In this case study, we evaluate a range of analytical techniques using a model Trastuzumab-siRNA conjugate, demonstrating why analytical strategies developed for conventional antibody drug conjugates (ADCs), can be less effective when dealing with the unique properties of oligonucleotides. By comparing orthogonal techniques including chromatography, electrophoresis and mass spectrometry, we identify the analytical toolbox required for robust antibody oligonucleotide conjugate (AOC) characterisation.
This study explores how a range of platform techniques perform when assessing critical quality attributes such as conjugation efficiency, oligonucleotide-to-antibody ratio (OAR), molecular integrity and site-specific conjugation. Readers will discover why some widely used ADC assays struggle to differentiate conjugated species, while other methods can provide clear quantitative information and direct confirmation of intact AOC structure.
The results highlight the importance of selecting analytical approaches that exploit the unique physicochemical properties of oligonucleotide payloads. Strong anion exchange chromatography (SAX-HPLC), for example, enables effective separation of conjugated species and provides robust OAR determination, meanwhile, native size exclusion chromatography-mass spectrometry (SEC-MS) delivers definitive confirmation of intact AOC structure and identity. Complementary techniques including capillary electrophoresis (CE-SDS), size exclusion chromatography (SEC-HPLC), reversed-phase chromatography (RP-HPLC) and mass spectrometry (RP-MS) contribute additional insight into conjugation site specificity, size distribution and molecular heterogeneity.
For scientists working in process development, analytical development, formulation, CMC programmes or regulatory preparation, understanding which methods provide meaningful data is becoming increasingly important as AOC pipelines continue to advance. Choosing the wrong analytical strategy can result in incomplete characterisation, reduced confidence in conjugation measurements or missed product attributes that could affect development decisions.
Download this case study from our ADC experts to discover which analytical platforms provide meaningful data and which legacy ADC methods may fail to identify essential information when it comes to AOC analysis.
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Frequently asked questions
Antibody-oligonucleotide conjugates (AOCs) are emerging as one of the fastest-growing classes of targeted RNA therapeutics. Their development is being driven by the rapid expansion of the broader oligonucleotide therapeutics market, which is forecast to grow at approximately 15-20% CAGR through 2030 . The ability of AOCs to combine the targeting precision of monoclonal antibodies with the gene-silencing power of oligonucleotide payloads is attracting significant investment and fuelling a growing pipeline of 235 clinical and preclinical AOC assets as of 15th July 2026 .
Small interfering RNA (siRNA) oligonucleotides induce target protein knockdown through the formation of the RNA-induced silencing complex (RISC) which cleaves messenger RNA (mRNA). Dosing of siRNA has historically been limited to the liver through GalNAc conjugation or lipid nanoparticle (LNP) formulations but conjugation to monoclonal antibodies allows for extrahepatic targeting.
Although AOCs and ADCs share a similar overall concept, their payloads possess fundamentally different properties. Traditional ADCs typically contain cytotoxic payloads which are small, hydrophobic and neutral, whereas oligonucleotides are large, hydrophilic and highly negatively charged. These differences can significantly affect analytical performance and may limit the usefulness of established ADC methods when applied to AOCs.
This distinction creates unique analytical challenges, such as those explored in our ‘Platform analysis of an antibody-oligonucleotide conjugate’ case study.
