Outsourced Pharma: Top 5 Challenges in Antibody-Oligonucleotide Manufacturing

The Hard Part of an AOC Isn’t the Biology. Here, our resident expert, Ian Glassford, discusses five places antibody-oligonucleotide (AOC) manufacturing breaks programs — and what it takes to get ahead of them.[IG1.1]

Antibody-oligonucleotide conjugates (AOCs) were a brilliant idea and one of those rare cases where the biological logic behind them was incredibly neat: take an oligonucleotide, a short, sequence-specific nucleic acid that can silence, splice, or redirect almost any gene you care to name, and attach it to an antibody that delivers it precisely to the cell type that needs it. What you end up with is targeted delivery of gene-modulating therapy where the antibody does the navigation and the oligonucleotide does the work.

And it’s an idea that’s led to real clinical momentum. Avidity’s AOC 1001, the first AOC to enter clinical trials, reached Phase III for myotonic dystrophy in 2024 and received FDA Breakthrough Therapy designation the same year (1), Dyne Therapeutics has advanced its lead Duchenne candidate, z-rostudirsen (DYNE-251), through the Phase 1/2 DELIVER trial (2) and, in 2026, both initiated the confirmatory Phase 3 FORZETTO trial and submitted a BLA to the FDA seeking accelerated approval in exon-51 DMD (3), and Novartis moved fast enough on the idea that it announced a $12 billion acquisition of Avidity – a company built almost entirely around AOCs – in October 2025, completing the deal in February 2026 (4).

But, however much promise AOCs hold, there’s no escaping the challenges inherent with manufacturing these therapeutics, as it’s far from simple to move from ‘elegant biological concept’ to ‘reproducible, regulatorily acceptable drug product.’ This is why it’s worth understanding the specific places where AOC development can break timelines and whole development programs if you’re not prepared for them.

1. Oligonucleotide synthesis creates impurities that are hard to remove

Let’s start with the oligonucleotide itself. The reason these are so therapeutically appealing (their ability to bind with sequence-specific precision to virtually any RNA target, including ones that small molecules can’t touch) is inseparable from the reason they’re so challenging to manufacture.

Oligos are synthesized by solid-phase phosphoramidite chemistry, where nucleotides are added one at a time to a growing chain anchored to a solid support, where each addition follows a four-step cycle of detritylation (deprotection), coupling, oxidation or sulfurization, and capping. This has been the industry standard for nearly forty years, and it works. But at each coupling step, a small fraction of chains may fail to extend, and when you multiply that fractional failure across twenty or more cycles, you accumulate a population of truncated sequences (n-1, n-2, missed incorporations) that are chemically similar enough to the target product to resist easy purification. Incomplete deprotection of intermediate groups adds another impurity category, which can affect target binding, immunogenicity, and toxicity in ways that matter to regulators.

The phosphorothioate (PS) backbone modifications that many therapeutic oligos carry (where a non-bridging oxygen in the phosphate group is replaced with sulfur to improve nuclease resistance) add a further wrinkle. Each PS linkage creates a chiral center at phosphorus and yields Rp and Sp diastereomers with measurably different pharmacological properties, such as nuclease sensitivity, stability, and interaction with enzymes involved in the antisense and RNAi pathways (5). A 20-mer with a fully PS backbone has the theoretical capacity to exist as 219 diastereomeric species, and while you don’t need to separate them all, you do need to characterize them, understand the distribution, and defend that distribution to regulators.

Then there are secondary structures, since oligos aren’t passive strings of nucleotides waiting to be conjugated. Under physiological buffer conditions they can fold into structures such as hairpins, G-quadruplexes and self-complementary duplexes. If the oligo folds into an unintended structure, target engagement, hybridization kinetics, conjugation efficiency, or potency can change. These secondary structures aren’t always detectable by standard analytical methods, and they don’t always show up at the conjugation step. Instead, they make themselves known later when you’re wondering why your potency data looks odd.

And these are issues you have to contend with before we’ve even discussed attaching the oligo to anything.

