From The Editor | July 11, 2025

Enzymatic Ligation: The Next Chapter In siRNA Manufacturing

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By Anna Rose Welch, Editorial & Community Director, Advancing RNA

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In May, I had the opportunity to attend my first ever TIDES. Having spent the first year-plus of my tenure with Advancing RNA learning the ins-and-outs of IVT for mRNA production, I figured it was high time I started demystifying the evolving manufacturing paradigm for oligonucleotides — namely siRNA. What I didn’t anticipate is that I’d find an industry on the cusp of transformation, albeit by building its future manufacturing paradigm around a common (but still multi-faceted) oxymoron: To go big, we must start small.

Of course, broadly speaking, we’re no stranger to the concept of starting small to make something bigger and better; the general trajectory of drug development (with smaller scale clinical development), as well as scale-down models being familiar examples. However, we can also look at this oxymoron through a different lens in the context of oligo manufacturing. Scott May, VP of chemistry-genetic medicines of Eli Lilly, said it best: To make a more high-quality and reproducible siRNA duplex, we need to “break the full strands apart into small pieces and handle those small pieces in more efficient ways.”

Enter enzymatic ligation.

During my three days at TIDES, I sat through several talks about the industry’s progress toward enzymatic ligation approaches for siRNA manufacturing. In the following article, I’ll share a few of the biggest takeaways I garnered from these different presentations, with a particular focus on the efficiencies the industry hopes such efforts unlock in the long-term.

Enzymatic Ligation: The Bridge Between The Old & “New” Manufacturing Worlds

As numerous speakers emphasized throughout the conference, we cannot underestimate the power of solid-phase synthesis (SPOS) considering it’s the backbone on which we’ve developed and commercialized our oligonucleotides to date. However, like most sectors that have come before ours, the goal of treating larger patient populations with oligos is making it increasingly necessary to improve the scalability of the manufacturing paradigm.

“SPOS is a powerful technology, and I would argue it’s maybe unbeatable in the discovery phase,” May explained. “It’s great for small volume products. But like many technologies, one approach isn’t great for everything. The more product we want to make, the more we start challenging our current manufacturing infrastructure.” 

Of course, the industry is already working on improving said-infrastructure, whether it be by refining current SPOS approaches, or by innovating with next-gen manufacturing approaches. If we look at SPOS, in particular, there is ongoing work being done to move from flow-through to batch reactors to help overcome production volume constraints. As May went on to describe, he and his colleagues at Lilly have been working on an oscillating fluidized bed reactor to promote more efficient solvent & reagent use (or reuse) and to operate at previously unimaginable bed heights to produce full strand oligos.

If we look beyond SPOS, solution-phase synthesis and enzymatic synthesis are both glimmering on the horizon, promising more efficient oligo manufacturing in the future.

However, I appreciated Hongene CTO David Butler’s breakdown of the industry’s progress thus far, in which he sees enzymatic ligation as the second generation of oligo manufacturing sitting firmly between SPOS and next- (next-) gen enzymatic synthesis.

“We believe that chemoenzymatic ligation of oligonucleotide fragments is going to play a huge part in oligo manufacturing over at least the next 10 years,” Butler said.

How Pure is Pure Enough? Exploring Fragment Quality & Process Efficiency

As you can imagine, the enzymatic ligation approach raises a few key questions. For example, whether the fragments should be treated as regulatory starting materials as opposed to an intermediate was raised multiple times throughout the conference. But overwhelmingly, each conversation I listened to on the topic of enzymatic ligation was seemingly centered around one big question: How pure is pure enough?

It was widely acknowledged throughout the conference that starting with oligo fragments and pursuing an enzymatic ligation approach promises a purer product. And, as Butler nicely summarized, there are two reasons for this.

“The SPOS-related impurities are generally greatly reduced in the fragments compared to what you would see in a typical full-length sequence manufactured with [SPOS] technology,” he explained. “We think the reason for this is quite simple; there’s less opportunity for these types of impurities to compound and accumulate for a shorter oligo compared to a longer oligo. Secondly, the oligo fragment impurities are not great substrates for the ligase.”

Similarly, May sees the fragment approach as a way for us to get even smarter about our overall processes. Rather than simply acknowledging that an impurity exists within the fragment, the more important work is to figure out how each impurity may react in the overall process and/or be incorporated into the final siRNA duplex.

Perhaps it goes without saying: The purer the starting fragments, the more efficient the ligation process and the purer the siRNA duplex will be post-ligation. In turn, the industry’s current manufacturing process for proof of concept and/or first-in-human studies relies on multiple chromatography steps. The pre-ligation fragments themselves are purified using a chromatography method, as is the resulting siRNA duplex post-ligation. (Butler abbreviated this as the P-to-P strategy — purified fragments and purified siRNA.)

However, it shouldn’t come as a surprise that we’re starting to question which units of operation (or rather just how many) units of operation are necessary to produce a high-quality product while also reducing cycle times and keeping COGS in mind. Though the P-to-P approach may be the go-to today, each conversation I sat in on broached the possibility of eliminating chromatography for fragment purification, using crude fragments to create the siRNA and purifying the final siRNA duplex using chromatography (a.k.a. the C-to-P approach — crude fragments, purified siRNA).

As Derek Gauntlett, head of ECO process development for Codexis, argued in his presentation/demonstrated via data: Crude RNA fragments can generate a high-quality siRNA duplex. Data shared across manufacturers demonstrates very close purity percentages between P-to-P [97 percent duplex purity] and C-to-P methods [95-98 percent duplex purity]. But why stop there; Butler even posed the question of whether a C-to-C strategy (crude fragments, UF/DF-purified siRNA) could be a viable future possibility for high-volume products.

“This could provide the advantages you need in terms of cost for these large volume cardiovascular disease drugs, while hopefully providing the necessary quality characteristics,” he concluded.