# Chapter 4 — Solid-Phase Remains the Default, but the Competitive Edge Is Shifting to Liquid-Phase and Enzymatic Ligation

Solid-phase phosphoramidite synthesis (SPOS) produced every approved GalNAc-siRNA drug to date and retains the only unambiguous GMP precedent for 2'-modified therapeutic oligonucleotides. Yet three converging developments are eroding that dominance for dual-target constructs specifically: the cumulative yield math of SPOS deteriorates sharply above ~40 nucleotides; Ajinomoto's AJIPHASE® liquid-phase platform has crossed into commercial-scale FDA-approved drug manufacturing; and Codexis's ECO Synthesis platform generated a verified 3 kg clinical siRNA batch in 2025, with three leading CDMOs validating the process transfer in their own facilities [src_B11, src_B12, src_B15]. The strategic question for suppliers serving dual-target pipelines is no longer whether to adopt alternatives, but which alternative fits which construct class and on what timeline.

## 4.1 Solid-Phase Phosphoramidite Synthesis: Where the Ceiling Is

Standard commercial coupling efficiency in well-controlled SPOS reaches 99.5% per cycle, with best-in-class IDT Ultramer™ chemistry achieving 99.6% [src_B02]. The 2'-acetal levulinic ester (ALE) phosphoramidite system — a recent chemistry-based advance, not enzymatic — demonstrated >99% coupling at 2–4 min cycle time for RNA up to 215 nt, the current published ceiling for chemical solid-phase RNA synthesis [src_B05].

The problem is cumulative yield decay. Maximum full-length product (FLP) = (coupling efficiency)^(n−1):

- 21-mer at 99.5%/cycle: 0.995^20 = **90.5%**
- 40-nt construct at 99.5%/cycle: 0.995^39 = **82.5%**
- 60-nt dual-target strand at 99.5%/cycle: 0.995^59 = **74.4%**
- 60-nt strand at 98.5%/cycle (common practical rate): 0.985^59 = **41.5%**

These are theoretical ceilings before cleavage losses, deprotection failures, and purification. In practice, a GalNAc-siRNA GMP campaign at WuXi AppTec reported an initial crude yield of 13% and purity of 18%, improved to 62% yield/75% purity after process development in a 500 g batch [src_E05]. The 60-nt threshold matters: covalent-linker tandem designs (as in Alnylam's US9187746) and GalNAc-loaded multivalent constructs routinely breach it. GalNAc phosphoramidite coupling in 500 Å CPG pores also reduces coupling efficiency and extends cycle time to approximately 6 minutes versus 2 minutes for standard bases [src_E07], eroding throughput on capital equipment costing $2–5 million per column-scale GMP synthesizer.

Environmental costs reinforce this ceiling. SPOS process mass intensity (PMI) for a 20-mer therapeutic oligonucleotide averages 4,299 (range 3,035–7,023), versus 168–308 for small molecules [src_C15]. Acetonitrile consumption reaches 100–1,000 kg per kg of API, with ~85% consumed during synthesis wash steps [src_E40]. This waste burden translates to direct cost, supply-chain risk, and increasing ESG pressure on facility design.

SPOS is the right tool for heavily-modified 21-mers with standard siRNA chemistry. For dual-target constructs combining GalNAc loading, multivalent scaffolding, and strand lengths ≥40 nt — the yield decay and waste economics push manufacturers toward alternatives.

## 4.2 Liquid-Phase Synthesis (AJIPHASE, Nitto CPOS) — Where It Already Wins

AJIPHASE® replaces the solid support with a soluble anchor (a phenyl core with >C10 alkyl chains). Reactions proceed homogeneously; at each cycle the product precipitates in an antisolvent and is filtered, eliminating intermediate separations [src_B14]. Scale becomes a function of vessel size, not column geometry.

