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WO2024083345A1 - Procédés et utilisations associés à des compositions liquides - Google Patents

Procédés et utilisations associés à des compositions liquides Download PDF

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Publication number
WO2024083345A1
WO2024083345A1 PCT/EP2022/079475 EP2022079475W WO2024083345A1 WO 2024083345 A1 WO2024083345 A1 WO 2024083345A1 EP 2022079475 W EP2022079475 W EP 2022079475W WO 2024083345 A1 WO2024083345 A1 WO 2024083345A1
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WO
WIPO (PCT)
Prior art keywords
liquid
rna
mixing chamber
lipid
liquid composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2022/079475
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English (en)
Inventor
Christian Reinsch
Steffen Panzner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biontech SE
Original Assignee
Biontech SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biontech SE filed Critical Biontech SE
Priority to PCT/EP2022/079475 priority Critical patent/WO2024083345A1/fr
Priority to EP23794304.8A priority patent/EP4604933A1/fr
Priority to PCT/EP2023/079374 priority patent/WO2024084089A1/fr
Publication of WO2024083345A1 publication Critical patent/WO2024083345A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3017—Mixing chamber
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F2215/00—Auxiliary or complementary information in relation with mixing
    • B01F2215/04—Technical information in relation with mixing
    • B01F2215/0413—Numerical information
    • B01F2215/0418—Geometrical information
    • B01F2215/0431—Numerical size values, e.g. diameter of a hole or conduit, area, volume, length, width, or ratios thereof

Definitions

  • the present disclosure relates to improvements associated with liquid compositions, in particular lipid nanoparticle compositions and their manufacture.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • DNA deoxyribonucleic acid
  • lipid nanoparticle compositions comprising or consisting of lipid nanoparticles (LNPs) is a key aspect for nucleic acid therapeutics.
  • Properties of the LNPs which are provided may influence the stability or quality of the therapeutic or intermediate products and/or have decisive influences on the yield during the production process.
  • the improvements may relate to the method of providing or producing liquid compositions, e.g. lipid nanoparticle compositions, as such, to components, e.g. components configured to be used in the method or configured to be used in the method, to uses associated with the method and/or preparations, e.g. comprising lipid nanoparticles, such as lipid nanoparticle compositions obtainable or obtained with the method.
  • One aspect of the present disclosure relates to a method of forming, providing or producing a liquid composition, e.g. by mixing a first liquid and a second liquid.
  • Another aspect of the present disclosure relates to a use of a mixing component for forming, providing or producing a liquid composition, e.g. by mixing a first liquid and a second liquid and/or with the method(s) described herein.
  • Yet another aspect of the present disclosure relates to a preparation comprising lipid nanoparticles, e.g. particles of the liquid composition obtainable or obtained with the method and/or with the use of the mixing component.
  • the preparation may be the liquid composition or be obtained or obtainable from the liquid composition.
  • the method and/or the use comprises:
  • a mixing chamber e.g. a region in the mixing component
  • the liquid composition is a lipid nanoparticle (LNP) composition.
  • the composition expediently comprises lipid nanoparticles, e.g. within a carrier liquid, such as a liquid comprising water and/or ethanol.
  • the liquid composition is a nucleic acid-LNP composition, e.g. an RNA-LNP composition or a DNA-LNP composition.
  • the mixing chamber is provided in a mixing component, the mixing component having a first inlet in fluid communication with the mixing chamber and a second inlet in fluid communication with the mixing chamber.
  • the first and second inlets may be fluidically separated from one another.
  • the first and second liquid may be separated from one another until they meet in the mixing chamber.
  • the mixing chamber may be that region of the mixing component where the two liquids meet.
  • the mixing chamber and/or the mixing component has an outlet.
  • the outlet of the mixing chamber may be a passage where the liquid composition flow, after mixing, enters a section of a flow path or conduit with constant cross sectional area or diameter.
  • the cross sectional area (or cross section) may be less than or equal to the (maximum, minimum and/or average) cross sectional area or diameter of the mixing chamber.
  • the outlet of the mixing component may be the passage to a region of the flow path of the liquid composition downstream of the mixing chamber where the cross section or diameter of the flow path increases (e.g. as compared to the outlet of the mixing chamber).
  • the outlet of the mixing component may be located at an interface between the mixing component and another component, e.g. a tubing.
  • the cross section or diameter of the outlet of the mixing component may be equal to the cross section of the outlet of the mixing chamber.
  • the flow path from the mixing chamber outlet to the mixing component outlet may have a constant cross section or diameter.
  • the method is performed such that the flow of the liquid composition away from the mixing chamber and/or at the outlet of the mixing chamber or of the mixing component has a Reynolds number of less than or equal to 10000 and/or greater than or equal to 800.
  • the mixing component is used to provide a liquid composition, e.g. a lipid nanoparticle (LNP) composition, by mixing a first liquid and a second liquid in a mixing chamber of the mixing component.
  • the mixing component may be used to provide a liquid flow, e.g. away from the mixing chamber and/or at the outlet of the mixing chamber or of the mixing component, with a Reynolds number of greater than or equal to 800 and/or less than or equal to 10000 at an outlet of the mixing chamber or of the mixing component.
  • the liquid flow may be a flow of the liquid composition.
  • Reynolds numbers are used to classify a liquid flow.
  • the Reynolds number R of a liquid flow can be calculated by using the following formula:
  • V V * D / Vis_kin
  • D a characteristic distance (e.g. the diameter of the flow path guiding the liquid flow, e.g. the inner diameter of a conduit) in m (meter)
  • Vis kin is the kinematic viscosity in m 2 /s.
  • the kinematic viscosity results from the (dynamic) viscosity (Vis dyn) of the liquid in Pascal seconds, Pa s, divided by the density D_L of the liquid, e.g. in kg/m 3 .
  • the velocity V can be derived from the flow rate (e.g. specified in ml/min, i.e.
  • the respective Reynolds number for the liquid composition flow discussed herein may relate to Reynolds numbers based on values for the relevant quantities which are calculated as set forth below or based on values for the relevant quantities which have been measured.
  • the Reynolds number may be calculated by using:
  • V (F l + F_2) / ((D/2) 2 * 71), with
  • D being the inner diameter of the flow path at the relevant location, e.g. at the outlet of the mixing chamber or of the mixing component,
  • F l being the flow rate of the first liquid into the mixing chamber
  • F_2 being the flow rate of the second liquid into the mixing chamber (the sum of F l and F_2_being the flow rate of the liquid composition at the outlet of the mixing chamber).
  • Vis_dyn F l / (F_l + F_2) * Vis_l + F_2 / (F_l + F_2) * Vis_2, with
  • Vis l being the (dynamic) viscosity of the first liquid
  • Vis_2 being the dynamic viscosity of the second liquid.
  • D_L F l / (F l + F_2) * D_1 + F_2 / (F_l + F_2) * D_2, with
  • Vis_kin Vis_dyn / D_L
  • Reynolds numbers are dimensionless quantities.
  • the Reynolds number can be used to qualify a liquid flow without having to specify dimensions of the conduit or other values which are characteristic for the flow like the flow rate, viscosity, density, etc..
  • the inventors attribute the positive effects for the nanoparticles to the liquid composition flow having Reynolds numbers less than 10000 and/or greater than 800 (e.g. right after the mixing of the first and second liquids, such as in the mixing chamber, at the outlet of the mixing chamber or at the outlet of the mixing component and/or before another substance is being added to the composition) when the mixture is in a state in which the nanoparticles are being formed from ingredients of the first liquid and the second liquid or the formation is being initiated. Having the liquid flow during the (initial) formation stage of the nanoparticles in the specified Reynolds number range resulted in the formation of advantageous nanoparticles.
  • Reynolds numbers of 2000 and above or 2500 and above may characterize liquid flow in a transitional regime between laminar flow and turbulent flow or mildly turbulent flow (usually the transition region between laminar and turbulent flow is around 2500).
  • a Reynolds number of 800 characterizes a laminar flow.
  • 10000 characterizes a turbulent but not yet very turbulent flow.
  • the range of 800 to 10000 covers laminar flow as well as its transition to turbulent and mild turbulent flow.
  • the liquid flow after mixing is maintained in the relevant Reynolds number range or the Reynolds number is changed after the outlet, e.g. due to an increase in diameter.
  • the method and/or the use is performed such that the flow of the liquid composition away from the mixing chamber and/or at the outlet of the mixing chamber or of the mixing component has a Reynolds number of less than or equal to any one of the following: 9950, 9900, 9850, 9800, 9750, 9700, 9650, 9600, 9550, 9500, 9450, 9400, 9350, 9300, 9250, 9200, 9150, 9100, 9050, 9000, 8950, 8900, 8850, 8800, 8750, 8700, 8650, 8600, 8550, 8500, 8450, 8400, 8350, 8300, 8250, 8200, 8150, 8100, 8050, 8000, 7950, 7900, 7850, 7800, 7750, 7700, 7650, 7600, 7550, 7500, 7450, 7400, 7350, 7300, 7250, 7200, 7150, 7100, 7050, 7000, 6950, 6900, 6850, 6800, 6750, 6700, 9650
  • the method and/or the use is performed such that the flow of the liquid composition away from the mixing chamber and/or at the outlet of the mixing chamber or of the mixing component has a Reynolds number of greater than or equal to any one of the following: 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200,
  • Reynolds numbers below 10000 and/or above 800 can yield advantageous lipid nanoparticles.
  • the method and/or the use is performed such that the flow of the liquid composition away from the mixing chamber and/or at the outlet of the mixing chamber or of the mixing component has a Reynolds number of between any one of the following Reynolds number pairs: 800 and 8500, 800 and 6500, 800 and 5000, 1000 and 8500, 1000 and 6500, 1000 and 5000, 2000 and 8500, 2000 and 6500, 2000 and 5000, 3000 and 8500, 3000 and 6500, 3000 and 5000, 4000 and 8500, 4000 and 6500, 4000 and 6500, 4000 and 5000.
  • the first liquid and/or the second liquid is guided into the mixing chamber and/or into the mixing component with a flow rate of greater than or equal to any one of the following: 10 ml/min, 20 ml/min, 30 ml/min, 40 ml/min, 50 ml/min, 60 ml/min, 70 ml/min, 80 ml/min, 90 ml/min, 100 ml/min, 110 ml/min, 120 ml/min, 130 ml/min, 140 ml/min, 150 ml/min, 160 ml/min, 170 ml/min, 180 ml/min, 190 ml/min, 200 ml/min, 210 ml/min, 220 ml/min.
  • the first liquid and/or the second liquid is guided into the mixing chamber and/or into the mixing component with a flow rate of less than or equal to any one of the following: 660 ml/min, 650 ml/min, 640 ml/min, 630 ml/min, 620 ml/min, 610 ml/min, 600 ml/min, 590 ml/min, 580 ml/min, 570 ml/min, 560 ml/min, 550 ml/min, 540 ml/min, 530 ml/min, 520 ml/min, 510 ml/min, 500 ml/min, 490 ml/min, 480 ml/min, 470 ml/min, 460 ml/min, 450 ml/min, 440 ml/min, 430 ml/min, 420 ml/min, 410 ml/min, 400 ml/min, 650
  • the flow rate of the first liquid and/or the flow rate of the second liquid may be between 10 and 660 ml/min.
  • Arbitrary sub-ranges may be formed by the disclosed values.
  • the flow may be driven by an associated flow driver, e.g. a pump.
  • One flow driver may be assigned to each liquid, i.e. the first liquid or the second liquid.
  • the liquid composition may be driven by the flow drivers in combination.
  • the flow rate with which the first liquid is guided or driven into the mixing chamber is different from, e.g. greater than, the flow rate with which the second liquid is guided or driven into the mixing chamber.
  • the ratio between the flow rate of the first liquid and the flow rate of the second liquid may be less than or equal to any one of the following: 5, 4, 3.
  • the ratio may be greater than 1 or greater than 2, e.g. 3.
  • the liquid composition is guided or driven away from the mixing chamber and/or leaves the mixing chamber or the mixing component via the respective outlet with a flow rate of greater than or equal to any one of the following: 10 ml/min, 20 ml/min, 30 ml/min, 40 ml/min, 50 ml/min, 60 ml/min, 70 ml/min, 80 ml/min, 90 ml/min, 100 ml/min, 110 ml/min, 120 ml/min, 130 ml/min, 140 ml/min, 150 ml/min, 160 ml/min, 170 ml/min, 180 ml/min, 190 ml/min, 200 ml/min, 210 ml/min, 220 ml/min, 230 ml/min, 240 ml/min, 250 ml/min, 260 ml/min, 270 ml/min, 280 ml/min, 290
  • the flow rate of the liquid composition away from the mixing chamber or at its outlet may be defined by, e.g. equal to, the sum of the flow rates with which the first liquid and the second liquid enter the mixing chamber.
  • the same flow drivers may be used to drive the first and second liquid flow and also the liquid composition flow.
  • the liquid composition is guided or driven away from the mixing chamber and/or leaves the mixing chamber or the mixing component via the outlet with a flow rate of less than or equal to any one of the following: 1000 ml/min, 950 ml/min, 900 ml/ min, 890 ml/min, 880 ml/min, 870 ml/min, 860 ml/min, 850 ml/min, 840 ml/min, 830 ml/min, 820 ml/min, 810 ml/min, 800 ml/min, 790 ml/min,
  • 140 ml/min, 130 ml/min, 120 ml/min, 110 ml/min, 100 ml/min, 90 ml/min, 80 ml/min, 70 ml/min, 60 ml/min, 50 ml/min, 40 ml/min, 30 ml/min, 20 ml/min, 10 ml/min.
  • the flow rate of the liquid composition may be between 10 ml/min and 1000 ml/min or be in any sub-range derived from the values stated above.
  • the nanoparticles of the lipid nanoparticle composition have a size of less than or equal to any one of the following: 195 nm, 190 nm, 185 nm, 180 nm, 175 nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145 nm, 140 nm, 135 nm, 130 nm, 125 nm, 120 nm, 115 nm, 110 nm, 105 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm.
  • the nanoparticles of the lipid nanoparticle composition have a size of greater than or equal to any one of the following: 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm
  • the nanoparticles of the lipid nanoparticle composition have a size, e.g. an average size, of greater than or equal to any one of the following: 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm (nm: nanometers).
  • the nanoparticles of the lipid nanoparticle composition have a size, e.g. an average size, of less than or equal to any one of the following: 100 nm, 99 nm, 98 nm, 97 nm, 96 nm, 95 nm, 94 nm, 93 nm, 92 nm, 91 nm, 90 nm, 89 nm, 88 nm, 87 nm, 86 nm, 85 nm, 84 nm, 83 nm, 82 nm, 81 nm, 80 nm, 79 nm, 78 nm, 77 nm, 76 nm, 75 nm, 74 nm, 73 nm, 72 nm, 71 nm, 70 nm, 69 nm, 68 nm, 67 nm, 66 nm, 65 nm, 64 nm,
  • the size may be defined by the diameter of the nanoparticles, e.g. based on the maximum, minimum or average diameter of the particles.
  • the size of the nanoparticles may be between 20 nm and 195 nm or be in any sub-range derived from the values stated above, e.g. between 40 nm and 100 nm.
  • the size depends on whether and what substance the nanoparticles encapsulate. The greater the substance, the greater the nanoparticles, of course.
  • the outlet of the mixing chamber or of the mixing component has a diameter of greater than or equal to any one of the following: 0.1 mm, 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm (mm: millimeters).
  • the respective diameters for inlets, openings or flow paths specified herein are expediently inner diameters.
  • the diameter of the flow path or conduit varies, e.g. azimuthally or circumferentially, the diameter at a certain position of the flow path may be the maximum, minimum or average diameter at the certain position.
