The "Middle Ground" Advantage: Why Alnylam Is Betting Big On Chemoenzymatic Ligation
By Anna Rose Welch, Editorial & Community Director, Advancing RNA
Success breeds its fair share of challenges.
I’ll admit, on the surface, this sounds like a pessimistic way to start an article. But I daresay if you step back and consider it, you’ll realize there’s an element of truth to this statement — especially in the RNA space.
You don’t have to look too closely at the oligo market to realize we’ve had a few successful years. As this previous Advancing RNA column nicely laid out, we’ve seen a (rather big) handful of products approved that have generated billions in revenue across companies and have been an answer for patients in both small and large patient populations. However, the approval of Novartis’ Inclisiran for reducing cholesterol in hyperlipidemia patients was perhaps the industry’s biggest wake-up call that we were entering a new era. Though this was a noteworthy approval for many reasons, it also made us keenly aware of the manufacturing and supply chain barriers we are likely to face if we want to continue bringing oligos into the treatment armamentarium for large disease indications.
No one is seemingly more aware of these barriers than Alnylam, which has in its pipeline several potentially high-volume programs targeting obesity, Type 2 diabetes, and hypertension. This is why, in December of last year, Alnylam announced two major advancements — the first being a planned expansion of its Norton, MA manufacturing site to house a dedicated chemoenzymatic ligation operation; the second being that its enzymatic ligation platform siRELIS™ was a recipient of the FDA’s Emerging Technology Program. The facility, upon its opening in late 2027, is poised to be the RNA industry’s first facility dedicated to enzymatic ligation.
Though conversations are picking up around this hybrid approach, we aren’t exactly known as an industry that embraces risk no-holds barred. While there are a handful of manufacturers and CDMOs who are embracing the hybrid model today, there are many more that are likely to hold out for the next-next gen approach: Fully enzymatic oligo production. However, as Alynlam’s CTO and Chief Quality Officer Tim Maines argued, embarking into the “middle ground” of enzymatic ligation is essential for garnering the step-by-step learnings needed to help us unlock even bigger manufacturing wins in the future.
“Enzymatic ligation is the only scalable path for large-population RNAi programs today, and the work we’re doing now will shape how regulators evaluate and ultimately standardize next-generation manufacturing,” Maines offered.
Here, in part one of this two-part article, Maines and Alnylam’s Chief CMC Officer Lubo Nechev share the “why’s” behind the company’s strategic shift to enzymatic ligation. In part 2, we’ll tackle some of the questions that remain about this hybrid approach. Stay tuned!
A Hybrid Approach The Key To Democratizing RNAi
My conversation with Nechev and Maines served as a great reminder of several important facts:
One, solid phase synthesis isn’t going anywhere. For those with small programs requiring less than 10 kilograms of product a year, solid phase synthesis remains the gold standard.
Two, enzymatic ligation is hardly new. Back in 1968, a professor at Harvard penned the first paper articulating the benefit that would come from using ligase to stitch together shorter oligo fragments or blockmers, as opposed to making a full-length oligo.
“Enzymes have been used for research for a long time, particularly in the small molecule industry where, for example, an enzyme can be used to remove ester groups from a small molecule,” Nechev explained. “The least novel aspect of what we are doing now in the siRNA space is using an enzyme. The novelty is that we are coming up with a hybrid method using solid phase synthesis to create the blockmers and stitching them together using an enzymatic process. And the beauty of this method is that your final product is identical to that of a final product made by solid phase synthesis.”
As we’ve discussed in the past here on Advancing RNA, there are a handful of important benefits this paradigm promises siRNA makers — including the potential opportunity to reduce purification steps. While we are currently using chromatography on both the fragments and the finished siRNA molecule, there are of course visions of eliminating at least one — if not both — chromatography steps on the fragments and duplex. Though enzymatic ligation does promise marginally higher purity, especially when looking to create oligos comprising 100-150 nucleotides, as Nechev went on to explain, current siRNA purity is already high, coming in between 95-98%. As such, it’s unclear whether achieving a product boasting a 99.5% purity profile will provide any additional meaningful clinical benefit.
In turn, enzymatic ligation is predominantly being touted as the necessary step the industry must take to achieve high product volumes.
“With our current solid phase synthesis process now, the largest batch we can produce is 20 kilos of siRNA,” Nechev said. “We make 10 kilos of each single strand and put them together to create 20 kg. Though we haven’t achieved these production volumes yet using our new ligation process, our calculations tell us we will be able to make between 300-500 kg of product in a single batch using this new process.”
As Maines continued, producing such high quantities of product will promote a much lower COGS per unit of drug substance, and, as is also to be expected, will enable us to gravitate toward a much more sustainable manufacturing process (i.e., less solvent).
“Enzymatic ligation is the rare case where you achieve the holy trinity: Orders-of-magnitude higher volume, materially lower COGs, and a dramatically smaller environmental footprint,” Maines concluded. “The destination is fully solvent-free, liquid-phase synthesis enzymatically, and we’re already advancing both. But for now, hybrid ligation is the bridge to new markets & patient populations that previously could not access and afford such important therapies.”
Like what you’re reading? Maines & Nechev will be back in part 2, where we’ll discuss the biggest surprises and ongoing questions surrounding the implementation of this hybrid manufacturing paradigm.
For more on enzymatic ligation & related topics, please check out more of our articles: