OligonucleotideGLOS2009

© 2006 Girindus Solvay Organics ® Oligonucleotide Manufacturing at Girindus 1 © 2009 1 ® Outline Major Classes of Oligonucleotide Therapeutics Solid Phase Synthesis of Oligonucleotides ƒ Synthesis Cycle ƒ Critical Raw Materials Solvay Organics 3 Manufacturing Trains for Oligonucleotide Synthesis ƒ Train 1: AKTA 100 DNA-Synthesizer; AKTA Purifier ƒ Train 2: OP400 DNA-Synthesizer; BioProcess HPLC Skid ƒ Train 3: Technikrom 120 mmol Synthesizer and HPLC Skid ƒ Desalting and Freeze-Drying Equipment to Scale 2 © 2009 Antisense Therapeutics Solvay Organics ® Protein Synthesis in unaltered Cell ƒ Central Dogma of Biology ƒ DNA Æ mRNA Æ Protein 3 © 2009 Principle of Action of Antisense Oligonucleotides ® Solvay Organics ƒ Antisense Strand hybridizes to targeted mRNA ƒ Blocks translation of message into protein ƒ Typical Modifications: 2’-O-Alkyl, LNA, Morpholinos, N3’,P5’Amidates, Gapmers, Conjugates, etc. 4 © 2009 ® Solvay Organics Aptamers ƒ Nucleic acid macromolecule that binds tightly to a protein target ƒ Linear sequence of nucleotides, typically 20-45 nucleotides long ƒ Chain of nucleotides forms intramolecular interactions and folds into a complex three-dimensional shape ƒ Typical modifications: Amino end modified for post-conjugation with PEG; DNA, RNA, 2’-OMe, thioates, L-isomers (Spiegelmers) 5 © 2009 ® Immunostimulatory Oligonucleotides Solvay Organics From Krieg, A. M. 2006. Nat.Rev.Drug Discov. 5:471-484. ƒ Oligonucleotides containing unmethylated CpG motifs ƒ TLR9 agonists initiate both an innate and an adaptive immune response, generating cytotoxic T cells (CTLs) and disease-specific antibodies 6 ƒ Typical modifications: DNA thioates, spacer, 5’-3’, base modifications © 2009 ® RNA Interference (siRNA) Solvay Organics From Nobel Price Committee of Physiology and Medicine ƒ Nobel Prize winning Science ƒ Science Magazine: Breakthrough of the year 2002 ƒ Andrew Fire and Craig Mello - Nobel Prize 2006 ƒ Typical modifications: 2’-OMe, 2’-Fluoro, Thioates, Conjugates 7 © 2009 miRNA Solvay Organics ® miRNA RISC mRNA From RegulusRX.com ƒ One miRNA regulates expression of multiple genes ƒ miRNAs function by preventing translation of mRNAs into proteins ƒ miRNA work as single strands, “bubbles” are important for function 8 © 2009 ® miRNA Solvay Organics miRNA anti miR RISC ƒ anti miR Therapeutics hybridize to miRNA inhibiting formation of miRNA / mRNA duplex (upregulation) ƒ miRNA Therapeutics are being incorporated into the RISC complex and forming a miRNA / mRNA duplex (downregulation, not shown) 9ƒ Typical modifications: 2’-OMe, 2’-Fluoro, Thioates, DNA, LNA © 2009 ® Girindus’ Extensive Oligo Manufacturing Experience ƒ DNA, Phosphorodiesters And Phosphorothioates ƒ RNA, Phosphorodiesters And Phosphorothioates ƒ 2‘-modified RNA ƒ Duplex RNA (small interfering RNA or siRNA) ƒ Chimeras/Gapmers (DNA/RNA/LNA) ƒ Aptamers With Various Backbones ƒ Conjugates ƒ DNA/RNA With Modified Backbone ƒ Radiolabeling (14C, 3H) Solvay Organics 10 © 2009 ® Solid Phase Synthesis Of Oligonucleotides Starting Nucleoside on Solid