2. AOC conjugation has to keep both targeting and payload function intact

Although an AOC is effective once it’s assembled, the antibody and the oligo aren’t exactly natural companions. The antibody is a large, relatively hydrophobic protein evolved to sit comfortably in plasma, while the oligonucleotide is a polyanionic, hydrophilic molecule whose entire therapeutic rationale depends on getting inside a cell. Joining them stably and reproducibly, while preserving the function of both components, is the central technical challenge of AOC manufacturing, and it can fail in several places at once if you’re not careful.

The most common conjugation approach uses maleimide-thiol chemistry, where the maleimide group on the linker reacts with free cysteine residues on the antibody. The maleimide ring is reactive, reasonably site-selective, and well understood from years of Antibody-drug conjugate (ADC) development. It’s also susceptible to hydrolysis in aqueous environments, which is inconvenient for a molecule that will spend its working life in an aqueous environment. Hydrolysis of the maleimide linker before it has coupled to the cysteine can reduce conjugation efficiency and introduce unreacted linker-oligo intermediates as product-related impurities[IG3.1][KB3.2]. In addition, conjugated maleimides are susceptible to post-conjugation retro-Michael/thiol-exchange reactions and deconjugation in plasma. The payload can transfer to albumin and glutathione in circulation causing off-site toxicity. At Abzena, we have the option to use ThioBridge®[IG4.1], a site-specific conjugation chemistry that sidesteps the maleimide problem altogether. Rather than relying on a maleimide, it selectively reduces the antibody’s native interchain disulfides and re-bridges them with a bis-sulfone reagent across a stable three-carbon bridge, restoring the disulfide connectivity instead of leaving an unstable thiosuccinimide behind. The result is a stable, homogeneous conjugate with low, defined, site-specific loading—exactly the control you want at the point where the standard maleimide approach introduces instability.

Linker design involves choices that will affect the whole development process. Cleavable linkers (those that release the oligo in response to the endosomal pH drop, lysosomal enzymes, or the reductive environment of the cytoplasm) are essential if the payload needs to be released intracellularly to function. But a linker that cleaves efficiently inside a late endosome needs to be stable enough in plasma to survive the journey there without shedding oligo into circulation. Non-cleavable linkers solve the stability problem but need lysosomal antibody degradation to release the payload, which introduces a different timing question and makes endosomal escape even more challenging (more on that shortly). Linker length matters, too: too short and steric hindrance from the bulky antibody blocks the oligo from engaging its target; too long and you create new aggregation risks. There is no default setting and every linker decision is a set of trade-offs, and the right set of trade-offs for your antibody-oligo combination will have to come from empirical data with a structured panel of conditions to resolve differences.

The oligonucleotide-to-antibody ratio (OAR) – the AOC’s equivalent of DAR in an ADC – adds another layer. OAR directly governs efficacy, safety, and pharmacokinetics (6, 7), but controlling it is harder than controlling DAR. Crucially, the optimal OAR is payload-dependent: charged payloads such as siRNA and ASOs tend toward low, precisely defined loading, because each additional copy adds anionic charge and hydrophilicity that can accelerate clearance, whereas neutral chemistries such as the phosphorodiamidate morpholino oligomers (PMOs) used in exon-skipping programs can tolerate higher loading. This is why site-specific chemistry that delivers a low, homogeneous, well-defined OAR is so valuable: it gives you a single, characterizable species to develop around rather than a distribution to defend[IG5.1]. The standard DAR analytical method, hydrophobic interaction chromatography (HIC), works because small-molecule payloads create measurable hydrophobicity differences between DAR1, DAR2, and DAR4 species. Oligos, being large and negatively charged, contribute essentially nothing to hydrophobicity shifts between OAR species, meaning HIC can be less useful for OAR determination. Anion exchange chromatography (AEX), capillary zone electrophoresis-mass spectrometry (CZE-MS), reduced capillary gel electrophoresis, and size-exclusion chromatography-mass spectrometry (SEC-MS) are the validated alternatives, with the caveat that mass spectrometry in positive ion mode can underestimate OAR because the oligo’s negative charge suppresses the signal of high-OAR species. This means you need to rely on multiple orthogonal methods for every batch.