  • the outlet of the mixing chamber or of the mixing component has a diameter of less than our equal to any one of the following: 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, 0.95 mm, 0.9 mm, 0.85 mm, 0.8 mm, 0.75 mm, 0.7 mm, 0.65 mm, 0.6 mm, 0.55 mm, 0.5 mm.
  • the outlet of the mixing chamber or of the mixing component may have a diameter of between 0.1 mm and 4 mm or be in any sub-range derived from the values stated above.
  • the first inlet and/or the second inlet of the mixing chamber or of the mixing component has a diameter of greater than or equal to any one of the following: 0.1 mm, 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm.
  • the first inlet and/or the second inlet of the mixing chamber or of the mixing component has a diameter of less than our equal to any one of the following: 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, 0.95 mm, 0.9 mm, 0.85 mm, 0.8 mm, 0.75 mm, 0.7 mm, 0.65 mm, 0.6 mm, 0.55 mm, 0.5 mm.
  • the first inlet of the mixing chamber or of the mixing component may have a diameter of between 0.1 mm and 4 mm or be in any sub-range derived from the values stated above.
  • the second inlet of the mixing chamber or of the mixing component may have a diameter of between 0.1 mm and 4 mm or be in any sub-range derived from the values stated above.
  • the first inlet and the second inlet may have the same or different diameters.
  • the outlet may have the same diameter as one of or both of the inlets.
  • the outlet may have a diameter which is different from the diameter of the first and the second inlets.
  • a viscosity of the first liquid and/or or the second liquid is less than or equal to any one of the following values: 1.8 cP, 1.7 cP, 1.6 cP, 1.5cP, 1.4 cp, 1.3 cP, 1.2 cP, 1.1 cP, 1.0 cP, 0.9 cP.
  • the viscosity of the first liquid and/or the second liquid may be between 0.5 cP and 1.8 cP or be in any sub-range derived from the values stated above.
  • a viscosity of the liquid composition is less than or equal to any one of the following values: 1.8 cP, 1.7 cP, 1.6 cP, 1.5cP, 1.4 cp, 1.3 cP, 1.2 cP, 1.1 cP, 1.0 cP. In an embodiment a viscosity of the liquid composition is greater than or equal to any one of the following values: 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP.
  • the viscosity of the liquid composition may be between 0.5 cP and 1.8 cP or be in any sub-range derived from the values stated above.
  • the viscosity of the first liquid is lower than the one of the second liquid.
  • measurements of quantities mentioned herein may be performed according to what is specified in an associated standard, e.g. a DIN standard or EN standard, or documents having standard character.
  • a DIN standard or EN standard e.g. a DIN standard or EN standard
  • standards which are related to the determination of viscosities are: DIN 1319, DIN 1342, DIN 53019-1 or DIN 53019-2.
  • a density of the first liquid and/or of the second liquid is less than or equal to any one of the following values: 1200 kg/m 3 , 1190 kg/m 3 , 1180 kg/m 3 , I 170 kg/m 3 , 1160 kg/m 3 , 1150 kg/m 3 , 1140 kg/m 3 , 1130 kg/m 3 , 1120 kg/m 3 , 1110 kg/m 3 , 1100 kg/m 3 , 1090 kg/m 3 , 1080 kg/m 3 , 1070 kg/m 3 , 1060 kg/m 3 , 1050 kg/m 3 , 1040 kg/m 3 , 1030 kg/m 3 , 1020 kg/m 3 , 1010 kg/m 3 , 1000 kg/m 3 , 990 kg/m 3 , 980 kg/m 3 , 970 kg/m 3 , 960 kg/m 3 , 950 kg/m 3 , 940 kg/m 3 , 930 kg/m 3 ,
  • a density of the first liquid and/or of the second liquid is greater than or equal to any one of the following values: 500 kg/m 3 , 510 kg/m 3 , 520 kg/m 3 , 530 kg/m 3 , 540 kg/m 3 , 550 kg/m 3 , 560 kg/m 3 , 570 kg/m 3 , 580 kg/m 3 , 590 kg/m 3 , 600 kg/m 3 , 610 kg/m 3 , 620 kg/m 3 , 630 kg/m 3 , 640 kg/m 3 , 650 kg/m 3 , 660 kg/m 3 , 670 kg/m 3 , 680 kg/m 3 , 690 kg/m 3 , 700 kg/m 3 , 710 kg/m 3 , 720 kg/m 3 , 730 kg/m 3 , 740 kg/m 3 , 750 kg/m 3 , 760 kg/m 3 , 770 kg/m 3 , 780 kg
  • the density of the first liquid may be greater than the one of the second liquid.
  • the density of the first liquid may be between 500 kg/m 3 and 1200 kg/m 3 or be in any sub-range derived from the values stated above.
  • the density of the second liquid may be between 500 kg/m 3 and 1200 kg/m 3 or be in any sub-range derived from the values stated above.
  • the specified densities and/or viscosities are typical for liquids suitable for LNP formation when mixing the liquids.
  • the lipid nanoparticle composition has a polydispersity index (PDI) of the nanoparticles of less than or equal to any one of the following: 0.3, 0.29, 0.28, 0.27, 0.26, 0.25, 0.24, 0.23, 0.22, 0.21, 0.2, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.1, 0.09.
  • PDI polydispersity index
  • the lipid nanoparticle composition has a polydispersity index (PDI) of the nanoparticles of greater than or equal to any one of the following: 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22.
  • PDI polydispersity index
  • the polydispersity index (PDI) is a heterogeneity index.
  • An associated industry standard may be: ISO 22412:2017 (relating to the particle size analysis and dynamic light scattering).
  • ISO 22412:2017 relating to the particle size analysis and dynamic light scattering.
  • the system available as Malvern Zetasizer Ultra can be used to determine the size and/or the PDI of the LNPs.
  • the polydispersity index of the liquid composition may be between 0.008 and 0.3 or be in any sub-range derived from the values stated above.
  • lipid nanoparticles e.g. nucleic acid-LNPs
  • lipid nanoparticles may be formed in the liquid composition with a flow rate of the composition after mixing or at the outlet of the mixing chamber or mixing component of greater than or equal to 50 ml/min, 60 ml/min, 70 ml/min, 80 ml/min, 90 ml/min, 100 ml/min, e.g. of 200 ml/min or more.
  • the PDI of the nanoparticles may be 0.13 or less, 0.12 or less, or 0.11 or less.
  • the size of the nanoparticles may be 65 nm or less, or 60 nm or less.
  • the first inlet is used for the first liquid or the second liquid.
  • the second inlet may be used for the other liquid not being guided through the first inlet into the mixing chamber. It has been noted that particles with advantageous properties can be provided regardless of which inlet is used for the first liquid and which inlet is used for the second liquid.
  • the mixing component is an impingement jet mixer.
  • the impinging jets in the mixer may provide for some turbulence or agitation in the mixing chamber to enhance or promote mixing of the first and second liquids.
  • the mixing component is a T-mixer.
  • the T-mixer may be used for impingement jet mixing. Alternatively, a dedicated impingement jet mixing unit may be used.
  • the T-mixer may have its mixing chamber at the location where the three flow path sections (as defined by the "T") meet.
  • the first and second liquid may enter the T-mixer through the opposite inlets.
  • the flows of the first and second liquid may be oppositely directed in the T-mixer, may meet one another in the mixing chamber where the liquids can be mixed.
  • the liquid composition leaves the mixing chamber and/or the T-mixer with a flow direction at an angle, e.g. about 90° or 90°, with respect to the flow direction of the first and/or second liquid into the mixing chamber.
  • the first liquid comprises RNA or DNA.
  • the first liquid is an aqueous phase or an aqueous solution.
  • the first liquid has a pH-value below 7 and/or greater than or equal to 2.
  • the first liquid may have a pH-value of 4 or more, e.g. between 4 and 6.
  • the first liquid may be an acidic liquid.
  • the pH-value may be adjusted to the proper range by adding citric acid and/or citrate or acetic acid and/or acetate.
  • the second liquid comprises one or more lipids.
  • the second liquid comprises one or more or all of: a cationic lipid, a non-cationic lipid or helper lipid, a PEG-lipid (sometimes also termed: PEGylated lipid or PEG-conjugated lipid), and cholesterol.
  • a second liquid with such a configuration is particularly suitable for lipid nanoparticle formation, and, especially, for RNA-LNPs.
  • the second liquid is an organic phase.
  • the second liquid comprises an organic solvent.
  • the organic solvent is selected from the group of ethanol, propanol, isopropanol and acetone.
  • the first liquid comprises
  • the second liquid comprises
  • the first liquid and the second liquid are mixed in the mixing chamber to provide the liquid composition, the liquid composition having a flow rate of greater than or equal to 65 ml/min and optionally less than or equal to 300 ml/min at an outlet of the mixing chamber or of a mixing component comprising the mixing chamber, wherein a diameter of the flow path at the outlet is greater than or equal to 0.15 mm and, optionally, less than or equal to 1 mm or less than or equal to 0.85 mm.
  • the liquid composition comprises lipid nanoparticles, the respective lipid nanoparticle encapsulating nucleic acid, e.g. RNA or DNA.
  • the liquid composition is a dispersion.
  • the liquid composition may be a homogeneous dispersion. That is to say, the dispersed phase (e.g. the nanoparticles) is homogeneous, e.g. with a low PDI, such as below 0.13, or below 0.12 or below 0.11.
  • the first liquid is a solution and/or the second liquid is a solution.
  • Another aspect of the disclosure relates to a method of processing a liquid composition obtainable or obtained with the method of providing or forming the liquid composition described further above.
  • the processed liquid composition still comprises the LNPs.
  • the processed liquid composition may be the preparation set forth below.
  • the processed liquid composition may be a drug product and/or a pharmaceutical product.
  • a third liquid is added to the liquid composition downstream of the mixing chamber.
  • a further mixing chamber e.g. in a further mixing component, e.g. a T-mixer, is used for mixing the liquid composition and the third liquid.
  • a further mixing component e.g. a T-mixer
  • the length of a flow path fluidly connecting the outlet of the mixing chamber or the mixing component and an inlet of the further mixing chamber or of the further mixing component, the inlet being provided for the liquid composition is greater than or equal to one of the following: 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm (cm: centimeters).
  • the liquid composition may enter the further mixing chamber or mixing component through the inlet.
  • the length of a flow path fluidly connecting the outlet of the mixing chamber or the mixing component and an inlet of the further mixing chamber or of the further mixing component, the inlet being provided for the liquid composition is less than or equal to one of the following: 40 cm, 39 cm, 38 cm, 37 cm, 36 cm, 35 cm, 34 cm, 33 cm, 32 cm, 31 cm, 30 cm, 29 cm, 28 cm, 27 cm, 26 cm, 25 cm, 24 cm, 23 cm, 22 cm, 21 cm, 20 cm.
  • the length of the flow path may be between 5 cm and 40 cm. Arbitrary sub-ranges may be formed by the disclosed values.
  • the third liquid is a buffer and/or provided to provide quenching for the liquid composition.
  • the third liquid may be a buffer, e.g. a citrate buffer.
  • the liquid composition e.g. the processed or unprocessed liquid composition
  • a filter may be a 0.2 pm filter, i.e. a filter which is designed to allow particles with a size or diameter below 200 nm to pass through the filter.
  • the pore size may be 0.2 pm.
  • a filter area of the filter is less than or equal to A cm 2 per gram of RNA in the lipid nanoparticles, where A is any one of the following values: 180, 170, 160, 150, 140, 130, 120.
  • the first liquid comprises RNA, of course.
  • a filter area of the filter is greater than or equal to A cm 2 per gram of RNA in the lipid nanoparticles, where A is any one of the following values: 80, 90, 100, 110, 120.
  • A may be between 80 and 180.
  • Arbitrary sub-ranges may be formed by the disclosed values.
  • the polydispersity index (PDI 2) of the nanoparticles in the filtered liquid composition deviates from the polydispersity index (PDI l) of the nanoparticles in the unfiltered liquid composition by less than or equal to any one of: 25 %, 24%, 23 %, 22 %, 21 %, 20 %, 19 %, 18 %, 17 %, 16 %, 15 %, 14 %, 13 %, 12 %, 11 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %.
  • an absolute value of the difference between the polydispersity index (PDI 2) of the nanoparticles in the filtered liquid composition and the polydispersity index (PDI l) of the nanoparticles in the unfiltered liquid composition is less than or equal to any one of: 0.030, 0.025, 0.020, 0.015, 0.010, 0.009, 0.008, 0.007, 0.006, 0.005.
  • the liquid composition e.g. the filtered or unfiltered liquid composition
  • a predetermined temperature e.g. to - 20 °C or - 70 °C.
  • the frozen liquid composition may be thawed, e.g. until the thawed liquid composition has reached room temperature.
  • the frozen liquid may be thawed after a predetermined time. That is to say, the liquid composition is kept frozen for the predetermined time. After that time, the frozen composition may be allowed to thaw at room temperature, e.g. without applying additional heat.
  • the predetermined time is greater than or equal to any one of: one week, two weeks, three weeks, four weeks, five weeks, six weeks, one month, two months, three months, six months, twelve months, 24 months.
  • the predetermined time is less than or equal to any one of: one week, two weeks, four weeks, five weeks, six weeks, one month, two months, three months, six months, twelve months, 24 months, 36 months.
  • multiple freeze and thaw cycles are conducted with the liquid composition, e.g. in the predetermined time.
  • the liquid composition may be frozen again until a predetermined number of cycles has been completed, e.g. frozen x times and thawed x times; x may be 3, 4, or 5, for example.
  • the freeze and thaw cycles may be performed between - 20° C and room temperature or between - 70°C and room temperature, for example.
  • the temperature to which the liquid composition is frozen is kept constant between different cycles of the same set.
  • the polydispersity index (PDI 2) of the nanoparticles in the thawed liquid composition which has been thawed after the predetermined time or after the last thawing process of the multiple freeze and thaw cycles has been completed, deviates from the polydispersity index (PDI l) of the nanoparticles in the not yet once frozen liquid composition by less than or equal to any one of: 25 %, 24%, 23 %, 22 %, 21%, 20 %, 19 %, 18 %, 17 %, 16 %, 15 %, 14 %, 13 %, 12 %, 11 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %.
  • an absolute value of the difference between the polydispersity index (PDI 2) of the nanoparticles in the thawed liquid composition, which has been thawed after the predetermined time or after the last thawing process of the multiple freeze and thaw cycles has been completed, and the polydispersity index (PDI l) of the nanoparticles in the not yet once frozen liquid composition is less than or equal to any one of: 0.030, 0.025, 0.020, 0.015, 0.010, 0.009, 0.008, 0.007, 0.006, 0.005.
  • the respective deviation or difference mentioned above may be greater than zero or zero.
  • the respective deviation in percentage shown above may be determined by (PDI l - PDI 2) / PDI l x 100 %. If the value is negative, the absolute value is used to yield a positive result.
  • the composition may be the one with or without addition of the third liquid or a further processed composition.
  • the liquid composition e.g. before filtering, may be purified and/or the organic solvent may be reduced or removed.
  • a preparation e.g. a pharmaceutical preparation, the preparation comprising lipid nanoparticles, the lipid nanoparticles or the preparation being obtainable or obtained with any one of the methods described herein above or below or with the use as described herein above or below.
  • the preparation may be the (unprocessed or processed) liquid composition.
  • features described for the composition or its nanoparticles also apply for the preparation and its nanoparticles.
  • administration typically refers to the administration of a composition to a subject or system.
  • routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be ocular, oral, parenteral, topical, etc.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, intradermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • bronchial e.g., by bronchial instillation
  • buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, intradermal, transdermal, etc.
  • enteral intra-arterial, intradermal, intragas
  • administration may be intramuscular.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • agent is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc.
  • the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof.
  • the term may be used to refer to a natural product in that it is found in and/or is obtained from nature.
