Codexis : TIDES Europe 2023 Presentation

Revolutionizing Nucleic Acid Synthesis with Engineered Enzymes

David Entwistle Ph.D., Sr. Director, Codexis Inc.

TIDES EU November 1st 2023

ECO Synthesis™ Platform: Positioned to Deliver in RNAi Market

RNAi Demand is Coming

Chemical synthesis (phosphoramidite chemistry) alone cannot meet

projected future wave of demand for RNAi therapeutics

Customers are asking us for a scalable, more sustainable enzymatic solution to complement chemical synthesis

1 kg of siRNA requires ~ 1000 kg of MeCN (BioSpring)

PMI per nucleotide added ~200 kg/kg ( Org. Proc. Dev, 2021, 86, 1, 49-61)

Codexis positioned to deliver based on 20-yearhistory of enzyme

engineering and directly relevant Pharmaceutical Manufacturing commercial expertise

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Codexis ECO Synthesis™ Technology

Enzyme Catalyzed Oligonucleotide Synthesis

Core process:

Oligonucleotide synthesis by iterative, single nucleotide extension

• Extend: Controlled, enzymatic addition of modified, 3'-phosphate blocked ribonucleotides

• Deblock: Enzymatic cleavage of 3'-phosphate blocking group & excess ribonucleotides

… Repeat

Supply processes:

• Enzyme cascade for synthesis of modified, 3'- phosphate blocked ribonucleotides (NQPs)

• Enzymatic synthesis of starter oligonucleotide

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Codexis ECO Synthesis™ Technology - Key Platform Traits

Enzyme Performance

• High incorporation efficiency

• Robustness & manufacturability

Scalable & Economical Manufacturing

• High volumetric productivity

• "Oligo in solution, enzyme immobilized"

• Controlled addition of monomers

• Low impurity production

• Smaller infrastructure/facilities footprint

• Established reagents supply

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Supplying Critical NQP Reagents For ECO Synthesis™ Platform

Building a "one-pot,two-step" enzyme cascade with engineered kinases

Targeted Key Performance Indicators

Robustness/Manufacturability - soluble expression & stability

Productivity - >98% conversion at millimolar substrate concentration

Substrate Tolerance - accepts ribonucleotides with 2' modified sugars & phosphorothioate backbone modifications

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Supplying Critical NQP Reagents For ECO Synthesis™ Platform

Building a "one-pot,two-step" enzyme cascade with engineered kinases

Step 1: N→NTP conversion via three consecutive phosphorylation steps, using three kinases

		A	C	G	U
Initial (wild type)	2'-OH	99	22	1	7
kinases	2'-dF	64	0	0	0
	2'-OMe	0	0	0	0

	A	C	G	U
2'-OH	98	96	93	91	Current engineered
2'-dF	93	19	93	29	kinases
2'-OMe	55	0	0	0

Percent conversion for individual nucleosides to the corresponding NTPs

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Supplying Critical NQP Reagents For ECO Synthesis™ Platform

Building a "one-pot,two-step" enzyme cascade with engineered kinases

Step 1: N→NTP conversion via three consecutive phosphorylation steps, using three kinases

Status: NTP Forming Cascade

• Full base promiscuity & emerging activity for 2'-modified nucleosides
• Operational at process-relevant substrate concentration
• Engineering on track to deliver full base and 2'-modification promiscuity for NTP formation

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Supplying Critical NQP Reagents For ECO Synthesis™ Platform

Building a "one-pot,two-step" enzyme cascade with engineered kinases

Step 2: NTP→NQP percent conversion with current 3'-O kinase

		A	C	G	U			A	C	G	U
Initial (wild type)	2'-OH	2	0	0	0		2'-OH	99	18	38	36	Current engineered
kinase	2'-dF	0	0	0	0		2'-dF	12	0	0	0	kinase
	2'-OMe	0	0	0	0		2'-OMe	0	0	0	0

Percent conversion for individual NTPs to the corresponding NQPs

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Supplying Critical NQP Reagents For ECO Synthesis™ Platform

Building a "one-pot,two-step" enzyme cascade with engineered kinases

Step 2: NTP→NQP percent conversion with current 3'-O kinase

Status: 3'-O-kinase engineering

• Engineering challenge as desired activity is non-natural (& potentially cytotoxic to expression host)
• Break through from initial strict A selectivity
• Ongoing engineering of 3'O-kinase starting to deliver activity on 2'-modification for NQP formation

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Terminal Deoxynucleotidyl Transferase (TdT)

A high engineered enzyme for catalyzing the extension reaction

PDB ID: 4i27

Targeted Key Performance Indicators

Robustness/Manufacturability - soluble expression & stability

Productivity - >99% conversion at millimolar substrate concentration

Substrate Tolerance - accepts ribonucleotides with 2'- and 3'-modifications & backbone phosphorothioate modifications

Promiscuity - minimal oligonucleotide sequence bias

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