Support DMTO Base O DMT (yellow) O O Base O O O O R 1 1 Solvay Organics + R 1. Detritylation CHCl2COOH HO O O R Cleavage Base1 R: OTBDMS (RNA) OCH3 (2'-OMe-RNA) H (DNA) AcO 5'-OH-Oligomer 4. Capping Chain Termination DMTO Base O O NCH2CH2CO P X O X=O S O NCH2CH2CO R O P O O O O R R Base1 O R Base O DMTO O 1 2 DMTO Base O O P N R 2 2. Coupling OCH2CH2CN Base2 + Ethylthio-tetrazole, Benzyl-mercapto-tetrazole or Dicyanoimidazole 3. Oxidation ƒ 4 synthetic steps per base (21-mer = 80 chemical reactions) ƒ No internal quality control until after cleavage and deprotection 11 © 2009 Key Raw Materials Solvay Organics ® Phosphoramidites: NH-Bz N N DMTO O P NCCH2CH2O NiPr2 NCCH2CH2O R= H: deoxy adenosine R= OMe: 2'-OMe adenosine R= OTBDMS: adenosine O R N DMTO O P NiPr2 NCCH2CH2O R= H: deoxy cytidine R= OMe: 2'-OMe cytidine R= OTBDMS: cytidine O R N N NH-Ac N O DMTO O P NiPr2 NCCH2CH2O R= H: deoxy guanosine R= OMe: 2'-OMe guanosine R= OTBDMS: guanosine O R N N N O R' NH NH-iBu DMTO O P NiPr2 R= H, R'=Me: thymidine R= OMe, R'= H: 2'-OMe uridine R= OTBDMS, R'=Me: uridine O R N O NH O Solid Supports: CPG Si H N O H N O N H B DMTO O O O O R DMTO O O O R B or PS N H Abbreviations: OCH3 O DMT: OCH3 Bz: Ac: O CH3 iBu: CH3 CH3 H3C CH3 Si TBDMS: H3C H3C CH3 O 12 © 2009 ® Solvay Organics Critical and Non-Critical Impurities Critical Impurities ƒ Phosphitylation reagent and all other related P(III) impurities ƒ Different Isomers of Nucleoside Phosphoramidite ƒ Other P(III) compounds resulting from phosphitylated nucleoside impurities ƒ Water and all Alcohols (residual solvents) Non-Critical Impurities ƒ All Oxidized and Hydrolyzed P(III) impurities ƒ Non-alcohol residual solvents 13 © 2009 Quality of 1e+005 31P-NMR 1e+005 Spectra Solvay Organics ® 1024 scans 0e+000 6e+004 4e+004 2e+004 0e+000 152.0 3e+004 2e+004 1e+004 0e+000 152.0 15000 10000 5000 0 152.0 5000 151.5 151.0 150.5 150.0 149.5 149.0 148.5 ppm 151.5 151.0 150.5 150.0 149.5 149.0 148.5 ppm 151.5 151.0 150.5 150.0 149.5 149.0 148.5 ppm 152.0 151.5 151.0 150.5 150.0 149.5 149.0 148.5 ppm 0e+000 6e+004 4e+004 2e+004 0e+000 35 3e+004 35 30 25 20 15 10 5 ppm 512 scans 30 25 20 15 10 5 ppm 256 scans 2e+004 1e+004 0e+000 35 15000 30 25 20 15 10 5 ppm 128 scans 10000 5000 0 35 5000 30 25 20 15 10 5 ppm 64 scans 0 0 14 152.0 151.5 151.0 150.5 150.0 149.5 149.0 148.5 ppm 35 30 25 20 15 10 5 ppm © 2009 Quality of Spectra continued No. of No. of Scans 1024 512 256 128 64 S/N ratio 6565 4691 3158 2343 1633 Purity 98.1% 97.8% 98.1% 98.0% 98.3% P(III) Impurities 0.82% 0.86% 0.62% 0.72% 0.41% P(III) Impurities 9 7 5 3 2 P(V) No. of P(V) Impurities Impurities 1.11% 6 1.33% 7 1.26% 6 1.31% 5 1.25% 5 31P-NMR ® Solvay Organics 15 © 2009 Amidite Quality Differences by 1.0e+005 31P-NMR Solvay Organics ® P(III): 0.8% 1e+005 Manufacturer A, Lot A1, 2y 9m at 20 C P(V): 1.4% 45 40 35 30 25 20 15 10 5 0 ppm 5.0e+004 0e+000 0.0e+000 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 ppm 1.0e+005 P(III): 0.5% 1e+005 Manufacturer A, Lot A2, upon receipt P(V): 0.8% 45 40 35 30 25 20 15 10 5 0 ppm 5.0e+004 0e+000 