  • the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
  • potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
  • the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.
  • an analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
  • an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
  • antibody agent refers to an agent that specifically binds to a particular antigen.
  • the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies.
  • an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art.
  • an antibody agent utilized in accordance with the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.)-, antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (T)
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group [e.g., poly-ethylene glycol, etc.).
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it shows at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
  • Antibody agents can be made by the skilled person using methods and commercially available services and kits known in the art. For example, methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Charnes. Humana Press (August 21,
  • Antibodies may be produced by standard techniques, for example by immunization with the appropriate polypeptide or portion(s) thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing a desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • an immunogenic polypeptide bearing a desired epitope(s) optionally haptenized to another polypeptide.
  • various adjuvants may be used to increase immunological response.
  • Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are well known in the art.
  • Antigen- refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody.
  • an antigen elicits a humoral response (e.g., including production of antigenspecific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen).
  • an antigen binds to an antibody and may or may not induce a particular physiological response in an organism.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g. , other than a nucleic acid or amino acid polymer) etc.
  • an antigen is or comprises a polypeptide.
  • an antigen is or comprises a glycan.
  • an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source).
  • antigens utilized in accordance with the present disclosure are provided in a crude form.
  • an antigen is a recombinant antigen.
  • Binding- typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
  • Bioreactor refers to a vessel used for in vitro transcription described herein.
  • a bioreactor can be of any size so long as it is useful for in vitro transcription.
  • a bioreactor can be at least 0.5 liter, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters or more, or any volume in between.
  • the internal conditions of the bioreactor including, but not limited to pH and temperature, are typically controlled during in vitro transcription.
  • the bioreactor can be composed of any material that is suitable for in vitro transcription under the conditions as described herein, including glass, plastic or metal.
  • suitable bioreactor volume for use in practicing in vitro transcription.
  • cap refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate).
  • a cap is or comprises a guanine nucleotide.
  • a cap is or comprises a naturally-occurring RNA 5’ cap, including, e.g., but not limited to a N7-methylguanosine cap, which has a structure designated as "m7G.”
  • a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art).
  • ARCAs anti-reverse cap analogs
  • a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system).
  • a capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, wherein, in addition to the GTP, an IVT system also contains a cap analog, e.g., as known in the art.
  • Non-limiting examples of a cap analog include a m7GpppG cap analog or an N7-methyl-, 2’-O- methyl -GpppG ARCA cap analog or an N7-methyl-, 3'- O-methyl-GpppG ARCA cap analog, or any commercially available cap analogs, including, e.g., CleanCap (Trilink), EZ Cap, etc..
  • a cap analog is or comprises a trinucleotide cap analog.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • Complementary As used herein, the term “complementary” is used in reference to oligonucleotide hybridization related by base-pairing rules. For example, the sequence “C-A-G-T” is complementary to the sequence “G-T-C-A.” Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term “complementary” provided that the partial complementarity permits oligonucleotide hybridization. Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
  • detecting is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and/or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification.
  • Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.
  • determining involves manipulation of a physical sample.
  • determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis.
  • determining involves receiving relevant information and/or materials from a source.
  • determining involves comparing one or more features of a sample or entity to a comparable reference.
  • Dosage form or unit dosage form may be used to refer to a physically discrete unit of an active agent (e.g. , a therapeutic or diagnostic agent) for administration to a subject.
  • each such unit contains a predetermined quantity of active agent.
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • Encapsulate The term “encapsulate” or “encapsulation” is used herein to refer to at least a portion of a component is enclosed or surrounded by another material or another component in a composition. In some embodiments, a component can be fully enclosed or surrounded by another material or another component in a composition.
  • Excipient As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired property or effect (e.g, desired consistency, delivery, and/or stabilizing effect, etc ⁇ .
  • suitable pharmaceutical excipients to be added to a LNP composition may include, for example, salts, starch, glucose, lactose, sucrose, gelatin, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like.
  • Encode refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids.
  • a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme).
  • An RNA molecule can encode a polypeptide (e.g, by a translation process).
  • a gene, a cDNA, or a single-stranded RNA encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system.
  • a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent.
  • a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Fed-batch process refers to a process in which one or more components are introduced into a vessel, e.g., a bioreactor, at some time subsequent to the beginning of a reaction.
  • a vessel e.g., a bioreactor
  • one or more components are introduced by a fed-batch process to maintain its concentration low during a reaction.
  • one or more components are introduced by a fed-batch process to replenish what is depleted during a reaction.
  • RNA Five prime untranslated region: As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region of an RNA.
  • Functional As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized. In some embodiments, a biological molecule may have two functions (i.e., bifonctional) or many functions (z.e., multifunctional).
  • a gene refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and/or a polypeptide product).
  • a gene includes coding sequence (i.e., sequence that encodes a particular product); in some embodiments, a gene includes non-coding sequence.
  • a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences.
  • a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).
  • Gene product or expression product generally refers to an RNA transcribed from the gene (pre-and/or post-processing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from the gene.
  • homolog refers to the overall relatedness between polynucleotide molecules (e.g. , DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.
  • Host cell' refers to a cell into which exogenous material (e.g., DNA such as recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g. , a recombinant nucleic acid sequence).
  • Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas.
  • bacterial cells e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.
  • mycobacteria cells e.g., fungal cells, yeast cells (e.g., S.
  • a host cell is a human, monkey, ape, hamster, rat, or mouse cell.
  • a host cell is eukaryotic.
  • an eukaryotic host cell may be CHO (e.g., CHO KI, DXB-1 1 CHO, Veggie-CHO), COS (e.g, COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g, BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, or a cell line derived
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g.
  • gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g. , nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Improved, increased or reduced indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent.
  • an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g. , in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
  • vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel (e.g., a bioreactor), in cell culture, etc., rather than within a multi-cellular organism.
  • in vitro transcription refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g. , production of protein or polypeptides.
  • synthetic RNA products can be translated in vitro or introduced directly into cells, where they can be translated.
  • synthetic RNA products include, e.g.
  • An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) as described and/or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase.
  • a DNA template e.g., a linear DNA template
  • ribonucleotides e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates
  • in vitro transcription RNA composition refers to a composition comprising target RNA synthesized by in vitro transcription.
  • a composition can comprise excess in vitro transcription reagents (including, e.g., ribonucleotides and/or capping agents), nucleic acids or fragments thereof such as DNA templates or fragments thereof, polypeptides or fragments thereof such as recombinant enzymes or host cell proteins or fragments thereof, and/or other impurities.
  • an in vitro transcription RNA composition may have been treated and/or processed prior to a purification processes that ultimately produces an RNA transcript preparation comprising RNA transcript at a desired concentration in an appropriate buffer for formulation and/or further manufacturing and/or processing.
  • an in vitro transcription RNA composition may have been treated to remove or digest DNA template (e.g. , using a DNase).
  • an in vitro transcription RNA composition may have been treated to remove or digest polypeptides (e.g., enzymes such as RNA polymerases, RNase inhibitors, etc.) present in an in vitro transcription reaction (e.g., using a protease).
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal.
  • Nanoparticle refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, a nanoparticle has a diameter of less than 80 nm as defined by the National Institutes of Health. In some embodiments, a nanoparticle comprises one or more enclosed compartments, separated from the bulk solution by a membrane, which surrounds and encloses a space or compartment.
  • nucleic acid refers to a polymer of at least 2 nucleotides or more, including, e.g., at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more .
  • a nucleic acid is or comprises DNA.
  • a nucleic acid is or comprises RNA.
  • a nucleic acid is or comprises peptide nucleic acid (PNA).
  • a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g, as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil).
  • a nucleic acid comprises on or more, or all, non-natural residues.
  • a non-natural residue comprises a nucleoside analog (e.g. , 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C- 5 propynyl-cytidine, 1-methyl-pseudouridine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inter
  • a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.
  • composition grade refers to standards for chemical and biological drug substances, drug products, dosage forms, compounded preparations, excipients, medical devices, and dietary supplements, established by a recognized national or regional pharmacopeia (e.g., The United States Pharmacopeia and The Formulary (USP-NF)).
  • polypeptide typically has its art-recognized meaning of a polymer of at least three amino acids or more.
  • polypeptide typically has its art-recognized meaning of a polymer of at least three amino acids or more.
  • polypeptide is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides.
  • polypeptides may contain L-amino acids, D-amino acids, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art.
  • polypeptides may comprise natural amino acids, nonnatural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).
  • a polypeptide may be or comprise an enzyme.
  • a polypeptide may be or comprise a polypeptide antigen.
  • a polypeptide may be or comprise an antibody agent.
  • a polypeptide may be or comprise a cytokine.
  • an agent or entity is “pure” or “purified” if it is substantially free of other components.
  • a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation.
  • an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure in a preparation.
  • Ribonucleotide encompasses unmodified ribonucleotides and modified ribonucleotides.
  • unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U).
  • Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) intemucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.)
  • base modifications
  • RNA Ribonucleic acid
  • an RNA refers to a polymer of ribonucleotides.
  • an RNA is single stranded.
  • an RNA is double stranded.
  • an RNA comprises both single and double stranded portions.
  • an RNA can comprise a backbone structure as described in the definition of “ Nucleic acid / Polynucleotide” above.
  • An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA).
  • mRNA messenger RNA
  • an RNA is a mRNA.
  • RNA typically comprises at its 3’ end a poly(A) region.
  • an RNA typically comprises at its 5’ end, an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation.
  • an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).
  • an RNA is a singlestranded RNA.
  • a single-stranded RNA may comprise self-complementary elements and/or may establish a secondary and/or tertiary structure.
  • encoding it can mean that it comprises a nucleic acid sequence that itself encodes or that it comprises a complement of the nucleic acid sequence that encodes.
  • a single-stranded RNA can be a self-amplifying RNA (also known as self-replicating RNA).
  • Recombinant is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide
  • one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silica. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g. , of a human, a mouse, etc.).
  • reference describes a standard or control relative to which a comparison is performed.
  • an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
  • a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
  • a reference or control is a historical reference or control, optionally embodied in a tangible medium.
  • a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • RNA polymerase refers to an enzyme that catalyzes polyribonucleotide synthesis by addition of ribonucleotide units to a nucleotide chain using DNA or RNA as a template.
  • the term refers to either a complete enzyme as it occurs in nature, or an isolated, active catalytic or functional domain, or fragment thereof.
  • an RNA polymerase enzyme initiates synthesis at the 3'-end of a primer or a nucleic acid strand, or at a promoter sequence, and proceeds in the 5'-direction along the target nucleic acid to synthesize a strand complementary to the target nucleic acid until synthesis terminates.
  • RNA transcript preparation refers to a preparation comprising RNA transcript that is purified from an in vitro transcription RNA composition described herein.
  • an RNA transcript preparation is a preparation comprising pharmaceutical-grade RNA transcript.
  • an RNA transcript preparation is a preparation comprising RNA transcript, which its one or more product quality attributes are characterized and determined to meet a release and/or acceptance criteria (e.g., as described herein).
  • product quality attributes include, but are not limited to appearance, RNA length, identity of drug substance as RNA, RNA integrity, RNA sequence, RNA concentration, pH, osmolality, residual DNA template, residual double stranded RNA, bacterial endotoxins, bioburden, and combinations thereof.
  • room temperature refers to an ambient temperature.
  • a room temperature is about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C.
  • sample typically refers to an aliquot of material obtained or derived from a source of interest, e.g., as described herein.
  • a source of interest is a biological or environmental source.
  • a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a mouse).
  • a source of interest is or comprises biological tissue or fluid.
  • a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
  • a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
  • a sample is or comprises cells obtained from a subject.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample.
  • a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
  • Stable when applied to nucleic acids and/or compositions comprising nucleic acids, e.g., encapsulated in lipid nanoparticles, means that such nucleic acids and/or compositions maintain one or more aspects of their characteristics (e.g., physical and/or structural characteristics, function, and/or activity) over a period of time under a designated set of conditions (e.g., pH, temperature, light, relative humidity, etc.).
  • such stability is maintained over a period of time of at least about one hour; in some embodiments, such stability is maintained over a period of time of about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, such stability is maintained over a period of time within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc.
  • such stability is maintained under an ambient condition (e.g. , at room temperature and ambient pressure). In some embodiments, such stability is maintained under a physiological condition (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline). In some embodiments, such stability is maintained under cold storage (e.g., at or below about 4 °C, including, e.g., -20 °C, or -70 °C). In some embodiments, such stability is maintained when nucleic acids and/or compositions comprising the same are protected from light (e.g, maintaining in the dark).
  • an ambient condition e.g. , at room temperature and ambient pressure
  • a physiological condition e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline.
  • cold storage e.g., at or below about 4 °C, including, e.g., -20 °C, or -70 °C
  • the term “stable” is used in reference to a nanoparticle composition (e.g., a lipid nanoparticle composition).
  • a stable nanoparticle composition e.g., a stable nanoparticle composition
  • component(s) thereof maintain one or more aspects of its characteristics (e.g., physical and/or structural characteristics, function(s), and/or activity) over a period of time under a designated set of conditions.
  • a stable nanoparticle composition e.g.
  • a lipid nanoparticle composition is characterized in that average particle size, particle size distribution, and/or polydispersity of nanoparticles is substantially maintained (e.g., within 10% or less, as compared to the initial characteristic(s)) over a period of time (e.g, as described herein) under a designated set of conditions (e.g., as described herein).
  • a stable nanoparticle composition e.g. , a lipid nanoparticle composition
  • no detectable amount of degradation products e.g., associated with hydrolysis and/or enzymatic digestion
  • a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis.
  • the term “synthetic” refers to an entity that is made outside of biological cells.
  • a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g, an RNA) that is produced by in vitro transcription using a template.
  • Three prime untranslated region' refers to the sequence of an mRNA molecule that begins following the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after the stop codon of the coding region of an open reading frame sequence. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence Threshold level (e.g., acceptance criteria)-.
  • Threshold level e.g., acceptance criteria
  • a threshold level refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay.
  • a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria).
  • a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population.
  • a threshold level can be determined based on one or more control samples or across a population of control samples.
  • a threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken.
  • a threshold level can be a range of values.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
  • viral vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • expression vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g. , electroporation, lipofection).
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.
  • Figure 1 illustrates a system for and a method of forming or providing a liquid composition comprising lipid nanoparticles.
  • Figure 2 illustrates a system for and a method of forming or providing a liquid composition comprising lipid nanoparticles using a T-mixer as mixing component.
  • Figures 3 A and 3B depict results obtained for the setting shown in figure 2 for different flow rates and two different first liquids.
  • Figure 4 depicts an exemplary process flow charts of manufacturing an RNA (e.g., for encapsulation in LNPs).
  • Figure 5 depicts an overview of an exemplary LNP drug product manufacturing process.
  • Figure 6 depicts an overview of an exemplary process of DNA template manufacture via a PCR-based process.
  • Figure 7 depicts an exemplary LNP manufacturing process.
  • Figure 8 depicts an exemplary process by which a drug product composition can be filled/finished.
  • Figure 9 depicts a Pareto effects chart illustrating relative influences of various factors on LNP particle size and stability.
  • Figure 10 depicts an exemplary process for LNP manufacturing (e.g., of RNA-LNPs), according to aspects of the present embodiments.
  • Figure 11 depicts an exemplary system for LNP manufacturing (e.g., of RNA-LNPs), according to aspects of the present embodiments.
  • FIG. 1 schematically illustrates an embodiment of a system 300 for forming or providing a liquid composition comprising lipid nanoparticles.
  • the system is expediently provided for conducting the methods described herein above and below.
  • the system 300 comprises a mixing chamber 302.
  • the mixing chamber has a first inlet 304 and a second inlet 306.
