High-Resolution AOC Analysis with Proteomix® IEX

The analytical challenge of AOC characterization

Antibody-oligonucleotide conjugates (AOCs) combine the target specificity and favorable pharmacokinetic properties of antibodies with the unique biological function of oligonucleotides. This combination is opening new possibilities for targeted delivery, therapeutics, diagnostics and other emerging applications.

However, AOC synthesis can produce a complex mixture. Depending on the conjugation strategy, the final sample may contain unconjugated antibody, AOCs with different degrees of conjugation and residual free oligonucleotide. Accurately determining the degree of conjugation, commonly expressed as the Oligonucleotide-to-Antibody Ratio (OAR), is therefore important for assessing product consistency, conjugation efficiency and purification performance.

An effective analytical method must distinguish these closely related species while accommodating the different physicochemical properties of the antibody, oligonucleotide payload and linker.

Separating AOC variants by charge

Ion-exchange high-performance liquid chromatography (IEX-HPLC) provides a direct, charge-based approach to AOC characterization. Each attached oligonucleotide generally increases the overall negative charge of the conjugate. As a result, AOCs with different OAR levels interact differently with an ion-exchange stationary phase.

Strong anion-exchange (SAX) chromatography is particularly useful for exploiting these charge differences. Conjugates with higher oligonucleotide loading generally bind more strongly to the positively charged SAX stationary phase and elute later during a salt or pH gradient.

This retention mechanism makes SAX suitable for profiling AOC conjugation variants and monitoring changes in OAR during method development, production and purification.

The Proteomix IEX platform offers four important advantages for AOC characterization:

• High-resolution charge separation: Proteomix SAX resolves unconjugated antibody, AOC conjugation variants and free oligonucleotide in one single analysis.
• Biomolecule-oriented surface chemistry: The nonporous PS/DVB particles feature a hydrophilic surface layer designed to reduce nonspecific interactions and support efficient, reproducible separations.
• Flexible separation modes: Proteomix SAX can be used independently or combined with Proteomix SCX in a tandem configuration for samples requiring complementary charge selectivity and milder conditions. Together, these modes allow researchers to adapt the analytical strategy to the antibody charge, oligonucleotide payload, linker chemistry and stability requirements of each AOC.
• Scalable purification formats: Semi-preparative and preparative columns, with dimensions up to 30 × 250 mm (internal diameter × length), support higher sample loads and larger-scale AOC purification, extending the Proteomix IEX platform from analytical characterization to purification workflows.

High-Resolution AOC profiling with Proteomix® SAX

Sepax Technologies evaluated a Proteomix® SAX-NP5 column for separating an unconjugated antibody, two AOC conjugation levels and free oligonucleotide.

Under Tris buffer conditions at pH 8.5, the method provided clear retention and separation of all four sample components. The unconjugated antibody eluted first, followed by AOC OAR-1 and AOC OAR-2. The free oligonucleotide eluted last and displayed additional charge-variant peaks.

The retention order correlated well with the increasing contribution of the negatively charged oligonucleotide payload. This demonstrates how Proteomix SAX converts differences in conjugation level into distinct chromatographic profiles, enabling direct comparison of the unconjugated antibody, individual AOC variants and free oligonucleotide within a single analysis. These capabilities support both qualitative assessment of sample heterogeneity and quantitative OAR analysis.

For researchers developing or optimizing an AOC, this profile helps answer practical questions:

• Does the reaction yield the target conjugate, and are unconjugated precursors fully consumed?
• What is the conjugation efficiency, OAR distribution, and relative abundance of heterogenous species?
• Does the chromatographic resolution provide sufficient peak capacity to support method translation to semi-preparative and preparative-scale purification?

Extend AOC analysis to milder conditions

AOC stability reflects the combined behavior of the antibody, oligonucleotide and linker. Although strongly basic conditions may improve SAX retention for some samples, prolonged exposure can affect antibody structure, linker stability or sensitive RNA-based payloads.

To expand method flexibility, Sepax combined Proteomix SCX and SAX columns in tandem. The two stationary phases provide complementary charge-based interactions. The SCX segment provides interaction with positive charge domains on the antibody component, while the SAX segment strengthens retention as the negative-charge contribution from the oligonucleotide increases.

The tandem method therefore complements the strong performance of standalone SAX: Proteomix SAX provides effective separation of the complete AOC mixture, while SCX + SAX extends the platform to AOCs requiring milder analytical conditions.

From conjugation development to purification monitoring

Proteomix IEX can support AOC development from initial conjugation studies through purification optimization and product characterization. Potential applications include:

• Monitoring conjugation reaction progress
• Resolving OAR distribution profiles and product heterogeneity
• Detecting unconjugated antibody and residual free oligonucleotide
• Screening and comparing conjugation reaction conditions
• Evaluating fraction purity during downstream processing
• Ensuring batch-to-batch process consistency

By resolving the major components of an AOC mixture, the method provides actionable information for optimizing reaction conditions, selecting purification strategies and monitoring changes throughout development.

Explore Proteomix IEX for your AOC

Reliable AOC characterization requires more than detecting the conjugated product. Separating the unconjugated antibody, individual conjugation levels and free oligonucleotide provides deeper insight into conjugation efficiency, product heterogeneity and downstream purification.

Proteomix SAX provides a direct, high-resolution approach for separating unconjugated antibody, AOC conjugation variants and free oligonucleotide in a single analytical run.

Download the complete Sepax AOC application note to review the chromatograms, column configurations and experimental conditions.

Need help selecting an IEX column or developing a method for your AOC? Contact your Bio-Connect account manager or our technical support team to explore the most suitable Proteomix IEX column and method-development strategy for your sample.