0.0e+000 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 ppm 1.0e+005 P(III): 2.6% 1e+005 0e+000 Manufacturer A, Lot A3,, upon receipt P(V): 0.7% 45 40 35 30 25 20 15 10 5 0 ppm 5.0e+004 0.0e+000 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 ppm 1.0e+005 P(III): 0.8% 1e+005 0e+000 Manufacturer B, Lot B1, upon receipt P(V): 0.0% 45 40 35 30 25 20 15 10 5 0 ppm 5.0e+004 0.0e+000 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 ppm 16 © 2009 Amidite Quality Differences by HPLC Solvay Organics 8 .2 4 7 ® 8 .2 4 7 9 .1 4 5 0.40 0.35 0.30 0.25 A U 0.20 0.15 0.10 0.05 0.00 2.00 4.00 6.00 8.00 0.050 3 .3 9 5 9 .8 7 7 3 .6 9 1 8 .0 0 0 0.000 -0.010 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Minutes 9.00 10.00 11.00 1 1 .1 7 6 0.010 7 .1 1 7 7 .2 1 5 2 .4 1 3 5 .2 2 9 5 .5 0 6 5 .7 1 1 6 .4 7 0 7 .7 8 2 1 0 .8 0 5 Lot A2 97.1% 0.040 0.030 A U 0.020 9 .1 4 5 12.00 13.00 14.00 15.00 10.00 1 0 .8 0 5 1 1 .1 7 6 12.00 3 .3 9 5 3 .6 9 1 7 .7 8 2 8 .0 0 0 7 .1 1 7 7 .2 1 5 5 .2 2 9 5 .5 0 6 5 .7 1 1 6 .4 7 0 2 .4 1 3 9 .8 7 7 14.00 16.00 Minutes 18.00 20.00 22.00 24.00 26.00 28.00 30.00 8 .2 8 5 9 .1 9 0 8 .2 8 5 0.50 0.040 0.40 A U 0.30 Lot A3 98.5% 5 .2 4 5 5 .5 2 3 5 .7 3 4 7 .1 2 2 7 .2 0 0 7 .8 1 2 6 .4 9 9 0.030 A U 0.020 5 .2 4 5 9 .9 2 9 1 0 .8 6 3 0.20 0.000 -0.010 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Minutes 9.00 10.00 11.00 1 1 .2 3 8 0.010 5 .5 2 3 5 .7 3 4 7 .1 2 2 7 .2 0 0 6 .4 9 9 7 .8 1 2 9 .1 9 0 0.050 12.00 13.00 14.00 15.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Minutes 18.00 20.00 22.00 24.00 26.00 28.00 30.00 8 .2 9 2 0.50 9 .1 9 4 1 0 .8 6 3 1 1 .2 3 8 9 .9 2 9 0.10 8 .2 9 2 0.050 0.40 0.30 A U 0.010 0.000 -0.010 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Minutes 9.00 10.00 11.00 1 0 .9 9 2 Lot B1 99.7% 0.040 0.030 A U 0.020 9 .1 9 4 12.00 13.00 14.00 15.00 0.20 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Minutes 18.00 20.00 22.00 24.00 26.00 28.00 30.00 1 0 .9 9 2 1 9 .6 4 0 2 0 .3 1 2 0.10 17 © 2009 Amidite Quality Lot A2 A3 B1 Vendor A A B 31P-NMR P NMR 96.7 98.7 99.2 vs HPLC Solvay Organics ® HPLC 97.1 98.5 99.7 P(III) 2.6 0.5 0.8 P(V) 0.7 0.8 0.0 18 © 2009 ® Girindus’ Manufacturing Site Cincinnati, Ohio, USA Solvay Organics ƒ Contract Manufacturer with strong GMP culture ƒ FDA inspected ƒ Wide range of licensing deals ensure Freedom to Operate ƒ Pre Clinical through Commercial Scale cGMP Manufacturing of Oligonucleotides ƒ 3 Manufacturing Trains available from Gram to Multi Kilogram 19 © 2009 ® Manufacturing Train 1 – Smaller Scales AKTA 100 DNA-Synthesizer (0.1 – 8 mmol) AKTA Purifier (max. flow rate 100 mL/min) Manual Desalting system ƒ FMI Metering Pump (1.2 L/min max flow) ƒ Total Membrane Area 0.1 – 0.4 m2 Solvay Organics Virtis Freeze Drier (6 lyo guard trays up to 1.8 L each) 20 © 2009 AKTA 100 Oligopilot DNA/RNA Solvay Organics ® Synthesizer (Scale 0.1 - 8 mmoles) 21 © 2009 ® Purification - AKTA 100 Purifier (max. flow rate 100 mL/min) Solvay Organics 22 © 2009 ® VirTis Genesis 35 XL Freeze Dryer Solvay Organics Space for 6 Lyoguard Freeze-Drying Trays of up to 1.8 L each Gore membrane allows for water or solvent removal without chance for spills or product contamination of freeze drier. Optional controls with vacuum gauges Barometric endpoint and moisture analysis Additional product probes and security systems. Shelf pull-down to -40°C in 30 minutes or less, with efficient, utility saving compressors. 