Lipid Nanoparticle LNP Systems for RNA Delivery Photo

Lipid Nanoparticle (LNP) Systems for RNA Delivery Photo by Kyriakos Kaziras

Lipid Nanoparticle (LNP) System Nucleic Acid Delivery by LNP Photo from Precision Nanosystems

Lipid Nanoparticle (LNP) System General Properties Size: diameters less than 100 nm Basic constituents • Structural lipid • Ionizable amino lipid • PEG-lipid conjugate • Cholesterol • Nucleic acid (si. RNA, m. RNA, etc) p. Ka of ionizable amino lipid less than p. H 7 Prepared using microfluidics (ethanol/water mixing in flow system) Advantages • Increased half life • Protection from nucleases • Aids in endocytosis and endosomal escape Disadvantages • Elevated risk of toxicity with introducing excipient materials

Lipid Nanoparticle (LNP) System Basic Constituents & Structural Elements Photo from Arbutus Biopharm

Lipid Nanoparticle (LNP) System Basic Constituents: Cationic Lipid Pros Efficient delivery Condense plasmid Cons Toxicity Dissociation from DNA Pros Lower toxicity Cons Dissociation from DNA Pros Low/no toxicity Dissociates from DNA Cons Low efficiency

Lipid Nanoparticle (LNP) System Basic Constituents: Cationic Lipid Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol Ther. 2017 Jul 5; 25(7): 1467 -1475.

Lipid Nanoparticle (LNP) System Effect of Cationic Lipid Structure on p. Ka Jayaraman et al. Maximizing the potency of si. RNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012 Aug 20; 51(34): 8529 -33.

Lipid Nanoparticle (LNP) System Effect of p. Ka on LNP Delivery One of the key findings from these studies was the optimum lipid p. Ka value of 6. 2– 6. 5 as a dominating factor in determining hepatic gene-silencing activity in vivo. Wan C, Allen TM, Cullis PR. Lipid nanoparticle delivery systems for si. RNA-based therapeutics. Drug Deliv Transl Res. 2014 Feb; 4(1): 74 -83.

Photo by Kyriakos Kaziras

c. GMP Manufacturing API, Excipient, & Contract Manufacturing c. GMP manufacturing initiated in 1985 Supplied the first semi-synthetic phospholipid for an approved drug product in 1990 Dipalmitoyl phosphatidylcholine (DPPC) Exosurf® Neonatal, synthetic lung surfactant replacement for neonates with respiratory distress syndrome Currently support ~25 active drug master files for clinical trials and commercial products Manufacture c. GMP quality materials from 50 g to 25 kg batches Maximum annual capacity: 2 MT per shift Manufacturing suites available for production: General lipid production 10 Small scale lipid production 1 Adjuvant production 2 Formulations 1

Applications In general, lipids are non-toxic, flexible, biocompatible, completely biodegradable, and non-immunogenic for systemic and non-systemic administrations. Classical drug delivery systems (drug depot) Encapsulation of hydrophilic drugs (passive and remote loading) Sequestration and dispersion of hydrophobic drugs Targeted drug delivery systems via coupling with site-specific ligands Delivery of genetic material (cationic lipids) Plasmid si. RNA m. RNA Liposomal adjuvant systems for vaccine development and immunotherapy Contrast agents for MRI (targeted and untargeted) Nasal and pulmonary delivery of drugs Topical delivery of drugs (enhanced penetration) Opthalmic delivery of drugs Lipid-coated microbubbles for ultrasound imaging or treatment

Approved Liposomal Drug Products Drug Product Patent Expires Active Drug Lipid Components Dauno. Xome® 2009 Daunorubicin DSPC, Cholesterol Doxil® 2010 Doxorubicin HSPC, Cholesterol, DSPE-m. PEG(2000) Epaxal® 2012 Inactivated hepatitis A virus DOPC: DOPE Inflexal® V 2012 Inactivated hemagglutinin of influenza virus strains A and B DOPC: DOPE Depocyt® 2015 Cytarabin/Ara-C DOPC, DPPG, Triolein, Cholesterol Ambisome® 2016 Amphotericin B HSPC, DSPG, Cholesterol Amphotec® 2016 Amphotericin B Cholesteryl sulphate Visudyne® 2016 Verteporphin DMPC, EPG Depo. Dur™ 2017 Morphine sulfate DOPC, DPPG, Triolein, Cholesterol Mepact® 2019 Mifamurtide DOPS, POPC Abelcet® 2019 Amphotericin B DMPC, DMPG Marqibo® 2020 Vincristine SM, Cholesterol Exparel® 2021 Bupivacaine DEPC, DPPG, Tricaprylin, Cholesterol Onivyde™ 2028 Irinotecan DSPC, DSPE-m. PEG(2000), Cholesterol Myocet® N/A Doxorubicin EPC, Cholesterol

c. GMP Lipids Currently Available Phospholipids (Semi-synthetic, plant-derived) DMPC DLPE DMPG DPPC DMPE DPPG DSPC DPPE DSPG DEPC DSPE POPG DOPC DOPE POPC Phospholipids (Natural) Egg PC (>95%) Adjuvants (Synthetic MPLA) PHAD® 3 D-PHAD® 3 D(6 -acyl)-PHAD® Other Tricaprylin Triolein DOTAP Semi-synthetic Cholesterol (plant-derived) DSPE-m. PEG(2000)-Maleimide

Adjuvants Vaccine Development & Immunotherapy Synthetic Monophosphoryl Lipid A (MPLA) PHAD® 3 D-PHAD® 3 D(6 -acyl)-PHAD® Equivalent to bacterial-derived MPLA (native) Toll-like receptor 4 (TLR 4) agonist Elicits a strong TH 1 immune response Used in multiple clinical trials Adjuvant system formulations available (research & c. GMP) Available in gram to kilogram scale

Formulations Liposomes & Nanoparticles From research to early phase clinical Products Liposomes Emulsion Lyophilized lipid formulations Scaled from milliliters to > 100 L Most Avanti lipids can be incorporated into liposomal formulations Custom synthesis lipids can also be used in formulations

Contract Manufacturing Proven Partner for Development to Commercialization Over 150 combined years of lipid synthesis experience Route scouting and process development for proprietary lipids Small-scale batches available (<100 g) Scale-up support for multi-kilo production Custom packaging options Full analytical support with method development and validation Regulatory support for clinical trial and commercial filings

People do amazing things with our lipids! What will you do?