization behavior of ARCs during mass spectrometry analysis.

Main Methods:

• Development of an intact multi-attribute method (iMAM) utilizing native size exclusion chromatography-mass spectrometry (SEC-MS).
• Evaluation and qualification of the iMAM as an identity release assay for ARC drug substance (DS) and drug product (DP).
• Investigation of mass spectrometry operating polarity effects on siRNA integrity and ARC ionization.

Main Results:

• The iMAM successfully monitored various CQAs including identity, purity, HMWS, LMWS, N-glycosylation, and drug-to-antibody ratio (DAR).
• Positive MS polarity preserved more siRNA duplexes, while negative polarity showed more single-stranded RNA on ARCs.
• Antibody presence protected siRNA duplexes during ionization, with duplex ratio correlating to siRNA GC content and Tm.

Conclusions:

• The qualified iMAM provides a simple, effective, and GMP-compliant method for ARC characterization and release testing.
• Understanding MS polarity effects on ARCs aids in optimizing analytical strategies for complex bioconjugates.
• These findings contribute to the fundamental knowledge of ARC ionization, facilitating improved analytical method development.