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

Generated: 2026-04-21  
Researcher: dr-analyst  
Word count: ~2,050 words / Quota 1,800 words (114% — within ±15% upper bound)

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## Core Claims Evidence Table

| Claim ID | Claim Summary (≤30 words) | Supporting Evidence 1 | Supporting Evidence 2 | Confidence | Notes |
|---|---|---|---|---|---|
| C01 | SPOS at 99.5%/cycle yields 90.5% max for 21-mer; drops to 74.4% for 60-nt construct | [src_B02] Nucleic Acids Review — coupling efficiency tables, Tier 1 | [ATDBio Oligo Synthesis textbook via search; src_C15] yield calculation confirmed independently | High | Calculation is standard textbook math; independently verifiable |
| C02 | ALE phosphoramidite: >99% coupling efficiency, 2–4 min cycle, up to 215 nt RNA | [src_B05] PMC 2024 paper on ALE chemistry, Tier 1, score 8.3 | Confirmed in ResearchGate summary of same paper | High | Pure chemistry platform (SPOS-based, not enzymatic) |
| C03 | Practical SPOS PMI for 20-mer: 3,035–7,023 (avg ~4,299); acetonitrile 100–1,000 kg/kg API | [src_C15] J Org Chem 2021 sustainability review, Tier 2 | SynerG White Paper 2025 (PMI data) [src_E41] | High | 85% of MeCN in synthesis steps confirmed by ACS OPR&D paper src_E40 |
| C04 | Codexis ECO Synthesis ligase used to generate 3 kg clinical siRNA batch in 2025 | [src_B11] Codexis blog + DeciBio Q&A, Tier 2, score 7.6 | [src_B12] TIDES USA 2025 presentations + Bachem validation, Tier 2 | High | Multiple independent sources confirm the 3 kg milestone |
| C05 | ECO Synthesis platform exceeds 10 kg/run; GMP facility (Hayward CA) online late 2027 | [src_B11] Codexis ECO platform page + DeciBio interview | [src_E43] Codexis press release March 2026 (50 g commercial agreement) | High | Company disclosures; GMP timeline is forward-looking |
| C06 | Three CDMOs (Bachem, Nitto Avecia, ST Pharm) validated Codexis ligation in-house at TIDES USA 2025 | [src_B12] Bachem LinkedIn/Codexis press release | [src_B15] Codexis–Nitto Denko Avecia Oct 2025 press release | High | Three independent CDMO validations at same conference |
| C07 | Codexis–Nitto Denko Avecia evaluation agreement signed Oct 29, 2025; for licensing and broader ECO adoption | [src_B15] Codexis IR press release, Tier 2, score 7.5 | Manufacturing Chemist article corroborating | High | Both sides confirmed; still evaluation stage, not production stage |
| C08 | AJIPHASE® commercially produces PMOs at 200 kg batches; FDA approved commercial oligo drug via AJIPHASE | [src_B14] Ajinomoto press release + platform page, Tier 2 | SynerG white paper 2025 corroborating [src_E41] | High | Commercial-scale validated; specific drug undisclosed by Ajinomoto |
| C09 | AJIPHASE 21-mer siRNA: 60% yield, >90% purity after purification | SynerG White Paper 2025 [src_E41] citing Ajinomoto data | [src_B14] platform page confirming comparable purity to SPOS | Medium | Yield figure from vendor-allied white paper; primary Ajinomoto data not separately accessed |
| C10 | GreenLight Biosciences taken private July 24, 2023; now focused exclusively on agriculture RNA (Calantha™, Norroa) | Goodwin Law announcement 2023 [src_E44] | GreenLight Biosciences website 2025–2026 (Calantha/Norroa products) | High | Clear corporate trajectory; no therapeutic siRNA activity post-2023 |
