Slide 1: Process Development for Enzymatic Synthesis of RNAi Therapeutics
Process Development for Enzymatic Synthesis of RNAi Therapeutics Derek Gauntlett – Sr. Director ECO Process Development
Slide 2: RNA Synthesis – An Evolving Manufacturing Landscape
RNA Synthesis – An Evolving Manufacturing Landscape Phosphoramidite Chemistry Generation 2 Generation 3 Development status >40 years; mature w/ incremental Ready for manufacturing Operational prototype w/ path improvements to manufacturing Product quality Lower Higher Higher w/ potential for more Pr
Slide 3: RNA Synthesis – An Evolving Manufacturing Landscape
RNA Synthesis – An Evolving Manufacturing Landscape Phosphoramidite Chemistry Enzymatic Ligation Generation 3 Development status >40 years; mature w/ incremental Ready for manufacturing Operational prototype w/ path improvements to manufacturing Product quality Lower Higher Higher w/ potential for m
Slide 4: RNA Synthesis – An Evolving Manufacturing Landscape
RNA Synthesis – An Evolving Manufacturing Landscape Phosphoramidite Chemistry Enzymatic Ligation Enzymatic Sequential Synthesis Development status >40 years; mature w/ incremental Ready for manufacturing today Operational prototype w/ path improvements to manufacturing Product quality Lower Higher H
Slide 5: Progress Achieved on the ECO Synthesis Manufacturing Platform
Progress Achieved on the ECO Synthesis Manufacturing Platform Demonstrating the Power of Combining Enzyme Engineering & Process Development in One Year Incorporation efficiency: ~92% Incorporation efficiency: >98% Incorporation efficiency: >98% FLP at 8mer (max. 6) = 72.3% FLP at 8mer (max. 14) = 92
Slide 6: ECO Synthesis Technology Enables Manufacturing Versatility
ECO Synthesis Technology Enables Manufacturing Versatility 4-Synthesis approaches to demonstrate ECO Synthesis toolbox for manufacturing RNA. 5’ Cm Um Am Gm Am Cm Cf Um Gf Um T Um Um Gm Cm Um Um Um Um Gm Um (GalNAc)3 3’ 3’ Am Am Gm Am Um Cf Um Gf Gm Af Cm Am Am Af Am Cf Gm Af Af Af Am Cf Am 5’ incli
Slide 7: ECO Synthesis Technology - Overview
Route 1: ECO Sequential Synthesis ECO Synthesis Technology - Overview Oligonucleotide synthesis by sequential incorporation of modified nucleotides via a two-step extend & deblock protocol Enabling Technology – NQP synthesis Key Performance Indicators for ECO enzymes: • Non-native Substrates • Enzym
Slide 8: Conjugation of Tissue Targeting Moieties
Route 1: ECO Sequential Synthesis Conjugation of Tissue Targeting Moieties L96 for inclisiran sense strand Um (GalNAc)3 • Codexis has engineered a manufacturable, highly active single-stranded O NH (ss)RNA ligase with broad tolerance for conjugated nucleotides. R''O O N O • Successful incorporation
Slide 9: ECO Synthesis Process Overview
Route 1: ECO Sequential Synthesis ECO SynthesisTM Process Overview In-line analytics In-line analytics Extension Reactor Deblock (40°C) Reactor (30°C) Tank 1 Tank 2 Process Characteristics: Process Conditions: • Enzymes immobilized within reactor • [Oligo]: 6mM • Oligo in solution with aqueous react
Slide 10: Sequential Enzymatic Synthesis - By the Numbers
Route 1: ECO Sequential Synthesis Sequential Enzymatic Synthesis - By the Numbers Sense and antisense strands synthesized using ECO Synthesis manufacturing platform RNA Starter Number Added Avg. Coupling Compound Length of Extensions Monomer Efficiency (%) Sense Strand 7 14 2’F / 2’OMe / L96 98.4 An
Slide 11: Ligation Disconnection Strategy