  • the inlets 304 and 306 are provided to permit entry of a first liquid 308 and a second liquid 310 into the mixing chamber.
  • the liquids may be provided in associated reservoirs which are in fluid communication with the associated inlet, e.g. via associated flow paths or the system may be connectable to such reservoirs.
  • a first reservoir 312 in the depicted embodiment holds the first liquid 308 and a second reservoir 314 holds the second liquid 310.
  • a first flow path 309 guides the first liquid 308 and a second flow path 311 guides the second liquid 310 towards the respective inlet.
  • the respective flow path may be defined by one or more conduits, tubings and/or other structures limiting the flow path laterally with respect to the flow direction (e.g. by the mixing component mentioned below).
  • the mixing chamber 302 is part of a mixing component, device or unit 316.
  • the respective inlet (first or second inlet) of the mixing chamber 302 may coincide with the inlet of the mixing component or an associated inlet of the mixing component (e.g. first inlet 318 or second inlet 320) may be arranged upstream of the mixing chamber (i.e. closer to the associated reservoir as seen along the flow path counter to the flow direction).
  • first liquid 308 can enter the mixing chamber or component via the first inlet (304, 318) and the second liquid (310) can enter the mixing chamber or component via the second inlet (306, 320).
  • a first flow driver 322 may be provided to move the first liquid 308 into the mixing chamber 302 or the component 316.
  • a second flow driver 324 may be provided to move the second liquid 310 into the mixing chamber 302 or the component 316.
  • the respective flow driver may be a pump.
  • the flow of the first liquid 308 towards and into the mixing chamber 302 or the mixing component 316 is highlighted by arrow 326
  • the flow of the second liquid 310 towards and into the mixing chamber 302 or the mixing component 316 is highlighted by arrow 328.
  • the flow of the first and second liquids may be continuously driven into the mixing chamber.
  • the first and second liquids can mix for the liquid composition 330.
  • the liquid composition 330 leaves the mixing chamber 302 via an outlet 332 of the mixing chamber.
  • the outlet of the mixing component 316 may coincide with the outlet of the mixing chamber or be arranged downstream of the outlet 332 of the mixing chamber 302 (see outlet 334, for example).
  • the flow rate of the liquid composition 330 at the respective outlet may be defined by the flow rates of the first liquid and the second liquid into the mixing chamber 302, e.g. be equal to the sum of these flow rates.
  • the liquid composition as indicated by arrow 336 continues its flow and can be further processed, e.g. buffered, purified, filtered and/or diluted.
  • a further liquid 338 may be added to the flow of the liquid composition downstream of the mixing component or mixing chamber, e.g. a buffer, such as a quench buffer.
  • the third liquid may be a citrate buffer.
  • the third liquid 338 can be added to the liquid composition flow 336 at an angle to the liquid composition flow 336, e.g. less than 160°, such as about 90°.
  • the liquid flow of the third liquid 338 is illustrated by arrow 340.
  • the third liquid can be continuously guided into the liquid flow.
  • the flow rate of the third liquid may be less than the one for the first liquid and/or the second liquid or less than the sum of these flow rates.
  • the processed lipid nanoparticle composition flow 342 may be guided towards a further processing step or unit and/or leave the system 300 via a system outlet (not explicitly shown).
  • the first and second liquids entering the mixing chamber 302 are chosen so as to, when mixed, provide a lipid nanoparticle composition comprising lipid nanoparticles, expediently lipid nanoparticles encapsulating a pharmaceutically active substance, e.g. comprising RNA, such as mRNA.
  • the respective liquids may be solutions.
  • the lipid nanoparticle composition expediently is a dispersed phase or, in other words, a dispersion with lipid nanoparticles being the dispersed phase in a liquid. Both, the nanoparticles and the liquid expediently result from mixing the first liquid and the second liquid with one another in the mixing chamber.
  • a preparation may comprise the processed lipid nanoparticle composition or the unprocessed lipid nanoparticle composition.
  • the first liquid 308 comprises the entity to be encapsulated by the nanoparticles, e.g. RNA, such as mRNA.
  • the first liquid expediently has a pH of between 2 and 7, e.g. between 4 and 7 or between 4 and 6 (e.g. adjusted via citric acid or acetic acid).
  • the first liquid may be an aqueous phase or solution. More detailed examples on the first liquid are given further below.
  • the second liquid 310 expediently comprises further ingredients for the nanoparticle formation.
  • the second liquid comprises one of, more of, or all of: a cationic lipid, a non-cationic or second cationic lipid or helper lipid, a PEG-lipid (sometimes also termed: PEGylated lipid), and cholesterol. More detailed examples on the second liquid are given further below.
  • the second liquid 310 may be an organic phase and/or comprise an organic solvent, e.g. ethanol, propanol, isopropanol or acetone.
  • lipid nanoparticles encapsulating RNA i.e. RNA-LNPs
  • the (average) size of the nanoparticles could be decreased (as compared to regimes with higher Reynolds numbers) and/or homogeneity of the dispersion with the nanoparticles could be increased (e.g. as the nanoparticles are more uniform in size which entails a smaller PDI).
  • Having smaller particles and/or a more homogeneous particle size distribution facilitates further processing of any preparation comprising the nanoparticles formed. For example, less particles are lost during a filtration step or finer filters can be used.
  • Figure 2 shows a setting which is very similar to the one shown in figure 1 but with more details on the flow paths and some associated data. Hence, features described in conjunction with figure l also apply for figure 2 and vice versa. Features from figure 1 are not repeated here.
  • a T-mixer (also referenced as “A” in the figure) is used as mixing component 316 for obtaining the liquid composition.
  • V and D specify the viscosity (V) and density (D) of the respective liquid or the liquid composition.
  • B For the addition of the third liquid another T-mixer is used (designated as B).
  • the inner diameters of the flow path sections e.g. provided by tube sections or the respective T-mixer) are specified as well as their lengths.
  • the flow rate of the first liquid may be greater than the flow rate of the second liquid.
  • a ratio of the flow rate of the first liquid to the one of the second liquid may be less than or equal to one of the following: 7, 6, 5, 4, 3.
  • the flow rate of the first liquid is about or equal to 3 times the flow rate of the second liquid.
  • the combined flow rate of the first and second liquids into the mixing chamber may then determine the flow rate of the liquid composition away from the mixing chamber 302.
  • the first and second liquids are mixed using a T-mixer in an impingement liquid setting.
  • the flow directions of the liquid flows are diametrically opposite and the liquids hit one another (frontally) in the mixing chamber.
  • the two impinging liquid streams may create some turbulences which may enhance the mixing in the chamber.
  • laminar flow in the mixing chamber may also be possible.
  • the liquid composition leaves the mixing component 316 at its outlet 334 in a flow direction which is at an angle of 90° or about 90° relative to the flow directions of the first and second liquid into the mixing chamber.
  • the densities (denoted “D” in kg/m 3 ) and viscosities (denoted “V” in centipoise) specified in figure 2 are typical values occurring when forming lipid nanoparticle compositions from mixing two liquids.
  • the diameters of the inlets and the outlet of the mixing chamber or the mixing component are equal.
  • the inlets have different diameters.
  • the inlet for the first liquid may have a greater diameter than the inlet for the second liquid.
  • the outlet may have a greater or smaller diameter than at least one of the inlets, e.g. greater than the first inlet and/or the second inlet.
  • T-mixer "A” it is also conceivable to use a dedicated impingement jet mixing unit for mixing the first and second liquid as is described further below.
  • Figures 3A and 3B show results obtained for the setting shown in figure 2 for different flow rates and two different first liquids (with RNA).
  • the first liquids employed differed only in the additives or buffers used, i.e. for Liquid 1 citric acid and/or citrate (e.g. natrium citrate) was used (the liquid comprises citrate, indicated by (Ci)) and for Liquid 2 acetic acid and/or acetate (e.g. natrium acetate) was used (the liquid comprises acetate, indicated by (Ac)).
  • the flow rate of the liquid composition with the nanoparticles encapsulating RNA at the outlet of the mixing chamber or of the mixing component was varied between 100 ml/min (via adjusting the flow rates for the first and second liquid appropriately while keeping their ratio at 3: 1) and 300 ml/min.
  • the measurements for the PDI and the average particle size were made using dynamic light scattering, e.g. using a Zetasizer available from Malvern.
  • the Zetasizer calculates the PDI and the average particle size.
  • the data relating to PDI and size were obtained using a Malvern Zetasizer Ultra, which is a system designed to measure and calculate particle properties, such as by using dynamic light scattering.
  • the (colloidal) parameters size and polydispersity (descriptive of the width of the size distribution) of LNPs produced were analyzed by dynamic light scattering (DLS) in the Malvern Panalytical Zetasizer Ultra. Samples were diluted to 2 pg/mL in phosphate-buffered saline (PBS) and were measured in PMMA cuvettes by back-scattering (173°) at 25°C.
  • PBS phosphate-buffered saline
  • the cuvette was set as ZEN0040, material was set as protein (refractive index RI 1.45, absorption 0.001) and RI and viscosity for PBS were 1.34 and 0.91 cP, respectively. Choice of all other measurement parameters was set to "automatic”. Measurement of each sample was repeated three times. Cumulants fit with the model “General Purpose” was used for data evaluation.
  • the “general purpose model” is a model which needs to be selected as a pre-setting for measurement of a “standard, non-deviating, known and expected” sample of nanoparticles and uses a certain cumulant fit for calculation of size and distribution in the Zetasizer defined by the software of the Zetasizer.
  • the data point at 240 ml/min with the Reynolds number of 9949 was qualified as likely resulting from an irregularity during the measurement.
  • the advantageous effects for the formed nanoparticles were achieved independent from the buffer used for the first liquid (citrate and acetate were used for Liquids 1 and 2, respectively) and also independent from the flow drivers which were used.
  • a syringe pump system or SPS was used (e.g. available from Cetoni)
  • a piston pump system e.g. available from Knauer
  • LNP compositions and associated processes for which the proposed concepts having a Reynolds number of the liquid composition flow (particularly after the initial mixing of the first and second liquids and/or before the liquid composition is further processed, e.g. before the third liquid is added) of 10000 and below are advantageous are set forth below.
  • Further processing of the liquid composition e.g. the unprocessed composition or the one to which the third liquid has been added or an even further processed liquid composition, with nanoparticles, may include filtering using a 0.2 pm filter, e.g. a Sartopore 2 filter.
  • a filter area of the filter can be less than or equal to A m 2 per gram of lipid nanoparticles in the liquid composition, where A is 120, for example.
  • Nucleic acid therapeutics, and particularly RNA therapeutics represent a particularly promising class of therapies for treatment and prevention of various diseases such as cancer, infectious diseases, and/or diseases or disorders associated with overabundance or deficiency in certain proteins.
  • RNA therapeutics in particular have proven remarkably effective as vaccines to address the COVID19 pandemic. Particularly given the promise of this technology, and its adaptability to a wide variety of clinical contexts, including massively large scale (e.g., vaccination and/or treatment on a global scale such as is trader development for SARS-CoV-2), improvements to manufacturing technologies, especially those applicable to large-scale production, are especially valuable.
  • massively large scale e.g., vaccination and/or treatment on a global scale such as is trader development for SARS-CoV-2
  • improvements to manufacturing technologies especially those applicable to large-scale production, are especially valuable.
  • lipid nanoparticle technologies have proven to be particularly effective (reviewed in, for example, Cullis et al. Molecular Therapy 25:1467, July 5, 2017; See also, US Patent 8058069), specifically including for RNA therapeutics (reviewed in, for example, Hou et al., Nat. Rev. Mater doi.org/10.1038/s41578-021-00358-0, August 10, 2021).
  • LNP preparations and/or compositions e.g., nucleic acid-LNP preparations, and specifically RNA- LNP preparations.
  • provided technologies permit and/or facilitate achievement of requirements unique to pharmaceutical-grade (and/or scale) production such as, for example, batch size and/or rate of production, pre-determined in-process controls and/or lot release specifications (e.g., high purity, integrity, potency, and/or stability, etc.), etc..
  • LNP compositions e.g., including RNA, e.g., therapeutic RNA such as therapeutic mRNA.
  • provided technologies are useful for manufacturing pharmaceutical-grade RNA-LNP therapeutics.
  • provided technologies are useful for large scale manufacturing of LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics, e.g., pharmaceutical-grade therapeutics.
  • technologies provided herein can be used to produce a pharmaceutical-grade batch throughput of at least 10,000 vials of LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics (including, e.g., at least 20,000 vials, at least 30,000 vials, at least 40,000 vials, at least 50,000 vials, at least 60,000 vials, at least 70,000 vials, at least 80,000 vials, at least 90,000 vials, at least 100,000 vials, at least 200,000 vials, at least 300,000 vials, at least 400,000 vials, at least 500,000 vials, or more).
  • LNP e.g., nucleic acid-LNP, e.g., RNA-LNP
  • therapeutics including, e.g., at least 20,000 vials, at least 30,000 vials, at least 40,000 vials, at least 50,000 vials, at least 60,000 vials, at
  • technologies provided herein can be used to produce a pharmaceutical-grade batch throughput of at least 50 L of LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics (including e.g., at least 50L, at least 60L, at least 70L, at least 80L, at least 100L, at least 110 L, at least 120 L, at least 130 L, at least 140 L, at least 150 L or more.
  • LNP e.g., nucleic acid-LNP, e.g., RNA-LNP
  • therapeutics including e.g., at least 50L, at least 60L, at least 70L, at least 80L, at least 100L, at least 110 L, at least 120 L, at least 130 L, at least 140 L, at least 150 L or more.
  • each vial can comprise an RNA drug product in an amount of 0.01 mg to 0.5 mg (e.g., 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg).
  • LNP e.g., nucleic acid-LNP, e.g., RNA- LNP
  • technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions that comprise or deliver (e.g., by comprising and/or delivering a nucleic acid, such as an RNA, that encodes it) a polypeptide.
  • technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for inducing an immune response to an antigen.
  • technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for treatment and/or prevention of coronavirus infection, e.g., SARS-CoV-2 infection, as described in Walsh et al.
  • RNA-based COVID-19 vaccine BNT162b2 selected for a pivotal efficacy study medRxiv preprint (2020), which is online accessible at: https://doi.0rg/lO.I 101/2020.08.17.20176651; and Milligan et al. “Phase MI study of COVID-19 RNA vaccine BNT162bl in adults” Nature (2020 August), which is online accessible at: https://doi.org/10.1038/s41586-020-2639-4, the contents of each of which are incorporated by reference in their entirety.
  • lipid nanoparticles have achieved successful clinical delivery of a wide range of therapeutic agents including, for example, small molecules, and various nucleic acids - e.g., oligonucleotides, siRNAs, and mRNAs (reviewed, for example, in Hu et al., Nat. Rev. Mater. https://doi.orgH August 10, 2021).
  • nucleic acids e.g., oligonucleotides, siRNAs, and mRNAs
  • LNPs are parenterally administered; most clinical studies have utilized parenteral administration, and particularly intravenous, subcutaneous, intradermal, intravitreal, intratumoral, or intramuscular injection. Intrautero injection has also been described.
  • topical administration is utilized.
  • intranasal administration is utilized.
  • administered LNPs are delivered to or accumulate in the liver.
  • liver delivery can prove useful for achieving delivery of an LNP-encapsulated agent (and/or, in the case of a nucleic acid agent such as an RNA agent, a polypeptide encoded thereby) into the bloodstream.
  • Such liver delivery has been proposed to be particularly useful, for example, for expression of proteins that are missing in certain metabolic or hematological disorders, or that are effective in provoking immune responses (e.g., particularly antibody responses), for example against infectious agents or cancer cells.
  • administered LNPs are delivered to and/or taken up by antigen-presenting cells (e.g., as may be present in skin, muscle, mucosal tissues, etc.)', such administration may be particularly useful or effective for induction of T cell immunity (e.g., for treatment of infectious diseases and/or cancers).