| C11 | GreenLight $1/g dsRNA claim applies only to unmodified agricultural dsRNA, not therapeutic 2'-modified siRNA | [src_B13] Axial blog — explicitly describes agricultural dsRNA | [src_B06] Biotech Adv 2025 review — IVT not suitable for 2'-modified therapeutic siRNA at GMP | High | Counter-factual is well-supported; concept technology proven but company pivoted |
| C12 | TdT 2'-OMe-ATP incorporation improving via directed evolution; 2'-OMe-UTP still rate-limiting | [src_B10] Cell Rep Methods 2025 — kinetic data table | [src_E45] Codexis TIDES EU 2023 presentation on TdT evolution rounds | Medium | Strong academic data; GMP readiness 3–5 yr is inference, not direct claim |
| C13 | NMPA/CDE Feb 28, 2026 guidance explicitly names enzymatic-catalysis fragment ligation synthesis as approved manufacturing method | [src_B18] NMPA CDE 2026 No. 21 announcement, Tier 1, score 8.2 | Chinese pharmaceutical site transcription of guidance text (m.xfdyb.com) corroborates specific Chinese text | High | First global regulator to enumerate chemoenzymatic ligation in oligo drug guidance |
| C14 | NMPA guidance requires additional risk controls for ligation (enzyme impurities, fragment intermediate controls) | [src_B18] same guidance document | CDE pharmaceutical website excerpt confirming specific control requirements | High | Well-documented; the requirement for controls does not prevent adoption |
| C15 | T4 RNA Ligase 1 requires 5'-phosphate, 3'-OH, and free 2'-OH; incompatible with 2'-OMe at ligation junction | Nucleic Acids Research review on RNA ligases [src_E42] | PMC biochemical insights paper on RNA ligase structure/mechanism | High | Mechanistic constraint is well-established in enzymology literature |
| C16 | Hongene (兆维) disclosed chemoenzymatic ligation in 2025 with >95% purity claim | [src_B16] 医药魔方 report, Tier 2, score 7.6 | [src_D09] 兆维 platform overview | Medium | Only one detailed primary source in Chinese media; purity figure unverified independently |
| C17 | Enzymatic ligation 60-nt construct yield math (~73.3%) matches SPOS (74.4%) with ≥95% ligation efficiency per junction | Calculated from fragment yield math (6×10-mer at 99.9%/cycle) combined with ligation yields | Consistent with DeciBio interview data: "higher yields and reduced impurities" [src_B11] | Medium | Math is internally consistent but specific per-junction ligation efficiency (95%) is derived from Codexis's >90–95% purity claim, not a direct published per-ligation-event yield |
| C18 | WuXi AppTec GMP GalNAc-siRNA campaign: initial yield 13%, improved to 62%/75% purity after process development in 500 g batch | [src_E05] TIDES 2024 WuXi AppTec case study, Tier 2, score 7.4 | No second source available — CDMO-authored but specific numbers suggest genuine disclosure | Low/Medium | Single CDMO-authored source; numbers reasonable for reported scale |
| F01 | Every FDA-approved siRNA therapeutic was manufactured by SPOS | Established fact across literature; [src_B02] review confirms | [src_E04] Molecular Therapy review pipeline table | High | Factual baseline for regulatory inertia argument |
| T01 | Enzymatic ligation will displace SPOS for >40-nt assembled dual-target constructs within 3–5 years | [src_B11, src_B12, src_B15] CDMO adoption wave | [src_B18] NMPA regulatory alignment | Medium | Trend projection; dependent on ECO GMP facility delivery and FDA guidance development |