3. AOC analytical characterization requires more than standard ADC methods

AOCs trend toward subQ administration and high concentration formulations which is a key capability that differs from ADCs. AOCs are analytically novel in a way that is more disruptive than it first appears. They straddle two fully developed analytical frameworks (the antibody characterization and the oligo characterization toolkits) that were each developed independently and are, in several respects, mutually incompatible when applied to the conjugate.

UV absorbance at 280 nm, the standard method for antibody quantification, is confounded by oligonucleotide absorbance at 260 nm. SEC retention times are altered by column-sample interactions specific to the conjugate that don’t occur with either component alone. Mass spectrometry for conjugation site mapping (a well-established technique for ADCs) is complicated by the oligo moiety, which requires nuclease pre-digestion of the oligo before the antibody can be analyzed, which in turn introduces its own impurity risks if the digestion is incomplete. Platform methods developed for monoclonal antibodies fail, in several cases, to accommodate RNA’s electrochemical properties at all.

The practical consequence is that method development for a new AOC is not a matter of selecting from an established menu. Instead, it’s genuine development work that needs dedicated time, material, and expertise, which translates directly into timeline costs and the quality of data available to support early manufacturing decisions. At Abzena, our analytical suite was built specifically for this dual-biology problem: SEC-MS, IEX, ICP-MS, UV correction for oligo absorbance, and hybridization-based bioassays for OAR and activity, all run as orthogonal verification rather than sequential steps. This is because no single method tells you the full picture, and acting on incomplete analytical data in AOC development tends to be expensive.

4. Intracellular delivery and endosomal escape can limit AOC potency

Even a perfectly manufactured AOC, with an ideal OAR, stable linker, and clean impurity profile, still has to get its payload into the right intracellular compartment to do anything useful. This is where AOC development confronts a problem that is partly biological and partly unsolved.

After binding its target antigen, the AOC is internalized by the cell, typically via clathrin-mediated endocytosis (8). It enters an early endosome, which acidifies as it matures toward the late endosome and eventually the lysosome. For the oligo to reach the RNA-induced silencing complex (RISC) (for siRNA), the nucleus (for splice-switching ASOs), or any other cytoplasmic target, it has to escape the endosome before lysosomal hydrolases degrade it. This endosomal escape step is the rate-limiting barrier to oligonucleotide delivery. Quantitative NanoSIMS microscopy studies have shown that only 1–2% of GalNAc-conjugated ASOs escape hepatocyte endosomes in vivo (9, 10). For AOCs, the escape rates are likely comparable since the lipid bilayer of the endosomal membrane sequesters roughly 99% of internalized RNA therapeutics (11).

Linker design is the primary manufacturing solution for this problem. Cleavable linkers responsive to endosomal acidification or enzymatic activity can help earlier payload release, giving the oligo a better chance of cytoplasmic escape before lysosomal degradation. This is why, at the lead characterization stage, it’s important to assess endosomal trafficking and escape, because they tell you whether your linker strategy is actually working in the biological context that matters, not just in plasma stability assays.

The honest caveat here is that endosomal escape is not fully solved at the field level (10). It’s a constraint that shapes what AOC targets work well (TfR1, which undergoes rapid recycling and efficient intracellular trafficking, is the dominant clinical target for this reason) and what targets remain difficult. No manufacturing capability addresses this entirely since it’s a biological reality of the endosomal pathway that AOC developers are forced to engineer toward and measure carefully. Acknowledging this is the kind of clarity that stops programs from failing for the wrong reasons.

5. AOCs need a product-specific regulatory strategy

Regulators have yet to issue AOC-specific CMC guidance. The FDA has issued oligo-focused guidance for clinical pharmacology and nonclinical development, while EMA published a draft guideline on the development and manufacture of oligonucleotides in 2024, which includes quality expectations for manufacturing, characterization, specifications, analytical control, and conjugation. [IG6.1]

But AOCs still sit across oligonucleotide, biologic, and bioconjugate precedent. This means that CMC packages for AOC programs need to be built from first principles, drawing simultaneously on ADC precedent, oligonucleotide precedent, and regulatory science that the field is still generating. Diastereomeric purity limits, co-eluting impurity thresholds, immunogenicity assessment requirements for novel chemical modifications, stability acceptance criteria – none of these have settled regulatory standards for AOC conjugates. Every program is, to some degree, negotiating these standards as it goes.

