# Polarity- and Sequence-Dependent Ionization of Therapeutic Antibody–siRNA Conjugates: Enabling Intact Multi-attribute Method for Comprehensive Characterization and Identity Release Assay

## TL;DR

This paper introduces a new method for analyzing antibody–siRNA conjugates using mass spectrometry, which helps in understanding and monitoring their quality during drug development.

## Contribution

The study introduces an intact multi-attribute method (iMAM) for characterizing antibody–siRNA conjugates using native SEC-MS.

## Key findings

- Ionization profiles of ARCs depend on mass spectrometry polarity, affecting siRNA preservation.
- Antibody presence protects siRNA duplexes during ionization compared to siRNA alone.
- siRNA duplex ratios correlate with GC content or melting temperature (Tm).

## Abstract

Antibody–siRNA conjugates (ARCs) represent a promising
approach
for delivering small interfering ribonucleic acids (siRNAs) to targeted
cells, tissues, or organs. The complexity of the molecules poses great
analytical challenges in identifying, characterizing, and monitoring
their critical quality attributes (CQAs) during process development
and manufacturing release. We developed a novel approach, intact multi-attribute
method (iMAM), for ARC characterization using native size exclusion
chromatography mass spectrometry (SEC-MS). The iMAM provides a simple
and effective approach for monitoring CQAs such as identity, purity,
higher molecular weight species (HMWS), lower molecular weight species
(LMWS), N-glycosylation, modifications on unconjugated cysteine, linker
hydrolysis, and drug-to-antibody ratio (DAR). The method was evaluated
and qualified in a Good Manufacturing Practice (GMP) environment as
an ARC drug substance (DS) and drug product (DP) identity release
assay. During the method development, we found that ionization profiles
of ARCs depended on the mass spectrometry operating polarity. Under
positive polarity, more duplex siRNAs are preserved on the ARC molecules,
whereas more ARC molecules with single-stranded RNA are present under
negative polarity. Meanwhile, the presence of the antibody in ARCs
provides a certain level of protection for the siRNA duplex during
the ionization compared with siRNA alone. The ratio of the siRNA duplex
on the ARC molecules during mass spectrometry detection was correlated
with its GC content or melting temperature (T
m). These findings provide a fundamental understanding of the
relationship between siRNA and the antibody during ARC ionization
and facilitate the development of effective methods for ARC characterization.