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## Source Details

**[src_B02]** — From liquid-phase synthesis to chemical ligation (Nucleic Acids Research 2025)  
DOI: 10.1093/nar/gkaf1084  Tier 1 | Score: 8.8 | Used in: Ch 4.1, Ch 4.2

**[src_B05]** — ALE phosphoramidite synthesis of long RNA (PMC 2024)  
PMID: 41548876  Tier 1 | Score: 8.3 | Used in: Ch 4.1, Ch 4.4

**[src_B06]** — Enzymatic de novo oligonucleotide synthesis (Biotechnol Adv 2025)  
ScienceDirect S0734975025000904  Tier 1 | Score: 8.7 | Used in: Ch 4.4

**[src_B10]** — TdT variants overcoming coupling bottleneck (Cell Rep Methods 2025)  
PMC11747941  Tier 1 | Score: 8.1 | Used in: Ch 4.4

**[src_B11]** — Codexis ECO Synthesis blog + DeciBio Q&A (2025)  
URL: codexis.com/blogs; decibio.com/insights/codexis  Tier 2 | Score: 7.6 | Used in: Ch 4.3

**[src_B12]** — Codexis–Bachem enzymatic ligation TIDES 2025 (LinkedIn/Bachem)  
URL: bachem.com/knowledge-center; linkedin.com/posts/codexis  Tier 2 | Score: 7.7 | Used in: Ch 4.3

**[src_B13]** — GreenLight Biosciences cell-free RNA (Axial blog, 2023–25)  
URL: medium.com/@axialxyz  Tier 2 | Score: 7.8 | Used in: Ch 4.4 (with correction re: company status)

**[src_B14]** — Ajinomoto AJIPHASE® platform page + news 2025  
URL: ajibio-pharma.ajinomoto.com/ajiphase/  Tier 2 | Score: 7.9 | Used in: Ch 4.2

**[src_B15]** — Codexis–Nitto Denko Avecia evaluation agreement press release (Oct 2025)  
URL: ir.codexis.com; prnewswire.com  Tier 2 | Score: 7.5 | Used in: Ch 4.3

**[src_B16]** — Shanghai Hongene chemoenzymatic ligation (医药魔方 2025)  
URL: 163.com/dy/article/KKOQIDFB0532CO9S  Tier 2 | Score: 7.6 | Used in: Ch 4.3

**[src_B18]** — NMPA CDE 化学合成寡核苷酸药物技术指导原则 (2026 No. 21)  
URL: pharmwyp.com/posts/56814/  Tier 1 | Score: 8.2 | Used in: Ch 4.3

**[src_C01]** — Liquid-phase assembly GalNAc-siRNA (PMC 2024)  
PMID: 41683454  Tier 1 | Score: 9.2 | Used in: Ch 4.2

**[src_C15]** — Sustainability challenges in oligonucleotide manufacturing (J Org Chem 2021)  
DOI: 10.1021/acs.joc.0c02291  Tier 2 | Score: 7.8 | Used in: Ch 4.1

**[src_D09]** — Hongene Shanghai platform (医药魔方 2025)  
URL: bydrug.pharmcube.com  Tier 2 | Score: 7.4 | Used in: Ch 4.2, Ch 4.3

**[src_E05]** — WuXi AppTec GMP siRNA case study (TIDES 2024)  
URL: tides.wuxiapptec.com  Tier 2 | Score: 7.4 | Used in: Ch 4.1

**[src_E07]** — BOC Sciences GalNAc coupling cycle time (vendor technical note)  
URL: bocsci.com  Tier 3 | Score: 5.5 | Used in: Ch 4.1 (directional only, flagged as unverified primary source)

**New sources in this chapter:**

**[src_E40]** — Acetonitrile regeneration from oligonucleotide production waste (ACS OPR&D 2024)  
URL: pubs.acs.org/doi/10.1021/acs.oprd.4c00188  Tier 1 | Score: 8.0 | Used in: Ch 4.1

**[src_E41]** — SynerG BioPharma SPOS and LPOS White Paper (2025)  
URL: synergbiopharma.com  Tier 2 | Score: 6.8 | Used in: Ch 4.1, Ch 4.2

**[src_E42]** — Structural and biochemical insights into RNA ligases (PMC + Nucleic Acids Res)  
PMC11071452; academic.oup.com/nar/40/7/e54  Tier 1 | Score: 8.5 | Used in: Ch 4.3

**[src_E43]** — Codexis signs agreement to manufacture 50 g siRNA, ECO Synthesis (March 4, 2026)  
URL: ir.codexis.com/news-events/press-releases/detail/442  Tier 2 | Score: 7.8 | Used in: Ch 4.3

**[src_E44]** — GreenLight Biosciences go-private merger with Fall Line (2023); post-2023 agriculture pivot  
Goodwin Law 2023 announcement; GreenLight website 2025–2026  Tier 2 | Score: 7.5 | Used in: Ch 4.4

**[src_E45]** — Codexis TIDES EU 2023 TdT engineering presentation  
URL: d1io3yog0oux5.cloudfront.net (Codexis TIDES EU PDF)  Tier 2 | Score: 7.0 | Used in: Ch 4.4

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## CRITICAL FINDING

**GreenLight Biosciences status**: The company did NOT go bankrupt. It was acquired in a go-private transaction at $45.5 million by Fall Line Endurance Fund, completed July 24, 2023. The surviving private entity continues as GreenLight Biosciences, Inc., but has pivoted to agriculture RNA exclusively. As of April 2026, the company raised a $25M Series C (Just Climate), launched Calantha™ (insecticide) and Norroa (varroa mite treatment), and has no publicly disclosed therapeutic siRNA manufacturing activity. The technology concept (cell-free IVT at scale) is proven for unmodified dsRNA, but the $1/g production cost claimed in src_B13 **cannot be used as a current reference for therapeutic siRNA manufacturing** — it is agricultural and unmodified. This is noted in ch04.md body text with appropriate caveats.

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## Unverified Claims

| Flag | Claim | Reason | Action |
|---|---|---|---|
| [Unverified-1] | GalNAc phosphoramidite cycle time: ~6 min (vs 2 min standard) | From src_E07 (vendor technical note, score 5.5, no primary reference given) | Acceptable as directional indicator; marked as single-source in notes |
| [Unverified-2] | Hongene >95% purity from chemoenzymatic ligation — specific enzyme, scale, construct length not disclosed | src_B16 (Chinese media, one source) | Flagged in evidence table as Medium confidence; acceptable given corroborating context |
| [Unverified-3] | Cost-per-gram advantage of enzymatic ligation vs SPOS at 1 kg scale | No peer-reviewed head-to-head published data found | Noted as limitation in ch04 body text |

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##反方证据 / Counter-Evidence (Pre-populated for dr-verifier)

1. **SPOS regulatory inertia is a genuine constraint**: Alnylam's Senior Director for Regulatory Affairs presented at OPT March 2026 on "Technical and Regulatory Considerations for Oligonucleotide Synthesis Using Enzymatic Ligation" — confirming that FDA does not yet have explicit guidance. This is counter-evidence against over-estimating the speed of enzymatic ligation adoption.

2. **Enzymatic ligation yield math does not clearly beat SPOS for 21-mers**: At 21-mer length, SPOS at 99.5% (90.5% max yield) outperforms simple 3×7-mer enzymatic ligation at 90% ligation efficiency (~79.5%). Enzymatic ligation's yield advantage only becomes clear at ≥40 nt constructs. The chapter correctly notes this.

3. **AJIPHASE purity claim for siRNA is sourced from a vendor-aligned white paper**: The 60%/90% yield/purity data for AJIPHASE 21-mer siRNA comes from SynerG BioPharma's white paper (which cites Ajinomoto). Independent peer-reviewed confirmation for siRNA (versus the confirmed PMO data) should be sought.

4. **Codexis ECO commercial timeline risk**: ECO GMP facility is not online until late 2027. Multiple CDMO evaluation agreements are still at evaluation (not production) stage. The 3 kg batch was at a leading CDMO, not at Codexis's own GMP facility. If the Hayward facility is delayed, the timeline projection in the chapter shifts.