23 © 2009 ® Manufacturing Train 2 – Mid Scale OP 400 DNA-Synthesizer (8 – 60 mmol) Purification Systems ƒ Bioprocess (max. flow rate 2 L/min) (low pressure) ƒ HyperSep Skid (max. flow rate 1 L/min) (high pressure) Solvay Organics UniFlux Automated Desalting System ƒ 10,000 mL/min max retentate flow ƒ Total Membrane Area 0.1 – 1.0 m2 Virtis Freeze Drier (12 lyo guards up to 1.8 L each) © 2009 24 ® OligoPilotTM 400 DNA/RNA Synthesizer (Scale 8 - 60 mmoles) Solvay Organics 25 © 2009 ® Purification BioProcess Low Pressure System Solvay Organics 26 © 2009 ® UniFlux 10 for HPUF Desalting Solvay Organics 27 © 2009 ® Solvay Organics Manufacturing Train 3 - Large Scale Technikrom DNA-Synthesizer (10 – 120 mmol) Technikrom HPLC Skid (3 L/min max flow at 1000 PSI) Manual Desalting System ƒ 30 L/min max retentate flow ƒ Total Membrane Area 0.5 – 4.0 m2 Virtis Freeze Drier (2 x 15 lyo guards up to 1.8 L each) 28 © 2009 ® Solvay Organics Technikrom DNA/RNA Synthesizer (Scale 20-120 mmoles) with 20 cm Dynamic Axial Compression Column 29 © 2009 ® Solvay Organics Purification: Technikrom HPLC Skid: • 3 L/min max flow at 1000 PSI • Pre and post column temperature control Varian Column • Jacketed, 20 cm 30 © 2009 ® VirTis 50L Ultra XL-70 Freeze Dryer Lyoguard Freeze-Drying Trays Gore membrane allows for water or solvent removal without chance for spills or product contamination of freeze drier. 15 shelves per chamber. Total of 2 chambers available. Optional controls with vacuum gauges Barometric endpoint and moisture analysis Additional product probes and security systems. Shelf pull-down to -40°C in 30 minutes or less, with efficient, utility saving compressors. Solvay Organics 31 © 2009 © 2006 Girindus ® Analytical Capabilities Purity (AEX-HPLC or RP-HPLC) Purity/impurity identification LC/MS (Electrospray ionisation) 31P-NMR Solvay Organics Capillary Gel Electrophoresis (CGE) Water Content by Karl Fischer Coulometry Residual Solvents Endotoxins Base Composition (out-of-house) Additional out-of-house testing (sodium content, metals testing, pH measurements, bioburden) 32 © 2009 ® Summary Girindus – Manufacturing Experience for all Major Types of Oligonucleotide Therapeutics Control of Critical Raw Materials of High Importance 3 Manufacturing Trains Available from Preclinical to Commercial Committed to Growth Full Service GMP Manufacturer ƒ QA ƒ QC ƒ Production Solvay Organics FDA Inspected 33 © 2009 ® Solvay Organics Acknowledgements Gary Burt, Chemist, QC Analytics Jose Bonilla, Section Head, QC Analytics Wayne Davis, Production Supervisor - Oligonucleotides Tad Wyrzykiewicz, Director – Oligonucleotides 34 © 2009 © 2006 Girindus Solvay Organics ® ® Solvay Organics 35 © 2009 2009 35