Route 2: PAC + dsRNA ligase Ligation Disconnection Strategy Two nick / four-fragment approach used for the generation of inclisiran S1 5’ Cm Um Am Gm Am Cm Cf Um Gf Um T Um Um Gm 3’ S2 5’ PO4 Cm Um Um Um Um Gm Um (GalNAc)3 3’ 3’ Gm Af Cm Am Am Af Am Cf Gm Af Af Af Am Cf Am 5’ 3’ Am Am Gm Am Um Cf Um
Slide 12: Ligation Using PAC Fragments - inclisiran
Route 2: PAC + dsRNA ligase Ligation Using PAC Fragments - inclisiran Low concentrations of Codexis dsRNA ligase with PAC fragments result in high conversion yields Sense Antisense Parameter Condition Substrate 99.7 99.5 99.4 6 to 98 98.5 98.4 97.8 (g/L) CDX Ligase 0.1 (mg/mL) Buffer % CONVERSION Aq
Slide 13: Ligation Using PAC + ECO Fragments - inclisiran
Ligation Using PAC + ECO Fragments - inclisiran Route 3: PAC / ECO + dsRNA ligase Two nick approach using a combination of PAC and ECO derived fragments for the generation of inclisiran Example: 1 PAC / 3 ECO Disconnection 5’ Cm Um Am Gm Am Cm Cf Um Gf Um T Um Um Gm 3’ S2 S1 5’ PO4 Cm Um Um Um Um Gm
Slide 14: Ligation Using PAC + ECO Fragments - inclisiran
Ligation Using PAC + ECO Fragments - inclisiran Route 3: PAC / ECO + dsRNA ligase Using low concentrations of Codexis dsRNA ligase with PAC + ECO fragments result in high conversion yields Sense Antisense Process Conditions 99.9 99.6 99.5 99.4 99.2 99.0 Parameter Condition Substrate 6 (g/L) CDX Liga
Slide 15: Ligation Using ECO Fragments - inclisiran
Ligation Using ECO Fragments - inclisiran Route 4: ECO + dsRNA ligase Two nick using 4 ECO derived fragment approach used for the generation of inclisiran Example: 4 ECO Disconnection S1 5’ Cm Um Am Gm Am Cm Cf Um Gf Um T Um Um Gm 3’ S2 5’ PO4 Cm Um Um Um Um Gm Um (GalNAc)3 3’ 3’ Gm Af Cm Am Am Af A
Slide 16: Ligation Using ECO Fragments - inclisiran
Route 4: ECO + dsRNA ligase Ligation Using ECO Fragments - inclisiran Low Codexis dsRNA ligase concentrations with ECO fragments result in high duplex yields 99.8 98.6 Parameter Condition Substrate 6 (g/L) CDX Ligase Sense 0.1 (mg/mL) %CONVERSION Antisense Buffer Composition Aqueous / TRIS Incubatio
Slide 17: Adaptable Ligation via ECO & PAC Fragments
Adaptable Ligation via ECO Antisense Sample Matrix Sense & PAC Fragments for high No Ligase Control S1, S2, AS1, AS2 yielding siRNA Duplex Ligation Performance Takeaway All PAC S1, S2, AS1, AS2 • Ligation using ECO and PAC fragments with Codexis dsRNA ligase yield high ligation efficiencies ≥ 98%. 1
Slide 18: Ligation Impurity Profile - % Comparison Summary
Ligation Impurity Profile - % Comparison Summary Codexis demonstrated ligation of siRNA using 4 different synthesis routes to meet your needs Conversion Antisense Strand Sense Strand Crude Duplex Notable Ligation Pathway (%) (%) (%) Purity (%) Impurities 4 PAC >98 53.3 41.6 91.5 AS1, S2 1 ECO / 3 PA
Slide 19: Forward Looking for ECO Synthesis Manufacturing Platform
Forward Looking for ECO Synthesis Manufacturing Platform Codexis is positioned to supply therapeutic siRNA in 2025 from our Innovation Lab • ECO Synthesis manufacturing platform has made significant progress since TIDES EU in November 2023 • Ligation Services are available today! • Continue process
Slide 20: Step into the future of manufacturing RNA
Step into the future of manufacturing RNA Learn more about enzymatic siRNA manufacturing Manufacturing – non-GMP Manufacturing – cGMP services GMP – GMP – GMP – Phase Commercial GMP – Phase II III Preclinical GLP material Phase I Codexis ECO Synthesis Coming soon Innovation Lab Booth #709 20
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