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Developing new RNA therapeutics requires years of dedicated effort. However, taking a program from the promise of preclinical models into the reality of clinical development presents the fundamental challenge of scale. Innovators must find a way to manufacture their drug, scaling up from grams to kilograms or even tons, all while maintaining quality, consistency and cost-effectiveness.

 

At this critical point, many innovators face a potential bottleneck because the very manufacturing methods that got them this far begin to hold them back. Conventional solid-phase oligonucleotide synthesis (SPOS), while reliable for discovery and development, was not designed for the large-scale production required for high-volume markets. This disconnect has driven renewed interest in enzymatic alternatives, such as chemoenzymatic ligation, which can overcome these limitations and ensure the supply of these life-saving medicines meets surging demand.

 

Limitations of traditional oligonucleotide synthesis

 

For decades, the standard for creating oligonucleotides has been SPOS, an intensive nucleotide-by-nucleotide approach. While precise for short oligonucleotide chains, using stepwise addition of nucleotide subunits for complex molecules has drawbacks, especially when scaling up for clinical and commercial needs.

 

Cumulative risk from stepwise addition means that with each added nucleotide, there’s a small chance of an error or a side reaction. For long molecules, such as sgRNA, errors accumulate and impurities are challenging to control during synthesis and difficult to remove during purification, increasing impurity levels and batch-to-batch variability.

 

Furthermore, as impurities rise, yields drop. While errors do accumulate, if an error occurs early on, the entire chain is compromised. The longer the chain, the greater the chance of error and the more yield declines, driving up cost and development timelines. 

 

Beyond product quality, the process faces structural and environmental limitations. High solvent use and atom economy are sustainability concerns for both the industry and investors if SPOS alone is used to meet the rising demand for RNA therapeutics. 

 

Finally, there is the challenge of replicating infrastructure to increase scale. Current SPOS processes use flow-through synthesizers that are limited to batch sizes of approximately 10 kg. To meet the multi-ton demand projected for high-volume therapeutics, manufacturing requires a “scaling-out” approach combining batches from multiple synthesis campaigns, increasing production complexity and costs.

 

While a fully enzymatic synthesis that mimics natural processes is a long-term goal, generation 2 oligonucleotide manufacturing technology offers a solution today. One that helps overcome the production bottleneck that lies between promising RNA programs and the end products reaching patients. 

 

The new science of synthesis: A chemoenzymatic alternative 

 

Chemoenzymatic ligation represents a hybrid approach that combines both chemical and enzymatic processes. Oligonucleotide fragments (also known as blockmers) are first synthesized using SPOS, then an enzyme called a ligase joins these fragments together to produce the full-length oligonucleotide. These smaller, more manageable fragments can be produced with much higher purity and yield, providing the high-quality building blocks needed for complex next-generation modalities, such as sgRNA. This elegant modular approach provides several key advantages.

 

Enhanced purity and consistency: Final product purity can be improved for two reasons. First, there is less opportunity for impurities to form and accumulate during the synthesis of shorter fragments. Second, the ligase enzyme is highly selective, helping to reduce the number of deletion impurities. It only accepts fragments with the correctly positioned 5′-phosphate and 3′-hydroxyl groups as substrates, meaning many common impurities are rejected and purged during downstream processing. 

 

Superior yield and cost-effectiveness: The production of fragments is more efficient and higher-yielding than the synthesis of a full-length oligonucleotide. Because the ligation reaction itself is highly efficient, often resulting in near-quantitative yields, the overall process is more cost-effective. 

 

A scalable solution: The ligation reaction is a solution-based process that is well-suited for large-scale stainless steel batch reactors. This approach fits seamlessly within an effective GMP CDMO infrastructure and can support the multi-ton volumes anticipated for future high-demand drugs.

 

A more sustainable footprint: Higher yields and lower reagent consumption inherently lead to less chemical waste, making chemoenzymatic ligation a greener alternative that aligns with modern environmental, social and governance (ESG) principles. 

 

Hongene applies distinct ligation workflows tailored for different oligonucleotide formats. For double-stranded siRNA, sticky-end ligation has already been successfully translated to GMP facilities to support clinical trials. For longer single-stranded sgRNA molecules, a splinted ligation process is used, demonstrating the versatility of the chemoenzymatic ligation for gene editing applications.

 

For greater detail on sticky end ligation and splinted ligation, download the Chemoenzymatic whitepaper here. 

 

Navigating regulatory considerations and future perspectives 

 

As Generation 2 synthesis platforms, such as chemoenzymatic ligation, advance into clinical development, they require careful consideration from a CMC (Chemistry, Manufacturing and Controls) and regulatory perspective. Hongene has proactively addressed key regulatory questions by developing specific control strategies for existing Critical Quality Attributes (CQAs) and implementing methods to control parameters that are unique to the ligation process. This includes: 

 

• Starting material designation: Working closely with partners and regulatory bodies to ensure a clear pathway for fragment classification.
• Phosphorothioate stereochemistry: Developing advanced analytical methods to ensure consistency and equivalence with SPOS-derived materials.
• Residual enzyme testing: Implementing robust assays to test for residual enzymes and conducting studies to assess the immunological impact, mitigating a potential new risk factor.

 

Rigorous development has established chemoenzymatic ligation as a clinically validated and commercially viable platform ready to scale with future RNA modalities. 

 

Powering progress and a sustainable future for RNA medicines

 

As the demand for RNA therapeutics grows, so must the ability to manufacture them efficiently, sustainably and at scale. Fully enzymatic generation 3 manufacturing technology may be the future, but generation 2 chemoenzymatic ligation continues to improve with work on chromatography-free purification to further reduce costs and waste and enzyme engineering programs to improve versatility by enabling more efficient ligation at elevated temperatures. Today, Hongene offers an approach to RNA manufacturing that is rooted in science and ready for the world.

 

To learn more about how Hongene can help bring your RNA to life, please contact our team to discuss your program.