The commercial record is established. Ajinomoto Bio-Pharma Services runs AJIPHASE at up to 200 kg batch for PMO synthesis in Japan and Belgium, and the FDA has approved commercial production of an undisclosed oligonucleotide API via AJIPHASE [src_B14]. For a standard 21-mer siRNA, AJIPHASE has delivered 60% yield with >90% purity after chromatographic purification — comparable to optimized SPOS performance [src_E41]. The Nucleic Acids Research 2025 LPOS review [src_B02] defines where LPOS wins: non-branched constructs in the 15–40 nt sweet spot at batch sizes exceeding ~100 g, where lower per-gram solvent cost justifies the development overhead.

LPOS has documented limits for dual-target work. Branched architectures and high-modification-density constructs (alternating 2'-F/2'-OMe with GalNAc phosphoramidite) require more robust coupling activators and longer precipitation cycles, and are more readily handled in SPOS. The 2026 Molecules paper on liquid-phase GalNAc-siRNA assembly confirmed gram-to-kilogram feasibility for standard PCSK9-targeting constructs [src_C01], but branched multivalent designs remain a challenge.

China's leading oligo CDMO, Hongene (兆维), operates 48 solid-phase synthesis lines at 1 kg/batch with NMPA/FDA/EMA qualification [src_D09]. Current public evidence does not confirm a validated LPOS offering at Hongene comparable to AJIPHASE; their platform is SPOS-centric, with enzymatic ligation as a disclosed add-on (Section 4.3). For Chinese pipelines requiring LPOS at >100 g single-strand scale, the domestic option set is narrow.

## 4.3 Enzymatic and Chemoenzymatic Ligation — The Breakout Track

Enzymatic ligation divides the full-length siRNA into short fragments (7–12 nt), synthesizes each at near-quantitative efficiency, then joins them using an engineered dsRNA ligase. This modular logic changes the yield mathematics for longer constructs.

**Yield comparison** (60-nt dual construct):
- **SPOS at 99.5%/cycle**: 0.995^59 = **74.4%**
- **Enzymatic ligation: 6×10-nt fragments** (each at 99.9%/cycle = 99.1%) + 5 ligations at 95% efficiency (Codexis engineered ligase): (0.999^9)^6 × 0.95^5 = 94.6% × 77.4% = **73.3%**

At 60 nt, enzymatic ligation with an optimized ligase essentially matches SPOS yield while delivering cleaner fragment inputs — reducing downstream purification burden. For constructs above 80 nt, the math inverts further in ligation's favor.

The enabling technology is the ligase. Wild-type T4 RNA Ligase 1 (T4 Rnl1) requires a 5'-phosphate, 3'-OH, and — critically — a free 2'-OH at the ligation junction, making it incompatible with 2'-OMe-modified termini [src_E42]. Wild-type T4 RNA Ligase 2 operates in a double-stranded context with broader tolerance but still performs poorly on 2'-F/2'-OMe substrates at manufacturing concentrations. Codexis supplies "optimized dsRNA ligases specifically developed to enable high-efficiency assembly of duplexed RNAi constructs under manufacturing-relevant conditions," with demonstrated higher volumetric productivity and substrate versatility over wild-type comparators [src_B11].

**The 2025–2026 proof points.** In 2025, Codexis's ECO Synthesis ligase generated a 3 kg siRNA clinical batch at a leading CDMO — the first publicly disclosed enzymatic ligation batch at clinical scale for a therapeutic siRNA [src_B11]. The ECO Synthesis platform is rated at >10 kg/run for technology transfer; a dedicated ECO GMP Manufacturing Center near Hayward, CA is targeted for late 2027 [src_B11]. In March 2026, Codexis signed a 50 g siRNA manufacturing agreement with an innovator company for a cardiovascular preclinical program, confirming commercial traction [src_E43]. Three CDMO validation signals underscore the platform's maturity:

1. **Bachem–Codexis** (TIDES USA 2025): Joint poster benchmarked Codexis ligases against wild-type enzymes in Bachem's own facility; Codexis enzymes showed superior volumetric productivity and substrate versatility [src_B12].
2. **Nitto Denko Avecia–Codexis** (October 29, 2025): Evaluation agreement signed; Nitto Avecia to assess the full ECO Synthesis platform toward licensing [src_B15].
3. **ST Pharm–Codexis** (TIDES USA 2025): Third CDMO to independently validate Codexis ligation in-house.

