Developing successful RNA therapeutics requires navigating a complex design space. Stability, expression kinetics, immunogenicity, and translational efficiency all depend on sequence features that interact in ways that are difficult to predict computationally. To overcome these in silico limitations, RNA therapeutic developers can instead turn to a lab-in-the-loop cycle where initial candidate sequences are designed, built, and tested. The validation data is then used to improve on the initial designs to achieve improved sequences.

Successful lab-in-the-loop approaches require that the design, build, and test phases are optimized both in terms of their individual processes and how they interact with one another. For example, many design approaches often treat manufacturing as a black box: A sequence is designed and later an RNA appears. This abstraction ignores a critical reality: Not all RNA sequences are equally manufacturable, and manufacturing problems have cascading consequences that corrupt downstream data quality. In this eBlog we review some of the common challenges that can arise with RNA manufacturing and how to overcome them.

DNA fragment synthesis and assembly

The first step in manufacturing custom RNA constructs involves synthesizing DNA fragments that encode your design and assembling them into plasmids for bacterial cloning. However, not every sequence can be effectively synthesized. Sequences with high GC content, repetitive elements, or strong secondary structures can increase error rates during chemical synthesis and can be challenging to assemble into full-length constructs.

If the sequence designs are not screened prior to synthesis, the issues may not be discovered until well into the manufacturing process, often when transformation plates yield no colonies or sequencing reveals incorrect assemblies. For programs trying to evaluate panels of 10-20 candidates, a 20-30% failure rate at this step increases both the costs and timelines.

Eclipsebio addresses these issues through upfront manufacturability assessment and vendor diversity. Our comprehensive design review process flags sequences with predicted assembly risks, problematic motifs, or cloning challenges before making DNA fragments into plasmids.

Plasmid manufacturing and bacterial transformation

Once DNA fragments are assembled into plasmids, the next step is bacterial transformation and clone selection. Specific designs may form toxic peptides in this step, which pressure the bacteria to mutate the plasmid or truncate problematic regions, including homopolymeric stretches such as poly(A) tails. These mutations often aren't discovered until after plasmid purification and sequencing, typically 2-3 days post-transformation. Mutations and problematic regions can negatively impact the entire manufacturing process, especially as drug developers scale up. This lengthens the production timeline and increases cost.

Even when plasmid identity remains intact, these design flaws lower plasmid prep yields while endotoxin levels remain fixed or even elevated. This directly increases the immunogenicity of the input material to the IVT reaction.

At Eclipsebio we use full-length plasmid nanopore-based sequencing at the clone screening stage to confirm 100% sequence identity before scale-up. In addition, we verify poly(A) tail length via Sanger sequencing to ensure no truncation has occurred. We also maintain clone libraries and utilize our design experience and datasets to navigate challenging sequences. The goal isn't just to produce a plasmid; it's to produce a verified plasmid that won't introduce additional variables into your downstream experiments once your process moves to scale.

In vitro transcription

The final manufacturing step, in vitro transcription (IVT), converts linearized plasmid templates into RNA. Here, sequence-dependent effects on transcription efficiency, RNA secondary structure during synthesis, and susceptibility to degradation can reduce yield and percent full-length product. These issues force developers to scale up reaction volumes, using extra DNA that is potentially contaminated with residual endotoxin, reaction proteins, and dsRNA that can carry into the drug substance.

To overcome these challenges, Eclipsebio's IVT workflow includes process selection based on application requirements (precipitation-based purification for standard research applications, oligo-dt based purification for primary cells or in vivo work) and fragment analyzer assessment of integrity to ensure percent of full-length product (%FLP) is greater than 75% ( >80-90% for most constructs). We also provide advanced drug substance characterization through our eMERGE analytics platform to obtain actionable insights into different dimensions of RNA quality, including dsRNA levels and RNA integrity.

Quality control as experimental variable isolation

In every step of manufacturing, quality control is essential. By incorporating analytics at each stage, developers can better distinguish if issues are related to their sequence or artifacts in the manufacturing process.

As an example, consider a common scenario: Two candidate mRNA constructs (Candidate A and Candidate B) are developed that differ in UTR structure. Both sequences are sent to a contract manufacturer, and the Certificates of Analysis from both show acceptable concentration and purity.

During subsequent testing, Candidate A shows 60% of Candidate B's expression level and produces a stronger innate immune response. A reasonable conclusion is that Candidate B's UTR design is superior, and it advances to the next round of optimization.

However, there are several unmeasured attributes that could show the differences are due to manufacturing rather than UTR efficacy:

• Candidate A's plasmid underwent poly(A) tail truncation during bacterial culture. The shortened poly(A) tail reduced translational efficiency and RNA stability, confounding your expression comparison.
• Candidate A had 78% FLP while Candidate B had 91% FLP. (Both passed the manufacturer's ">70% FLP" spec, so it wasn't flagged.) This would then lead to changes in protein production and immunogenic responses.
• In this scenario, you're not comparing UTR designs, you're instead comparing manufacturing runs. The conclusion that Candidate B is superior might be correct, or it might be an artifact of differential manufacturing quality.

This is the core problem with treating manufacturing as a black box: Without rigorous quality control at each process step, you cannot isolate sequence-dependent effects from manufacturing-dependent effects.

At Eclipsebio our multidimensional quality control approach is designed for variable isolation:

• Sequence verification: Oxford Nanopore sequencing of plasmid clones confirms 100% identity to design across all functional regions (5' UTR, ORF, 3' UTR) and verifies poly(A) tail integrity before scale-up. You know you're testing the sequence you designed, not a mutated variant.
• Integrity assessment: Fragment Analyzer measurement of %FLP ensures material meets specifications (>75% minimum, typically >80-90%). Truncated products are quantified, not hidden.
• Purity characterization: Spectrophotometry (A260/A280, A260/A230) provides baseline assessment of contaminants. Advanced quality control with our eMERGE analytics platform provides detailed insights into other aspects of RNA quality, capturing details that are missed by other approaches such as changes in dsRNA content between batches.
• The result is that when you test Candidate A versus Candidate B, you're making a controlled comparison. Differences in expression or immunogenicity can be attributed to sequence features with confidence because manufacturing variables have been measured and minimized.

Manufacturing and design knowledge compounds over iterations

Through manufacturing hundreds of constructs, troubleshooting challenges, and recognizing patterns that predict problems before synthesis, Eclipsebio has built a knowledge base for RNA design. We use this knowledge throughout our manufacturing process, especially in our upfront sequence assessment.

This knowledge allows us to complete the manufacturing cycle quickly, generating a greater number of high-quality mRNA sequences for functional RNA-based therapeutics.

Manufacturing-informed design allows for:

• Rapid prototyping with data confidence: Since we know that every construct meets sequence identity, integrity, and purity specifications, we can attribute differences in functional readouts between constructs to sequence features, not manufacturing artifacts.
• Assay readout clarity: Our advanced quality analysis assays quantify residual contaminants including dsRNA and proteins, enabling us to interpret results with confidence that unexpected immune activation or toxicity isn't due to manufacturing carryover.
• Exploration of design space: Knowing where the boundaries of manufacturability lay allows us to further explore the sequence space, gaining a broader understanding of their RNA’s effects.

Eclipsebio's approach integrates manufacturability expertise at the design stage, implements QC gates throughout the build process, and provides full traceability for every batch. The result is research-grade RNA that enables confident iteration to know what is tested, attribute differences between constructs to sequence, and inform new designs with data from each cycle.

Interested in how manufacturing-informed design can accelerate your program? Contact Eclipsebio today.