The practical requirement is regulatory foresight built into manufacturing process development from the start. If your conjugation chemistry produces a diastereomeric distribution you can’t defend, you need to know that at process development stage, not at pre-IND meeting. If your analytical methods don’t resolve OAR species with the resolution a reviewer will expect, that gap needs to be identified and addressed before it becomes a deficiency letter. At Abzena, our global CMC and regulatory teams are embedded in program development from early process design, because the regulatory strategy and the manufacturing strategy are, for AOCs, the same strategy.

6. Sustainability pressures are emerging in AOC manufacturing

Beyond our top five manufacturing challenges, there’s another issue that sits at the edge of most AOC development discussions, which is exactly why we think it’s worth discussing. Oligo manufacturing has a sustainability problem that, as production scales up, is increasingly unavoidable.

Process mass intensity (PMI), the total mass of raw materials consumed per kilogram of API produced, sits between 3,000 and 7,000 for therapeutic oligos (12). The median for small-molecule APIs is 170–300 (13). Oligo synthesis is, by this measure, roughly ten to forty times more material-intensive than small-molecule manufacture, driven primarily by the wash solvents required at each synthesis cycle and the preparative chromatography needed for purification, which alone accounts for approximately half of all process materials used. The solvents involved include toluene, acetonitrile, dichloroacetic acid, pyridine, and triethylamine, several of which carry REACH classification concerns and require specialist waste management.

These inefficiencies are inherent to the phosphoramidite synthesis cycle, being stoichiometric rather than catalytic reagents, with extensive protecting group usage and cumulative yield losses that scale with oligo length. Enzymatic synthesis alternatives, which would offer genuinely greener process chemistry, don’t yet scale to the chemically modified nucleotides that therapeutic oligos require. And lyophilization, the standard isolation method, consumes more than 2 kWh per kilogram and represents a multi-day production bottleneck per batch.

While this isn’t a regulatory hurdle for AOCs right now, as clinical programs scale, oligo API demand will increase, and so solvent use, PMI, waste handling, and lyophilization bottlenecks will become harder to ignore. Developers should ask oligo suppliers and CDMOs about PMI and waste strategy early, because sustainability is also a cost and supply-chain question.

The benefit of overcoming AOC manufacturing challenges

Despite the challenges around manufacturing AOCs, the biological upsides are very real. AOCs can silence disease-driving genes in cell types, from skeletal muscle to the cardiac tissue, that other conjugates simply don’t reach. They can address targets that small molecules can’t touch and complement the ADC toolkit with a fundamentally different mechanism of action.

The path to a successful AOC in the clinic rests on the integration of oligonucleotide chemistry expertise, bioconjugation science, analytical method development, formulation capability[IG7.1], and regulatory understanding into a development program where these functions are actually talking to each other. That integration is what we do at Abzena, across a platform built out of more than 400 conjugate programs, and specifically adapted for the dual-biology demands that make AOCs different from everything that came before them.

Interested in how Abzena approaches AOC conjugation process development and analytical characterization? Get in touch or take a look at our on-demand webinar, Solving the AOC Puzzle: Strategies for Chemistry, Manufacturing and Regulatory Success.

References

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2. DYNE-251 for DMD, Dyne Therapeutics. https://www.dyne-tx.com/dyne-251-for-dmd/.
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4. Novartis successfully completes acquisition of Avidity Biosciences, strengthening late-stage neuroscience pipeline and advancing xRNA strategy, Novartis. https://www.novartis.com/news/media-releases/novartis-successfully-completes-acquisition-avidity-biosciences-strengthening-late-stage-neuroscience-pipeline-and-advancing-xrna-strategy.
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