**Hongene chemoenzymatic ligation (China).** Hongene disclosed in 2025 a chemoenzymatic ligation process claiming >95% purity for assembled oligonucleotides [src_B16]. Short fragments are made by SPOS on Hongene's existing 48-line infrastructure, then joined enzymatically. This preserves sunk capital while extending the synthesis envelope. Specific constructs, scales, and enzymes remain undisclosed, but the >95% purity figure aligns with TIDES data for fragment-ligation approaches.

**NMPA regulatory de-risking.** The NMPA/CDE "Technical Guidance for Pharmaceutical Research of Chemically Synthesized Oligonucleotide Drugs (Innovative Drugs) (Trial Implementation)", issued February 28, 2026 as CDE Announcement No. 21 [src_B18], explicitly enumerates three manufacturing methods: solid-phase synthesis, liquid-phase synthesis, and "enzymatic-catalysis fragment ligation synthesis" (酶催化片段连接合成). This is the first major global regulatory authority to formally recognize chemoenzymatic ligation in oligonucleotide drug guidance, predating any equivalent FDA or EMA statement. The guidance requires specific risk controls (enzyme-introduced impurities, fragment intermediate purity, coupling efficiency monitoring), but does not demand that ligation prove superiority to SPOS. For Chinese CDMOs and developers, this 12–24 month regulatory head-start over Western timelines is a material competitive advantage.

**Residual limitations.** Three constraints remain. The sequence constraint at ligation junctions — the requirement for a ligation-compatible (typically 2'-OH or 2'-F, not 2'-OMe) nucleotide at the −1 position — constrains fragment design and cannot yet be fully bypassed even by engineered ligases. Cost-per-gram comparisons between enzymatic ligation and SPOS at commercial scale have not been published in peer-reviewed form. And the GMP precedent gap — the 3 kg batch is non-GMP clinical-material grade, and the ECO GMP facility is ~18 months from commissioning — means that Phase 3 programs needing >10 kg batches in 2026–2027 will default to SPOS.

## 4.4 Cell-Free IVT and Template-Free Enzymatic Synthesis — Promise vs. Current Reality

**GreenLight Biosciences requires a correction.** The company did not go bankrupt. GreenLight Biosciences Holdings, PBC was taken private on July 24, 2023, in a $45.5 million go-private transaction led by Fall Line Endurance Fund [src_E44]. The surviving private entity pivoted fully to agriculture RNA, launching Calantha™ (EPA-registered RNA insecticide, 2023) and Norroa (RNA varroa mite treatment, October 2025), and raised a $25 million Series C from Just Climate in March 2025 for agricultural commercialization. The company has no disclosed therapeutic siRNA manufacturing activity. The claimed <$1/g production cost applied exclusively to unmodified dsRNA for agricultural use — it is not a valid cost benchmark for 2'-F/2'-OMe modified therapeutic siRNA, and should not be cited as such.

**IVT's fundamental barrier.** T7 RNA polymerase-based IVT produces unmodified or minimally modified RNA. Therapeutic siRNA requires alternating 2'-F and 2'-OMe modifications at virtually every position to resist nuclease degradation in vivo. T7 RNAP can incorporate 2'-F-UTP and 2'-F-CTP at reduced rates, but full alternating 2'-F/2'-OMe pattern synthesis has not been demonstrated at GMP scale. The Biotechnology Advances 2025 review explicitly concludes IVT is suitable for unmodified dsRNA (agriculture, vaccines) but not for 2'-modified therapeutic siRNA at GMP scale [src_B06].

**TdT template-free synthesis.** Engineering of terminal deoxynucleotidyl transferase (TdT) for de novo RNA synthesis continues. The Cell Reports Methods 2025 paper on TdT variants demonstrated progressive improvements: engineered murine TdT achieved kcat/Km of 47.49 mM⁻¹min⁻¹ for 2'-OMe-ATP versus 19.51 for earlier variants, but 2'-OMe-UTP incorporation (kcat/Km = 2.66) remains severely rate-limiting [src_B10]. Codexis's TIDES EU 2023 data showed iterative TdT evolution toward 2'-modified RNA synthesis with increasing efficiency across evolution rounds [src_E45], confirming progress but not GMP readiness. For DNA synthesis, TdT platforms reach 600–750 nt; for full alternating 2'-F/2'-OMe 21-mer RNA synthesis at therapeutic quality, a 3–5 year timeline is realistic.

**ALE platform (chemistry, not enzyme).** The ALE system is a solid-phase chemistry improvement — not enzymatic. Its significance is in demonstrating that chemistry-based SPOS, with the right 2'-protecting group, can efficiently produce RNA up to 215 nt at >99%/cycle [src_B05]. For a 200-nt sequence, improving coupling efficiency from 98% to 99.4% increases theoretical FLP yield from 1.8% to 30.2% — a 17-fold gain [src_B05]. ALE extends SPOS's practical range for guide RNAs and mRNA vaccine candidates but does not address SPOS's solvent waste or capital-intensity constraints.

## Synthesis Modality Comparison

| Modality | Max practical length | 2'-mod incorporation | GMP precedent | Cost/g at 1 kg scale | Green score | Dual-target suitability |
|---|---|---|---|---|---|---|
| Solid-phase (SPOS) | 60–80 nt; ~215 nt with ALE | ✅ Mature | ✅ Established | $$$$ | Low | Good for ≤21-mer simple constructs; declines for multivalent/tandem |
| LPOS (AJIPHASE) | 15–40 nt sweet spot | ✅ Validated | ✅ Partial (commercial for PMO) | $$$ | Medium | Limited for branched; strong for high-volume single-strand |
| Enzymatic ligation | 40–120 nt assembled | ✅ Fragments (engineered ligase) | 🔶 Emerging (3 kg clinical 2025; GMP 2027) | $$ | High | Excellent for complex/long dual-target once GMP capacity onlines |
| Cell-free IVT | Unlimited | ❌ Minimal (no therapeutic-grade 2'-mods) | ❌ | $ | Very high | Not yet — agricultural dsRNA only |
| TdT template-free | 600+ nt (DNA) | ❌ RNA 2'-mods rate-limiting | ❌ | $$ | High | Future (3–5 yr) |

## Counter-Evidence: Why SPOS Will Not Decline Quickly

Three forces constrain the transition pace. First, regulatory inertia: every approved siRNA therapeutic used SPOS, and Alnylam's Senior Director for Regulatory Affairs CMC presented at OPT March 2026 on "Technical and Regulatory Considerations for Oligonucleotide Synthesis Using Enzymatic Ligation" — confirming FDA has no explicit guidance yet, and that the industry is still defining the regulatory pathway. Second, scale capacity: Codexis's ECO GMP facility is not online until late 2027; the three CDMO validation partners (Bachem, Nitto Avecia, ST Pharm) are still at evaluation stage for commercial GMP runs. A Phase 3 program needing >10 kg batches in 2026–2027 has no validated commercial enzymatic ligation source and will default to SPOS. Third, construct diversity: cocktail approaches (two 21-mers co-administered, no covalent linker) present no length challenge for SPOS and remain the simplest CMC path, representing a substantial fraction of the current dual-target pipeline.

The transition will be construct-class-specific. Enzymatic ligation will first claim >40 nt assembled constructs and complex scaffolds. LPOS will take high-volume single-strand commercial production. SPOS will hold the heavily-modified short-strand segment indefinitely and the majority of the current pipeline through at least 2028.