  • antigen-presenting cells e.g., as may be present in skin, muscle, mucosal tissues, etc.
  • T cell immunity e.g., for treatment of infectious diseases and/or cancers.
  • lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 50 nm to about 130 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 50 to about 90 nm, or about 60 nm to about 80 nm, or about 60 nm to about 70 nm.
  • lipid nanoparticles that may be useful in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm.
  • lipid nanoparticles may have an average size (e.g., mean diameter) of less than 80 nm, less than 75 nm, less than 70 nm, less than 65 nm, less than 60 nm, less than 55 nm, less than 50 nm, or less than 45 nm.
  • lipid nanoparticles that may be useful in accordance with the present disclosure can have an average size (e.g.
  • lipids that form lipid nanoparticles described herein comprise: a polymer- conjugated lipid; a cationic lipid; and a helper neutral lipid.
  • total polymer- conjugated lipid may be present in about 0.5-5 mol%, about 0.7-3.5 mol%, about 1-2.5 mol%, about 1.5-2 mol%, or about 1.5-1.8 mol% of the total lipids. In some embodiments, total polymer-conjugated lipid may be present in about 1-2.5 mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipid to total polymer-conjugated lipid (e.g., PEG-conjugated lipid) may be about 100:1 to about 20:1, or about 50:1 to about 20:1, or about 40:1 to about 20:1, or about 35:1 to about 25:1. In some embodiments, the molar ratio of total cationic lipid to total polymer-conjugated lipid may be about 35: 1 to about 25:1.
  • total cationic lipid is present in about 35-65 mol%, about 40-60 mol%, about 41-49 mol%, about 41-48 mol%, about 42-48 mol%, about 43-48 mol%, about 44-48 mol%, about 45-48 mol%, or about 46-49 mol% of the total lipids.
  • total cationic lipid is present in about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol% of the total lipids.
  • total neutral lipid is present in about 35-65 mol%, about 40-60 mol%, about 45-55 mol%, or about 47-52 mol% of the total lipids. In some embodiments, total neutral lipid is present in 35-65 mol% of the total lipids. In some embodiments, total non-steroid neutral lipid (e.g., DPSC) is present in about 5-15 mol%, about 7-13 mol%, or 9-11 mol% of the total lipids.
  • DPSC total non-steroid neutral lipid
  • total non-steroid neutral lipid is present in about 9.5, 10 or 10.5 mol% of the total lipids.
  • the molar ratio of the total cationic lipid to the non-steroid neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0.
  • total steroid neutral lipid e.g., cholesterol
  • total steroid neutral lipid e.g., cholesterol
  • molar ratio of total cationic lipid to total steroid neutral lipid is about 1.5:1 to 1: 1.2, or about 1.2: 1 to 1: 1.2.
  • a lipid composition comprising a cationic lipid, a polymer-conjugated lipid, and a neutral lipid can have individual lipids present in certain molar percents of the total lipids, or in certain molar ratios (relative to each other) as described in WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; the cationic lipid is present in 35-65 mol% of the total lipids; and the neutral lipid is present in 35-65 mol% of the total lipids.
  • lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; and the neutral lipid is present in 45-55 mol% of the total lipids.
  • a polymer-conjugated lipid e.g., PEG-conjugated lipid
  • lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid comprising a non-steroid neutral lipid and a steroid neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; the non-steroid neutral lipid is present in 9-11 mol% of the total lipids; and the steroid neutral lipid is present in about 36-44 mol% of the total lipids.
  • a PEG-conjugated lipid is or comprises a structure as described in WO
  • a PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000
  • a cationic lipid is or comprises a chemical structure selected from 1-1 to 1-10 of Table 1 herein or a derivative thereof.
  • a cationic lipid is or comprises ((4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate).
  • a neutral lipid comprises DSPC and cholesterol, wherein DSPC is a non-steroid neutral lipid and cholesterol is a steroid neutral lipid.
  • lipid nanoparticles include one or more cationic lipids (e.g., ones described herein).
  • cationic lipid nanoparticles may comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g. , at least one neutral lipid).
  • a lipid nanoparticle described herein comprises at least one helper lipid, which may be a neutral lipid, a positively charged lipid, or a negatively charged lipid.
  • a helper lipid is a lipid that are useful for increasing the effectiveness of delivery of lipid-based particles such as cationic lipid-based particles to a target cell.
  • a helper lipid may be or comprise a structural lipid with its concentration chosen to optimize LNP particle size, stability, and/or encapsulation.
  • a lipid nanoparticle described herein comprises a neutral helper lipid.
  • neutral helper lipids include, but are not limited to phosphotidylcholines such as 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, cholesterol, steroids such as sterols and their derivatives.
  • DOPE 1,2- dioleoyl-sn-glycero-3-phospho
  • Neutral lipids may be synthetic or naturally derived.
  • Other neutral helper lipids that are known in the art, e.g., as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein, can also be used in lipid nanoparticles described herein.
  • a lipid nanoparticle for delivery of RNA(s) described herein comprises DSPC and/or cholesterol.
  • a lipid nanoparticle described herein comprises at least two helper lipids (e.g., ones described herein).
  • a lipid nanoparticle may comprise DSPC and cholesterol.
  • a lipid nanoparticle described herein comprises a cationic lipid.
  • a cationic lipid is typically a lipid having a net positive charge.
  • a cationic lipid may comprise one or more amine group(s) which bear a positive charge.
  • a cationic lipid may comprise a cationic, meaning positively charged, headgroup.
  • a cationic lipid may have a hydrophobic domain (e.g., one or more domains of a neutral lipid or an anionic lipid) provided that the cationic lipid has a net positive charge.
  • a cationic lipid comprises a polar headgroup, which in some embodiments may comprise one or more amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperizine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan.
  • a polar headgroup of a cationic lipid comprises one or more amine derivatives.
  • a polar headgroup of a cationic lipid comprises a quaternary ammonium.
  • a headgroup of a cationic lipid may comprise multiple cationic charges. In some embodiments, a headgroup of a cationic lipid comprises one cationic charge.
  • monocationic lipids include, but are not limited to 1,2-dimyristoyl-sn- glycero-3-ethylphosphocholine (DMEPC), 1 ,2-di-O-octadecenyl- 3 -trimethylammonium propane (DOTMA) and/or 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl)azanium bromide (DDAB), 1 ,2-dioleyloxypropyl-3 -dimethyl -
  • a positively charged lipid structure described herein may also include one or more other components that may be typically used in the formation of vesicles (e.g. for stabilization).
  • other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or cholesterol esters or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
  • sterols include cholesterol, cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
  • the at least one cationic lipid comprises DMEPC and/or DOTMA.
  • a cationic lipid is ionizable such that it can exist in a positively charged form or neutral form depending on pH. Such ionization of a cationic lipid can affect the surface charge of the lipid particle under different pH conditions, which in some embodiments may influence plasma protein absorption, blood clearance, and/or tissue distribution as well as the ability to form endosomolytic non- bilayer structures. Accordingly, in some embodiments, a cationic lipid may be or comprise a pH responsive lipid. In some embodiments a pH responsive lipid is a fatty acid derivative or other amphiphilic compound which is capable of forming a lyotropic lipid phase, and which has a pKa value between pH 5 and pH 7.5.
  • a pH responsive lipid may be used in addition to or instead of a cationic lipid for example by binding one or more RNAs to a lipid or lipid mixture at low pH.
  • pH responsive lipids include, but are not limited to, 1,2- dioieyioxy-3 -dimethylaminopropane (DODMA).
  • a lipid nanoparticle may comprise one or more cationic lipids as described in WO 2016/176330, WO 2017/075531 (e.g., as presented in Tables 1 and 3 therein) and WO 2018/081480 (e.g, as presented in Tables 1-4 therein), the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • a cationic lipid that may be useful in accordance with the present disclosure is an amino lipid comprising a titratable tertiary amino head group linked via ester bonds to at least two saturated alkyl chains, which ester bonds can be hydrolyzed easily to facilitate fast degradation and/or excretion via renal pathways.
  • an amino lipid has an apparent pKa of about 5.5-6.5 (e.g., in one embodiment with an apparent pKa of approximately 6.1), resulting in an essentially fully positively charged molecule at an acidic pH (e.g., pH 5).
  • such an amino lipid when incorporated in LNP, can confer distinct physicochemical properties that regulate particle formation, cellular uptake, fusogenicity and/or endosomal release of RNA(s).
  • introduction of an aqueous RNA solution to a lipid mixture comprising such an amino lipid at pH 4.0 can lead to an electrostatic interaction between the negatively charged RNA backbone and the positively charged cationic lipid. Without wishing to be bound by any particular theory, such electrostatic interaction leads to particle formation coincident with efficient encapsulation of RNA drug substance.
  • RNA encapsulation After RNA encapsulation, adjustment of the pH of the medium surrounding the resulting LNP to a more neutral pH (e.g., pH 7.4) results in neutralization of the surface charge of the LNP.
  • a more neutral pH e.g., pH 7.4
  • charge-neutral particles display longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are rapidly cleared by the reticuloendothelial system.
  • the low pH of the endosome renders LNP comprising such an amino lipid fusogenic and allows the release of the RNA into the cytosol of the target cell.
  • a cationic lipid that may be useful in accordance with the present disclosure has one of the structures disclosed in WO 2017/075531, some of which are set forth in Table 1 below:
  • a cationic lipid that may be useful in accordance with the present disclosure is or comprises a chemical structure selected from 1-1 to I- 10 as shown in Table 1 above.
  • a cationic lipid is or comprises a chemical structure of 1-3 shown in Table 1 above.
  • a cationic lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate).
  • a cationic lipid that may be useful in accordance with the present disclosure is or comprises a chemical structure selected from A-F as shown in Table 2 below.
  • a cationic lipid is or comprises a chemical structure of B shown in Table 2 above.
  • a cationic lipid is or comprises a chemical structure of D shown in Table 2 above.
  • a cationic lipid that may be useful in accordance with the present disclosure is an ionizable lipid-like material (lipidoid).
  • lipidoid ionizable lipid-like material
  • Cl 2-200 which has the following structure:
  • Cationic lipids may be used alone or in combination with neutral lipids, e.g., cholesterol and/or neutral phospholipids, or in combination with other known lipid assembly components.
  • neutral lipids e.g., cholesterol and/or neutral phospholipids
  • a lipid nanoparticle may comprise at least one polymer-conjugated lipid.
  • a polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.
  • a polymer-conjugated lipid is a PEG-conjugated lipid.
  • a PEG-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer.
  • a PEG-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.
  • PEG-conjugated lipids include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S- DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetrade
  • PEG-conjugated lipids also known as PEGylated lipids
  • PEG-conjugated lipids are known to affect cellular uptake, a prerequisite to endosomal localization and payload delivery.
  • the present disclosure provides an insight that the pharmacology of encapsulated nucleic acid can be controlled in a predictable manner by modulating the alkyl chain length of a PEG-lipid anchor.
  • the present disclosure provides an insight that such PEG-conjugated lipids may be selected for an RNA/LNP drug product formulation to provide optimum delivery of RNAs to the liver.
  • such PEG-conjugated lipids may be designed and/or selected based on reasonable solubility characteristics and/or its molecular weight to effectively perform the function of a steric barrier.
  • a PEGylated lipid does not show appreciable surfactant or permeability enhancing or disturbing effects on biological membranes.
  • PEG in such a PEG-conjugated lipid can be linked to diacyl lipid anchors with a biodegradable amide bond, thereby facilitating fast degradation and/or excretion.
  • a LNP comprising a PEG- conjugated lipid retain a full complement of a PEGylated lipid. In the blood compartment, such a PEGylated lipid dissociates from the particle over time, revealing a more fusogenic particle that is more readily taken up by cells, ultimately leading to release of the RNA payload.
  • a lipid nanoparticle may comprise one or more PEG-conjugated lipids or pegylated lipids as described in WO 2015/199952, WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • a PEG-conjugated lipid that may be useful in accordance with the present disclosure can have a structure as described in WO 2017/075531, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Rs and Rg are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • R8 and R9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • w has a mean value ranging from 43 to 53. In other embodiments, the average w is about 45.
  • a PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000
  • nucleic acid agent may be single stranded; in some embodiments, a nucleic acid agent may be double stranded.
  • a nucleic acid agent may be or comprise DNA; in some embodiments, a nucleic acid agent may be or comprise RNA.
  • nucleic acids may include one or more non-natural features (e.g., residues, modifications, intra-nucleoside linkages, etc.).
  • a nucleic acid is a non-coding in that its nucleotide sequence does not include an open reading frame (or complement thereof).
  • a nucleic acid has a nucleotide sequence that is or includes a sequence that encodes (or is the complement of a sequence that encodes) a polypeptide as described herein.
  • a nucleic acid e.g., and RNA
  • a relevant nucleic acid includes a polypeptide-encoding portion.
  • such a portion may encode a polypeptide that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc.
  • an encoded polypeptide may be or include one or more neoantigens or neoepitopes associated with a tumor.
  • an encoded polypeptide may be or include an antigen (or epitope thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.).
  • an encoded polypeptide may be a variant of a wild type polypeptide
  • technologies described herein may utilize a nucleic acid having a length of at least 500 residues (such as, e.g., at least 600 residues, at least 700 residues, at least 800 residues, at least 900 residues, at least 1000 residues, at least 1250 residues, at least 1500 residues, at least 1750 residues, at least 2000 residues, at least 2500 residues, at least 3000 residues, at least 3500 residues, at least 4000 residues, at least 4500 residues, at least 5000 residues, or longer).
  • technologies described herein may utilize a nucleic acidhaving a length of about 1000 residues to 5000 residues.
  • nucleic acids e.g., RNAs
  • when present in provided lipid nanoparticles are resistant in aqueous solution to degradation with a nuclease.
  • the present disclosure relates to production and/or use (e.g., handling, processing, transporting, etc.) of LNP compositions that include RNA.
  • an RNA amenable to technologies described herein is a single-stranded RNA.
  • an RNA as disclosed herein is a linear RNA.
  • a singlestranded RNA is a non-coding RNA in that its nucleotide sequence does not include an open reading frame (or complement thereof).
  • a single-stranded RNA has a nucleotide sequence that encodes (or is the complement of a sequence that encodes) a polypeptide or a plurality of polypeptides (e.g., epitopes) of the present disclosure.
  • a relevant RNA is an mRNA.
  • an RNA includes unmodified uridine residues; an RNA that includes only unmodified uridine residues may be referred to as a “uRNA”.
  • an RNA includes one or more modified uridine residues; in some embodiments, such an RNA ⁇ e.g., an RNA including entirely modified uridine residues) is referred to as a “modRNA”.
  • an RNA may be a self-amplifying RNA (saRNA).
  • an RNA may be a trans-amplifying RNA (see, for example, WO20I7/162461).
  • RNA e.g., a single stranded RNA
  • technologies described herein may be particularly useful for production of an RNA (e.g., a single stranded RNA) having a length of at least 500 ribonucleotides (such as, e.g., at least 600 ribonucleotides, at least 700 ribonucleotides, at least 800 ribonucleotides, at least 900 ribonucleotides, at least 1000 ribonucleotides, at least 1250 ribonucleotides, at least 1500 ribonucleotides, at least 1750 ribonucleotides, at least 2000 ribonucleotides, at least 2500 ribonucleotides, at least 3000 ribonucleotides, at least 3500 ribonucleotides, at least 4000 ribonucleotides, at least 4500 ribonucleotides, at least
  • a relevant RNA includes a polypeptide-encoding portion or a plurality of polypeptide-encoding portions.
  • such a portion or portions may encode a polypeptide or polypeptides that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc.
  • an encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with a tumor.
  • an encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g. , a bacterium, fungus, virus, etc.).
  • an encoded polypeptide may be a variant of a wild type polypeptide.
  • a single-stranded RNA may comprise a secretion signal-encoding region (e.g., a secretion signal-encoding region that allows an encoded target entity or entities to be secreted upon translation by cells).
  • a secretion signal-encoding region may be or comprise a non-human secretion signal.
  • such a secretion signal-encoding region may be or comprise a human secretion signal.
  • a single-stranded RNA may comprise at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency).
  • non-coding sequence elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure for co-transcriptional capping of mRNA, a poly adenine (polyA) tail, and any combinations thereof.
  • RNA pharmaceutical compositions e.g., immunogenic compositions or vaccines
  • uRNA non-modified uridine containing mRNA
  • modRNA nucleosidemodified mRNA
  • saRNA self-amplifying mRNA
  • trans-amplifying RNAs e.g., trans-amplifying RNAs.
  • non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.
  • modified uridine e.g., pseudouridine
  • pseudouridine may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses.
  • the present disclosure provides an insight that such strong antibody and CD4 T cell responses may be particularly useful for vaccination.
  • a self-amplifying platform may include, for example, long duration of polypeptide (e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.
  • a self-amplifying platform e.g., RNA
  • a self-amplifying platform e.g., RNA
  • a /ra/?.s -rcplication system comprises the presence of both nucleic acid molecules in a single host cell.
  • a nucleic acid encoding a replicase is not capable of self-replication in a target cell and/or target organism.
  • a nucleic acid encoding a replicase e.g., a viral replicase
  • a self-amplifying RNA comprises a 5 ’-cap.
  • a 5 ’-cap is important for high level expression of a gene of interest in trans.
  • a 5’-cap drives expression of a replicase.
  • a self-amplifying RNA does not comprise an Internal Ribosomal Entry Site (IRES) element.
  • IRES Internal Ribosomal Entry Site
  • translation of a gene of interest and/or replicase is not driven by an IRES element.
  • an IRES element is substituted by a 5 ’-cap. In some such embodiments, substitution by a 5 ’-cap does not affect the sequence of a polypeptide encoded by an RNA.
  • a self-amplifying platform does not require propagation of virus particles (e.g., is not associated with undesired virus-particle formation). In some embodiments, a self-amplifying platform is not capable of forming virus particles.
  • a polynucleotide (e.g., RNA) utilized in accordance with the present disclosure comprises a 5’-cap.
  • RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference.
  • a 5 ’-cap structure which may be suitable in the context of the present disclosure is a capO (methylation of the first nucleobase, e.g.
  • capl additional methylation of the ribose of the adjacent nucleotide of m7GpppN
  • cap2 additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN
  • cap3 additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN
  • cap4 additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN
  • ARCA anti-reverse cap analogue
  • modified ARCA e.g.
  • phosphothioate modified ARCA e.g., beta-S-ARCA
  • inosine N1 -methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosinc.
  • a utilized 5’ caps is a Cap-0 (also referred herein as “CapO”), a Cap-1 (also referred herein as “Capl”), or Cap-2 (also referred herein as “Cap2”). See, e.g., Figure 1 of Ramanathan A et al., and Figure 1 of Decroly E et al.
  • RNA refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5 '-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).
  • a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7- position) on a base (guanine), and/or by methylation at one or more positions of a ribose.
  • a guanosine nucleoside included in a 5’ cap comprises a 3’0 methylation at a ribose (3’0MeG). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OMeG)).
  • providing an RNA with a 5'-cap disclosed herein or a 5'-cap analog may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes.
  • co- transcriptional capping with a cap disclosed herein e.g., with a capl or a capl analog, improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator.
  • improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.
  • an RNA described herein comprises a 5 ’-cap or a 5’ cap analog, e.g., a CapO, a Capl or a Cap2.
  • a provided RNA does not have uncapped 5'-triphosphates.
  • an RNA may be capped with a 5'- cap analog.
  • an RNA described herein comprises a CapO.
  • an RNA described herein comprises a Capl, e.g., as described herein.
  • an RNA described herein comprises a Cap2.
  • alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.
  • a CapO structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G).
  • a CapO structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as m7Gppp or m7G(5')ppp(5').
  • a Capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine (TM 7 G or 7m G) and a 2'0 methylated first nucleotide in an RNA (2'OMeNi or Ni2'0Me or Ni 2 OMe ).
  • a Capl structure is connected to an RNA via a 5'- to 5'-triphosphate linkage; in some embodiments, a Capl structure may be represented as m7 Gppp(Ni 2OMe ) or m7 G(5')ppp(5')(Ni 2OMe ) or 7m G(5')ppp(5')Ni 2 ’ OMe ).
  • Ni is chosen from A, C, G, or U.
  • Ni is A.
  • Ni is C.
  • Ni is G.
  • Ni is U.
  • methylation of one or more positions in a cap structure may impact or reflect mode of incorporation (e.g., co-transcriptional vs post-transcriptional), as presence of a methyl group (e.g., a 2'0Me group) at certain positions (e.g., Ni) may interfere with elongation, e.g., by a particular polymerase (e.g., T7), as underlies the ARCA technology.
  • a methyl group e.g., a 2'0Me group
  • a particular polymerase e.g., T7
  • a m7 G(5')ppp(5')(Ni 2OMe ) Capl structure comprises a second nucleotide, N2 which is a cap proximal A, G, C, or U at position +2.
  • such Capl structures are represented as ( m7 G(5')ppp(5')(Ni 2 OMe )pN2).
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • a Capl structure is or comprises m7 G(5')ppp(5')(Ai 2 OMe )pG2 wherein Ai is a cap proximal A at position +1 and G2 is a cap proximal G at position +2 and has the following structure:
  • a Capl structure is or comprises m7 G(5')ppp(5')(Ai 2 OMe )pU2 wherein Ai is a cap proximal A at position +1 and U2 is a cap proximal U at position +2, and has the following structure:
  • a Capl structure is or comprises m7 G(5')ppp(5')(Gi 2 OMe )pG2 wherein Gi is a cap proximal G at position +1 and G2 is a cap proximal G at position +2, and has the following structure:
  • a Capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ( m7 G) and one or more additional modifications, e.g., methylation on a ribose, and a 2'0 methylated first nucleotide in an RNA.
  • a Capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine and a 3'0 methylation at a ribose (m7G3'OMe) or 2'0 methylated first nucleotide in an RNA (Ni 2OMe ).
  • a Capl structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m7G3'OMe)ppp(2'OMeNi) or ( m7 G 3OMe )(5')ppp(5')( 2OMe N 1 ).
  • Ni is chosen from A, C, G, or U. In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is G. In some embodiments, Ni is U.
  • Capl structure comprises a second nucleotide, N 2 which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or U
  • N2 is A. In some embodiments, N2 is C. In some embodiments, N 2 is G. In some embodiments, N 2 is U.
  • a Capl structure is or comprises ( m7 G 3 ' OMe )(5')ppp(5')(Ai 2OMe )pG2 wherein Ai is a cap proximal A at position +1 and G2 is a cap proximal G at position +2, and has the following structure:
  • a Capl structure is or comprises ( m7 G 3 OMe )(5')ppp(5')(G 1 2 OMe )pG 2 wherein Gi is a cap proximal G at position +1 and G2 is a cap proximal G at position +2, and has the following structure:
  • a second nucleotide in a Capl structure can comprise one or more modifications, e.g., methylation.
  • a Capl structure comprising a second nucleotide comprising a 2'0 methylation is a Cap2 structure.
  • an RNA polynucleotide comprising a Capl structure has increased translation efficiency, increased translation rate and/or increased expression of an encoded payload relative to an appropriate reference comparator.
  • an RNA polynucleotide comprising a Capl structure having ( m7 G 3 OMe )(5')ppp(5')(A 1 2 OMe )pG2 wherein Ai is a cap proximal nucleotide at position +1 and G2 is a cap proximal nucleotide at position +2, has increased translation efficiency relative to an RNA polynucleotide comprising a Capl structure having ( m7 G 3 OMe )(5')ppp(5')(G 1 2 OMe )pG2 wherein Gi is a cap proximal nucleotide at position 1 and G2 is a cap proximal nucleotide at position 2.
  • increased translation efficiency is assessed upon administration of an RNA polynucleotide having
  • a cap analog used in an RNA polynucleotide is m7 G 3 OMe Gppp(ml 2 OMe )ApG (also sometimes referred to as m2 7 ’ 3 ' OMe G(5’)ppp(5’)m 2 ’ OMe ApG or ( m7 G 3 ' OMe )(5')ppp(5')(A 2 ’ OMe )pG), which has the following structure:
  • Capl RNA which comprises RNA and m2 7 ’ 3 OMe G(5’)ppp(5’)m 2 OMe ApG:
  • 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element).
  • a 5’ UTR comprises multiple different sequence elements.
  • untranslated region or "UTR” is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'- UTR).
  • a 5'-UTR if present, is located at the 5' end, upstream of the start codon of a polypeptide- (e.g., protein)-encoding region.
  • a 5 -UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 5' UTR is a heterologous 5’ UTR, i.e., is a 5’ UTR found in nature associated with a different ORF.
  • a 5' UTR is a synthetic 5’ UTR, i.e., does not occur in nature.
  • aynthetic 5’ UTR may be utilized, such as a 5’ UTR whose sequence has been altered relative to a parental reference 5’ UTR. Those skilled in the art will be aware of various 5’ UTR sequence alterations that, for example, may have been reported to increase expression of an ORF with which the variant 5’ UTR is associated.
  • a utilized 5' UTRs may be or comprise a 5’ UTR from a gene such as: a-globin or p- globin, such as Xenopus or human a-globin, p ⁇ globin, or oc-globin (e.g., as described, for example, in US Patent 8278063 and/or US Patent 9012219) genes, human cytochrome b- 245 a polypeptide, hydroxysteroid (17b) dehydrogenase, Tobacco etch virus (e.g., as described, for example, in US Patent 8278063and/or US Patent 9012219).
  • a-globin or p- globin such as Xenopus or human a-globin, p ⁇ globin, or oc-globin (e.g., as described, for example, in US Patent 8278063 and/or US Patent 9012219) genes
  • human cytochrome b- 245 a polypeptide
  • IE1 immediate-early 1 gene
  • HSD17B4 RPL32, ASAHI , ATP5A1, MP68, NDUFA4, NOSIP, RPL31 , SLC7A3, TUBB4B, UBQLN2, PSMB3, RPS9, CASP1, COX6B1, NDUFA1, Rpl31, GNAS, ALB7.
  • a 5’ UTR is or comprises a 5’ UTR from an a- globin gene, or a variant thereof.
  • embodiment utilized 5' UTR is a 5’ UTR of a TOP gene, for example a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., as described, for example, in WO/2015/101414, W02015/101415, WO/2015/062738, WO2015/024667, WO2015/024667); a 5' UTR element of a ribosomal protein Large 32 (L32) gene (e.g., as described, for example, in WO/2015/101414, W02015/101415, WO/2015/062738), a 5' UTR element of an hydroxysteroid (17-P) dehydrogenase 4 gene (HSD17B4) (e.g., as described, for example, in WO2015/024667), or a 5' UTR element of ATP5A1 (e.g., as described, for example, in WO2015/024667) can be
  • an internal ribosome entry site (IRES) is used instead of or in addition to a 5' UTR.
  • a 5 ’ UTR utilized in accordance with the present disclosure is or comprises a sequence: gggaaauaag agagaaaaga agaguaagaa gaaauauaag accccggcgc cgccacc.
  • a 5’ UTR utilized in accordance with the present disclosure is or comprises a sequence: gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccacc.
  • a 5’ UTR may be or comprise a sequence GGGAUCCUACC (see, e.g., WO2014/144196).
  • a 5’ UTR may be or comprise a sequence as set forth in one of SEQ ID NOs: 231-252, or 22848-22875 of WO2021/156267, or a fragment or a variant of any of the foregoing.
  • a 5’ UTR may be or comprise a sequence as set forth in claim 9 of and/or of one or more of SEQ ID NOs: 1 -20 of W02019/077001 Al, or a fragment or variant of any of the foregoing.
  • a 5’ UTR may be or comprise one set forth in WO2013/143700, for example one or more of SEQ ID NOs: 1 -1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of W02013/143700, or a fragment or variant of any of the foregoing.
  • a 5’-UTR is or comprises a 5’ UTR as described in WO2016/107877, for example in SEQ ID NOs: 25-30 or 319-382 of WO2016/107877, or fragments or variants of any of the foregoing.
  • a 5 ’-UTR is or comprises a 5’ UTR as described in W02017/036580 for example in SEQ ID NOs: 1 -151 of W02017/036580, or fragments or variants of any of the foregoing.
  • a 5’ UTR is or comprises a 5 ’-UTR as described in WO2016/022914, for example in SEQ ID NOs: 3-19 of WO2016/022914, or fragments or variants of any of the foregoing
  • a 5' UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the source and/or from different sources (see, for example, the 5’ UTRs described in US Patent Application Publication No.2010/0293625 and PCT/US2014/069155).
  • a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a Cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +1 (Nl) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (Nl and N2) of an RNA polynucleotide.
  • one or more residues of a cap proximal sequence may be included in an RNA by virtue of having been included in a cap entity that (e.g., a Capl structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g, by a polymerase such as a T7 polymerase).
  • +1 and +2 are the (ml 2 °)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).
  • a cap proximal sequence comprises Nl and N2 of a Cap structure, wherein Nl and N2 are any nucleotide, e.g., A, C, G or U.
  • Nl is A.
  • Nl is C.
  • Nl is G.
  • Nl is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • Nl is A and N2 is A. In some embodiments, Nl is A and N2 is C. In some embodiments, Nl is A and N2 is G. In some embodiments, Nl is A and N2 is U.
  • Nl is C and N2 is A. In some embodiments, Nl is C and N2 is C. In some embodiments, Nl is C andN2 is G. In some embodiments, N 1 is C and N2 is U.
  • Nl is G and N2 is A. In some embodiments, Nl is G and N2 is C. In some embodiments, Nl is G and N2 is G. In some embodiments, Nl is G and N2 is U. In some embodiments, N1 is U and N2 is A. In some embodiments, N1 is U and N2 is C. In some embodiments, Nl is U and N2 is G. In some embodiments, Nl is U and N2 is U.
  • a cap proximal sequence comprises Nl and N2 of a Cap structure and N3, N4 and N5, wherein Nl to N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is A.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is C.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is G.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is U.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is A.
  • N4 is A.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • N 1 is A and N2 is G.
  • N3 is A.
  • N4 is G.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is C.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is U.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is A.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is C.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is G.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is A.
  • N4 is U.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is A.
  • N4 is A.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is A.
  • N4 is C.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is G.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is A.
  • N4 is U.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U. In some embodiments, Nl is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is A. In some embodiments, N5 is A. In some embodiments, Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U. In some embodiments, Nl is A andN2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is G.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is U.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is A.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is G.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is C.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is U.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is A.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U. In some embodiments, Nl is A andN2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is C. In some embodiments, Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U. In some embodiments, Nl is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is G. In some embodiments, N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is U.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is A.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is C.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is G.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is C.
  • N4 is U.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is A.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is C.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is G.
  • N4 is G.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is U.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is A.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is G.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is C.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is U.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is A.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is C.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is G.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is U.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is A.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is C.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is G.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is G.
  • N4 is U.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is A.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is C.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is G.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is U.
  • N5 is A.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is U.
  • N4 is A.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is G.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is C.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is U.
  • N5 is G.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is A.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is C.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is G.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is U.
  • N5 is C.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is A.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A andN2 is G.
  • N3 is U.
  • N4 is C.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is G.
  • N5 is U.
  • Nl, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G or U.
  • Nl is A and N2 is G.
  • N3 is U.
  • N4 is U.
  • N5 is U.
  • a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as disclosed herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap.
  • a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a Cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +1 (Nl) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (Nl and N2) of an RNA polynucleotide.
  • Nl and N2 are each independently chosen from: A, C, G, or U.
  • Nl is A.
  • Nl is C.
  • Nl is G.
  • Nl is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • Nl and N2 are each independently chosen from: A, C, G, or U.
  • Nl is A.
  • Nl is C.
  • Nl is G.
  • Nl is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • Nl is A and N2 is A. In some embodiments, Nl is A and N2 is C. In some embodiments, Nl is A and N2 is G. In some embodiments, Nl is A and N2 is U.
  • Nl is C and N2 is A. In some embodiments, Nl is C and N2 is C. In some embodiments, Nl is C andN2 is G. In some embodiments, N 1 is C and N2 is U.
  • Nl is G and N2 is A. In some embodiments, Nl is G and N2 is C. In some embodiments, Nl is G and N2 is G. In some embodiments, Nl is G and N2 is U. In some embodiments, N1 is U and N2 is A. In some embodiments, N1 is U and N2 is C. In some embodiments, N1 is U and N2 is G. In some embodiments, N1 is U and N2 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising: A3A4X5.
  • N1 and N2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • X5 is chosen from A, C, G or U.
  • X5 is A.
  • X5 is C.
  • X5 is G.
  • X5 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising: C3A4X5.
  • N1 and N2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • X5 is chosen from A, C, G or U.
  • X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising X3Y4X5.
  • N1 andN2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • X3 and X5 is each independently chosen from A, C, G or U.
  • X3 and/or X5 is A.
  • X3 and/or X5 is C.
  • X3 and/or X5 is G.
  • X3 and/or X5 is U.
  • Y4 is not C.
  • Y4 is A.
  • Y4 is G.
  • Y4 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising X3Y4X5.
  • N1 andN2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • X3 and X5 is each independently chosen from A, C, G or U.
  • X3 and/or X5 is A.
  • X3 and/or X5 is C.
  • X3 and/or X5 is G.
  • X3 and/or X5 is U.
  • Y4 is not G.
  • Y4 is A.
  • Y4 is C.
  • Y4 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising A3C4A5.
  • N1 and N2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • a cap proximal sequence comprises Nl and N2 of a Cap structure, and a sequence comprising A3U4G5.
  • Nl andN2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • a Cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +1 (Nl) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide.
  • a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (Nl and N2) of an RNA polynucleotide.
  • Nl andN2 are any nucleotide, e.g., A, C, G, or U.
  • Nl is A.
  • Nl is C.
  • Nl is G.
  • Nl is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • Nl and N2 are any nucleotide, e.g., A, C, G, or U.
  • Nl is A.
  • Nl is C.
  • Nl is G.
  • Nl is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • Nl is A and N2 is A. In some embodiments, Nl is A and N2 is C. In some embodiments, Nl is A and N2 is G. In some embodiments, Nl is A and N2 is U.
  • Nl is C andN2 is A. In some embodiments, Nl is C andN2 is C. In some embodiments, Nl is C and N2 is G. In some embodiments, Nl is C and N2 is U.
  • Nl is G and N2 is A. In some embodiments, Nl is G and N2 is C. In some embodiments, N 1 is G and N2 is G. In some embodiments, N 1 is G and N2 is U.
  • Nl is U and N2 is A. In some embodiments, Nl is U and N2 is C. In some embodiments, Nl is U and N2 is G. In some embodiments, Nl is U and N2 is U.
  • a cap proximal sequence comprises Nl and N2 of a Cap structure, and a sequence comprising: A3A4X5.
  • Nl and N2 are any nucleotide, e.g., A, C, G, or U.
  • Nl is A and N2 is G.
  • X5 is chosen from A, C, G or U.
  • X5 is A.
  • X5 is C.
  • X5 is G.
  • X5 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising: C3A4X5.
  • N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is any nucleotide, e.g., A, C, G or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising X3Y4X5.
  • N1 and N2 are any nucleotide, e.g., A, C, G, or U.
  • N1 is A and N2 is G.
  • X3 and X5 is any nucleotide, e.g., A, C, G or U.
  • X3 and/or X5 is A.
  • X3 and/or X5 is C.
  • X3 and/or X5 is G.
  • X3 and/or X5 is U.
  • Y4 is not C.
  • Y4 is A.
  • Y4 is G.
  • Y4 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising X3Y4X5.
  • N1 and N2 are any nucleotide, e.g., A, C, G, or U.
  • N1 is A and N2 is G.
  • X3 and X5 is any nucleotide, e.g., A, C, G or U.
  • X3 and/or X5 is A.
  • X3 and/or X5 is C.
  • X3 and/or X5 is G.
  • X3 and/or X5 is U.
  • Y4 is not G.
  • Y4 is A.
  • Y4 is C.
  • Y4 is U.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising A3C4A5.
  • N1 and N2 are any nucleotide, e.g., A, C, G, or U.
  • N1 is A andN2 is G.
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising A3U4G5.
  • N1 and N2 are any nucleotide, e.g., A, C, G, or U.
  • N1 is A andN2 is G.
  • Exemplary 5’ UTRs include a human alpha globin (hAg) 5’UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP70 5’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof.
  • hAg human alpha globin
  • an RNA disclosed herein comprises a hAg 5’ UTR or a fragment thereof.
  • an RNA as described herein comprises a 3 -UTR.
  • a “3 ’-untranslated region” or “3’-UTR” or “3’-UTR element” will be recognized and understood by the person of ordinary skill in the art.
  • a 3’ UTR typically is a part of a nucleic acid molecule that is located 3’ (i.e. downstream) of a coding sequence and is not translated into protein.
  • a 3 ’-UTR may located between a coding sequence and an (optional) terminal poly(A) sequence.
  • a 3 ’-UTR may comprise elements for controlling gene expression, such a what may be referred to as regulatory elements. Such regulatory elements may be or comprise, e.g., ribosomal binding sites, miRNA binding sites etc..
  • a 3 -UTR if present, is located at the 3' end, downstream of the termination codon of a polypeptide- (e.g., protein-) encoding region, but the term "3 -UTR" does preferably not include the poly(A) sequence. Thus, the 3 -UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.
  • an RNA disclosed herein comprises a 3’ UTR comprising an F element and/or an I element.
  • a 3’ UTR or a proximal sequence thereto comprises a restriction site.
  • a restriction site is a BamHI site.
  • a restriction site is a Xhol site.
  • an RNA construct comprises an F element.
  • a F element sequence is a 3 ’-UTR of amino-terminal enhancer of split (AES).
  • an RNA disclosed herein comprises a 3’ UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a 3’ UTR with the sequence comprising: CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUC CCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUA GUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACC CCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 15).
  • an RNA disclosed herein comprises a 3’ UTR provided in SEQ ID NO: 15.
  • a 3 ’UTR is an FI element as described in WO2017/060314.
  • a utilized 3 ’UTR may be or comprise a 3 ’UTR from a gene such as globin UTRs, including Xenopus p-globin UTRs and human p-globin UTRs are known in the art (see, for example, 8278063, 9012219, US2011/0086907).
  • a modified p- globin construct with enhanced stability in some cell types may be utilized; such a construct has been reported as having been made by cloning two sequential human
  • a2-globin, al-globin, UTRs and variants thereof are also known in the art (W02015/101415, W02015/024667).
  • Exemplary 3' UTRs described in the mRNA constructs in the non-patent literature include those from CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015).
  • exemplary 3' UTRs include that of bovine or human growth hormone (wild type or modified) (W02013/185069, US2014/0206753, W02014152774), rabbit p globin and hepatitis B virus (HBV), a-globin 3' UTR and Viral VEEV 3' UTR sequences are also known in the art.
  • the sequence UUUGAAUU (W02014/144196) is used.
  • 3' UTRs of human and/or mouse ribosomal protein are used.
  • examples include rps9 3'UTR (W02015/101414), FIG4 (W02015/101415), and human albumin 7 (W02015/101415).
  • a nucleic acid comprises at least one heterologous 3 ’-UTR, wherein the at least one heterologous 3 ’-UTR comprises a nucleic acid sequence derived from a 3 ’-UTR of a gene selected from PSMB3, ALB7, alpha-globin (referred to as “muag”), CASP1 , COX6B1 , GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or variant of any one of these genes.
  • a gene selected from PSMB3, ALB7, alpha-globin (referred to as “muag”), CASP1 , COX6B1 , GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or variant of any one of these genes.
  • a utilized 3 ’UTR may be as exemplified, for example, in published PCT application W02019/077001 Al , in particular, claim 9 of W02019/077001 Al .
  • a 3’ UTR may be or comprise one of SEQ ID NOs: 23-34 of W02019/077001 Al , or a fragment or variant thereof).
  • a 3’ UTR utilized in accordance with the present disclosure comprises a sequence: ugauaauagg cuggagccuc gguggccuag cuucuugccc cuugggccuc cccccagccc cuccuccccu uccugcaccc guacccccgu ggucuuugaa uaaagucuga gugggcggc.
  • a 3’ UTR of the present disclosure comprises a sequence: ugauaauagg cuggagccuc gguggccaug cuucuugccc cuugggccuc cccccagccc cuccuccccu uccugcaccc guacccccgu ggucuuugaa uaaagucuga gugggcggc.
  • a nucleic acid may comprise a 3’-UTR as described in WO2016/107877In some embodiments, suitable 3’-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016/107877, or fragments or variants of these sequences.
  • a 3 ’-UTR as described in W02017/036580 may be utilized.
  • suitable 3’-UTRs are SEQ ID NOs: 152-204 of WO2017/036580, or fragments or variants of these sequences.
  • a 3 ’-UTR as described in WO2016/022914 is utilized.
  • a 3’-UTRs is or comprises a sequence according to SEQ ID NOs: 20-36 of WO2016/022914, or fragments or variants of these sequences.
  • a polynucleotide e.g, DNA, RNA
  • a polyadenylate (PolyA) sequence e.g., as described herein.
  • a PolyA sequence is situated downstream of a 3 -UTR, e.g., adjacent to a 3 -UTR.
  • poly(A) sequence or "poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3 '-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of polypeptide (e.g., protein) that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).
  • polypeptide e.g., protein
  • a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length.
  • a poly(A) sequence comprises, essentially consists of, or consists of at least 10, at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 1000, up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly(A) sequence typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • nucleotide or “A” refers to adenylate.
  • a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in accordance with the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly (A) sequence is not masked or followed at its 3 '-end by a nucleotide other than A.
  • the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.
  • a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200.
  • a poly A tail of a string construct may comprise 200 A residues or less.
  • a poly A tail of a string construct may comprise about 200 A residues.
  • a poly A tail of a string construct may comprise 180 A residues or less.
  • a poly A tail of a string construct may comprise about 180 A residues.
  • a poly A tail may comprise 150 residues or less.
  • the poly(A) sequence may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides.
  • the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.
  • the nucleic acid comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides.
  • the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In some embodiments, the poly (A) sequence comprises about 100 adenosine nucleotides (A100). In some embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides.
  • the nucleic acid comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, preferably by 10 non adenosine nucleotides (A30-N10-A70).
  • an RNA produced in accordance with technologies provided herein comprises an Open Reading Frame (ORF), e.g., encoding a polypeptide of interest or encoding a plurality of polypeptides of interest.
  • ORF Open Reading Frame
  • an RN A produced in accordance with technologies provided herein comprises a plurality of ORFs (e.g., encoding a plurality of polypeptides).
  • an RNA produced in accordance with technologies herein comprises a single ORF that encodes a plurality of polypeptides.
  • polypeptides are or comprise antigens or epitopes thereof (e.g., relevant antigens).
  • an encoded polypeptide may be or comprise an antigen or epitope thereof, so that, when expressed in a subject to which a provided RNA is administered, an immune response (e.g., characterized by antibodies and/or T cells specifically directed to the antigen or one or more epitopes thereof); in some such embodiments, an encoded polypeptide may be polyepitopic, for example including multiple polypeptide elements, each of which includes at least one epitope, linked to one another and optionally separated by linkers. As is understood in the art, in some embodiments, a polyepitopic construct may include individual epitopes found in different portions of the same protein in nature.
  • a polyepitopic construct may include individual epitopes found in different proteins in nature.
  • Those skilled in the art will be aware of a variety of considerations relevant to selection of desirable polyepitopic constructs, and/or antigens and/or epitopes for inclusion therein, useful in accordance with the present disclosure (see, for example, WO2014082729, WO2012I59754, WO2017173321, WO2014180659, WO20161283762, W020I7194610, WO2011143656, W02015I03037, Nielsen JS, et al. J Immunol Methods. 2010 Aug 3 l;360(l-2): 149-56.
  • a relevant antigen may be or comprise comprise an infectious antigen (i.e., an antigen associated with an infectious agent such as an infectious virus, a bacterium, a fungus, etc.) and/or a cancer antigen (e.g., an antigen associated with a class of tumors or a specific tumor; in some embodiments, a cancer-associated antigen may be or comprise a neoantigen or neoepitope), or epitope thereof.
  • infectious antigen i.e., an antigen associated with an infectious agent such as an infectious virus, a bacterium, a fungus, etc.
  • a cancer antigen e.g., an antigen associated with a class of tumors or a specific tumor
  • a cancer-associated antigen may be or comprise a neoantigen or neoepitope, or epitope thereof.
  • an ORF may encode, for example, an antibody or portion (e.g., antigen-binding portion) thereof, an enzyme, a cytokine, a therapeutic protein, etc. (see, for examp/e,WO20I7186928, WO2017191274, US10669322, Dammes et al Trens Pharmacol Sci 4:755, 2020-10-01, Wang et al Nature Reviews Drug Discovery 19, 441-442 (2020), Damase et al Front.
  • an antibody or portion e.g., antigen-binding portion
  • an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells.
  • a utilized signal sequence is “intrinsic” in that it is , in nature, it is associated with (e.g., linked to) the encoded polypeptide.
  • a utilized signal sequence is heterologous to the encoded polypeptide - e.g., is not naturally part of a polypeptide (e.g., protein) whose sequences are included in the encoded polypeptide.
  • signal peptides are sequences, which are typically characterized by a length of about 15 to 30 amino acids.
  • signal peptides are positioned at the N-terminus of an encoded polypeptide as described herein, without being limited thereto.
  • signal peptides preferably allow the transport of the polypeptide encoded by RNAs of the present disclosure with which they are associated into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • a signal sequence is selected from an S1S2 signal peptide (aa 1-19), an immunoglobulin secretory signal peptide (aa 1-22), an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV-2 gD signal peptide (MGRLTSGVGTAALLWAVGLRWCA); a human SPARC signal peptide, a human insulin isoform 1 signal peptide, a human albumin signal peptide, etc.
  • an RNAsequence encodes an epitope that may comprise or otherwise be linked to a signal sequence (e.g., secretory sequence), such as those listed in Table 1, or at least a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto.
  • a signal sequence such as MFVFLVLLPLVSSQCVNLT, or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.
  • a sequence such as
  • MFVFLVLLPLVSSQCVNLT or a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto, is utilized.
  • a signal sequence is selected from those included in the Table 1 below and/or those encoded by the sequences in Table 2 below: Table 1: Exemplary signal sequences
  • an RNAutilized as described herein encodes a multimerization element (e.g., a heterologous multimerization element).
  • a heterologous multimerization element comprises a dimerization, trimerization or tetramerization element.
  • a multimerization element is one described in WO2017/081082 (e.g., SEQ ID NOs: 1116-1167, or fragments or variants thereof).
  • Exemplary trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.
  • a provided encoded polypeptide(s) is able to form a trimeric complex.
  • a utilized encoded polypeptide(s) may comprise a domain allowing formation of a multimeric complex, such as for example particular a trimeric complex of an amino acid sequence comprising an encoded polypeptide(s) as described herein.
  • a domain allowing formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein.
  • an encoded polypeptide(s) can be modified by addition of a T4-fibritin-derived “foldon” trimerization domain, for example, to increase its immunogenicity.
  • an RNAas described herein encodes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.
  • a membrane association element e.g., a heterologous membrane association element
  • a transmembrane domain can be N-terminal, C-terminal, or internal to an encoded polypeptide.
  • a coding sequence of a transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences of sequences (e.g., sequences encoding polypeptide(s)) with which it is to be linked.
  • a transmembrane domain comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.
  • HA Hemagglutinin
  • EIAV equine infectious anaemia virus
  • MMV murine leukaemia virus
  • VSV vesicular stomatitis virus
  • Rabies virus or a seven transmembrane domain receptor.
  • an ORF encoding polypeptide of the disclosure is codon optimized.
  • Various codon optimization methods are known in the art.
  • an ORF of any one or more of the sequences provided herein may be codon optimized.
  • Codon optimization in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove polypeptide trafficking sequences; remove/add post translation modification sites in encoded polypeptide (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the polypeptide to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art - non limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the open reading frame (ORF) sequence is optimized using optimization algorithms.
  • a codon optimized sequence shares less than 95% sequence identity to a naturally- occurring or wild- type sequence ORF (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wildtype mRNA sequence encoding a polypeptide).
  • a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide).
  • a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide).
  • a codon-optimized sequence encodes polypeptide (e.g., an antigen) that is as immunogenic as, or more immunogenic than (e.g, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than a polypeptide encoded by a non-codon- optimized sequence.
  • polypeptide e.g., an antigen
  • immunogenic e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more
  • the modified mRNAs when transfected into mammalian host cells, have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.
  • a codon optimized RNA may be one in which the levels of G/C are enhanced and/or A/U are enhanced.
  • the G/C-content of nucleic acid molecules may influence the stability of the RNA.
  • RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides.
  • WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region.
  • the modifications work by substituting existing codons for those that promote, for example greater RNA stability, without changing the resulting amino acid.
  • the approach is limited to coding regions of the RNA.
  • RNAs encoding viral antigen(s) (and/or epitope(s) thereof), for example coronavirus antigen(s) and/or epitope(s).
  • the present disclosure exemplifies use of a single-stranded RNA whose nucleotide sequence encodes a coronavirus polypeptide or a variant thereof.
  • a single-stranded RNA comprises a nucleotide sequence that encodes a prefusion coronavirus spike protein, e.g., as described in WO 2018081318, the entire contents of which are incorporated herein by reference for purposes described herein.
  • an RNA for use in accordance with the present disclosure encodes a SARS-CoV-2 spike protein with K986P and V978P mutations.
  • a single-stranded RNA comprises a nucleotide sequence that encodes a SARS- CoV-2 polypeptide (including, e.g., a spike (S) protein, a nucleocapsid (N) protein, envelope (E) protein, and a membrane (M) protein) or an immunogenic fragment thereof.
  • a singlestranded RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof (e.g., a receptor binding domain of a S protein).
  • such a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof may be a mutant protein.
  • such a SARS-CoV-2 S protein or an immunogenic fragment thereof may be one as described in Walsh et al. “RNA-based COVID-19 vaccine BNT162b2 selected for a pivotal efficacy study” medRxiv preprint (2020), which is online accessible at: https://d0i.org/lfi.l lOl/2O2O.O8.17.2O176651- and Milligan et al. “Phase I/II study of COVID-19 RNA vaccine BNT162bl in adults” Nature (2020 August), which is online accessible at: https://doi .oig/10.1038/s41586-020-2639-4, the contents of each of which are incorporated by reference in their entirety.
  • a single-stranded RNA comprises a nucleotide sequence that encodes a SARS- CoV-2 polypeptide as shown in Example 10.
  • a single-stranded RNA may comprise a secretion signal-encoding region (e.g. , a secretion signal-encoding region that allows an encoded target entity to be secreted upon translation by cells).
  • a secretion signal-encoding region may be or comprise a non-human secretion signal.
  • such a secretion signalencoding region may be or comprise a human secretion signal.
  • a single-stranded RNA e.g., mRNA as described herein
  • may comprise at least one non-coding sequence element e.g., to enhance RNA stability and/or translation efficiency).
  • non-coding sequence elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure for co-transcriptional capping of mRNA, a poly adenine (polyA) tail, and any combinations thereof.
  • UTR untranslated region
  • 5’ UTR a cap structure for co-transcriptional capping of mRNA
  • polyA poly adenine
  • RNA can comprise a nucleotide sequence that encodes a 5 ’UTR of interest and/or a 3’ UTR of interest.
  • untranslated regions e.g., 3’ UTR and/or 5’ UTR
  • mRNA sequence can contribute to mRNA stability, mRNA localization, and/or translational efficiency.
  • a single-stranded RNA can comprise a 5’ UTR nucleotide sequence and/or a 3’ UTR nucleotide sequence.
  • a 5’ UTR sequence can be operably linked to a 3’ of a coding sequence (e.g., encompassing one or more coding regions).
  • a 3’ UTR sequence can be operably linked to 5’ of a coding sequence (e.g., encompassing one or more coding regions).
  • 5' and 3' UTR sequences included in a single-stranded RNA can consist of or comprise naturally occurring or endogenous 5' and 3' UTR sequences for an open reading frame of a gene of interest.
  • 5’ and/or 3’ UTR sequences included in a singlestranded RNA are not endogenous to a coding sequence (e.g., encompassing one or more coding regions); in some such embodiments, such 5’ and/or 3’ UTR sequences can be useful for modifying the stability and/or translation efficiency of an RNA sequence transcribed.
  • a skilled artisan will appreciate that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, as will be understood by a skilled artisan, 3' and/or 5’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • a nucleotide sequence consisting of or comprising a Kozak sequence of an open reading frame sequence of a gene or nucleotide sequence of interest can be selected and used as a nucleotide sequence encoding a 5’ UTR.
  • Kozak sequences are known to increase the efficiency of translation of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation.
  • a single-stranded RNA can comprise a nucleotide sequence that encodes a 5' UTR derived from an RNA virus whose RNA genome is stable in cells.
  • RNA sequences can be used in the 3' and/or 5' UTRs, for example, to impede exonuclease degradation of the transcribed RNA sequence.
  • a 5’ UTR included in a single-stranded RNA may be derived from human a- globin mRNA combined with Kozak region.
  • a 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 12 as shown in Example 10.
  • a single-stranded RNA may comprise one or more 3’UTRs.
  • a single-stranded RNA may comprise two copies of 3’-UTRs derived from a globin mRNA, such as, e.g., alpha2-globin, alpha 1 -globin, beta-globin (e.g, a human beta-globin) mRNA.
  • two copies of 3 ’UTR derived from a human beta-globin mRNA may be used, e.g., in some embodiments which may be placed between a coding sequence of a single-stranded RNA and a poly(A)-tail, to improve protein expression levels and/or prolonged persistence of an mRNA.
  • a 3’ UTR included in a single-stranded RNA may be or comprise one or more (e.g., 1, 2, 3, or more) of the 3 ’UTR sequences disclosed in WO 2017/060314, the entire content of which is incorporated herein by reference for the purposes described herein.
  • a 3‘-UTR may be a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017/060314, herein incorporated by reference).
  • FI element comprises the nucleotide sequence of SEQ ID NO: 13 as shown in Example 10.
  • a single-stranded RNA can comprise a polyA tail.
  • a polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenosine nucleotides.
  • a polyA tail is a nucleotide sequence comprising at least 30 adenosine nucleotides or more, including, e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more adenosine nucleotides.
  • a polyA tail is or comprises a polyA homopolymeric tail.
  • a polyA tail may comprise one or more modified adenosine nucleosides, including, but not limited to, cordycepin and 8-azaadenosine.
  • a polyA tail may comprise one or more non-adenosine nucleotides.
  • a polyA tail may be or comprise a disrupted or modified polyA tail as described in WO 2016/005324, the entire content of which is incorporated herein by reference for the purpose described herein.
  • a polyA tail included in a single-stranded RNA described herein may be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 consecutive A nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 20 consecutive A nucleotides, which is 3’ of the linker sequence.
  • a modified polyA sequence may comprise: a linker sequence comprising at least ten nucleotides (e.g., U, G, and/or C nucleotides); a first sequence of at least 30 consecutive A nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 70 consecutive A nucleotides, which is 3’ of the linker sequence.
  • a polyA tail comprises the nucleotide sequence of SEQ ID NO: 14 as shown in Example 10.
  • a single-stranded RNA described herein may comprise a 5’ cap, which may be incorporated into such a single-stranded RNA during transcription, or joined to such a singlestranded RNA post-transcription.
  • a single-stranded RNA may comprise a 5’ cap structure for co-transcriptional capping of mRNA. Examples of a cap structure for co-transcriptional capping are known in the art, including, e.g., as described in WO 2017/053297, the entire content of which is incorporated herein by reference for the purposes described herein.
  • a 5’ cap included in a single-stranded RNA described herein is or comprises a capl structure.
  • a capl structure maybe or comprise m7G(5')ppp(5')(2'OMeA)pG, also known asm 2 73 ⁇ °Gppp(mi 2 °)ApG.
  • a single-stranded RNA described herein may comprise at least one modified ribonucleotide, for example, in some embodiments to increase the stability of such a single-stranded RNA and/or to decrease cytotoxicity of such a single-stranded RNA.
  • at least one of A, U, C, and G ribonucleotide of a single-stranded RNA may be replaced by a modified ribonucleotide.
  • some or all of cytidine residues present in a singlestranded RNA may be replaced by a modified cytidine, which in some embodiments may be, e.g.
  • uridine residues present in a single-stranded RNA may be replaced by a modified uridine, which in some embodiments may be, e.g., pseudouridine, such as, e.g., 1 -methylpseudouridine.
  • pseudouridine such as, e.g., 1 -methylpseudouridine.
  • all uridine residues present in a single-stranded RNA is replaced by pseudouridine, e.g., 1 -methylpseudouridine.
  • RNA preparations e.g. , pharmaceutical-grade RNA preparations, including large batch preparations
  • RNA preparations include, for example (i) synthesizing RNA by in vitro transcription e.g., in a bioreactor, to produce an in vitro transcription RNA composition; and (ii) removing one or more components (e.g., undesired components) from the in vitro transcription RNA composition, thereby producing an RNA transcript preparation; in some embodiments, such the RNA transcript is present in such RNA transcript preparation at a concentration (i.e., an adjusted concentration, in light of the removing) of at least 1 mg/mL (including, e.g., at least 1.5 mg/mL, at least 2 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, at least 4 mg/mL, at least 4.5 mg/mL, at least 5 mg/mL, at least 6 mg/mL, or
  • the RNA may be present at a concentration of 1.5 mg/mL to 5 mg/mL or 2 mg/mL to 4 mg/mL.
  • all unit operations described herein are performed at room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C), unless specified otherwise.
  • RNA e.g., single-stranded RNA as described herein
  • RNA can be synthesized from a DNA template by in vitro RNA transcription, e.g., in the presence of appropriate reagents comprising, e.g., at least one RNA-polymerase and appropriate ribonucleotide triphosphates or variants thereof (e.g., modified ribonucleotide triphosphates), e.g., in a bioreactor.
  • appropriate reagents comprising, e.g., at least one RNA-polymerase and appropriate ribonucleotide triphosphates or variants thereof (e.g., modified ribonucleotide triphosphates), e.g., in a bioreactor.
  • a bioreactor that is useful for in vitro transcription is large enough for an in vitro transcription reaction volume of at least 1 liter, including, e.g., at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 liters or more.
  • a bioreactor that is particularly useful for commercial-scale in vitro transcription is large enough for an in vitro transcription reaction volume of at least 20 liters, including, e.g., at least 25, 30, 35, 40, 45, 50 liters, or more.
  • a DNA template is used to direct synthesis of RNA (e.g., single-stranded RNA).
  • a DNA template is a linear DNA molecule.
  • a DNA template is a circular DNA molecule.
  • DNA can be obtained or generated using methods known in the art, including, e.g., gene synthesis, recombinant DNA technology, or a combination thereof.
  • a DNA template comprises a nucleotide sequence coding for a transcribed region of interest (e.g., coding for a RNA described herein) and a promoter sequence that is recognized by an RNA polymerase selected for use in in vitro transcription.
  • RNA polymerases are known in the art, including, e.g., DNA dependent RNA polymerases (e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, or a variant or functional domain thereof).
  • DNA dependent RNA polymerases e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, or a variant or functional domain thereof.
  • an RNA polymerase utilized herein may be a recombinant RNA polymerase, and/or a purified RNA polymerase, i.e., not as part of a cell extract, which contains other components in addition to the RNA polymerases.
  • a DNA template can comprise a promoter sequence for a T7 RNA polymerase.
  • a DNA template comprises a nucleotide sequence coding for an RNA described herein (e.g. , comprising a nucleotide sequence coding for an antigen of interest and optionally comprising one or more nucleotide sequences coding for characteristic elements of an RNA described herein, including, e.g., polyA tail, 3’ UTR, and/or 5’ UTR, etc.).
  • a coding sequence may be generated by gene synthesis.
  • such a coding sequence may be inserted into a vector by cold fusion cloning.
  • a DNA template may further comprise one or more of a recognition sequence for an appropriate restriction endonuclease (e.g., utilized for linearization), an appropriate resistance gene, and/or an appropriate origin of replication.
  • a DNA template may further comprise a recognition sequence for an appropriate restriction endonuclease (e.g., utilized for linearization such as, e.g., but not limited to a Class II restriction endonuclease), an appropriate resistance gene (e.g., but not limited to a kanamycin resistance gene), and an appropriate origin of replication.
  • a DNA template may be amplified via polymerase chain reaction (PCR) from a plasmid DNA.
  • PCR polymerase chain reaction
  • a plasmid DNA may be obtained, e.g., from bacterial cells (e.g., Escherichia coli (E. coli)) followed by an endotoxin- and animal product-free plasmid isolation procedure.
  • a DNA template may be a linearized plasmid DNA (pDNA) template in the absence of PCR-based amplification.
  • a cell bank or a cell stock for a pDNA of interest may be established.
  • such a cell bank or a cell stock may comprise a frozen stock of bacterial cells (e.g., E. coli cells, such as DH10B E. coli cells) that are genetically engineered to comprise a pDNA template of interest (e.g. , as described herein) with pre-determined specifications.
  • a pDNA contains a promoter sequence (e.g. T7 RNA polymerase).
  • a pDNA contains a recognition sequence for an endonuclease (e.g., for linearization). In some embodiments, a pDNA contains a resistance gene. In some embodiments, a pDNA contains an origin of replication. In some embodiments, a pDNA contains one or more of a promoter sequence, a recognition sequence for an endonuclease, a resistance gene, and/or an origin of replication.
  • a master cell bank or a master cell stock may be established.
  • a cell bank or cell stock may be established, for example, by transforming a stock of competent bacterial cells (e.g., E. coli cells) with a pDNA of interest.
  • a pure culture of transformed cells may be produced, for example, by growth on selective medium.
  • a single colony isolate may be selected and grown in liquid culture and, in some embodiments, used to inoculate larger cultures volumes.
  • culture growth is stopped at a predetermined threshold (e.g., optical density (OD) threshold).
  • OD optical density
  • cryoprotectant e.g., glycerol
  • cryoprotectant e.g., glycerol
  • the cell suspension is aliquoted into a container (e.g., tubes, vials, cryovials, etc.) and frozen using a controlled rate freezer.
  • cell bank aliquots are stored at least at -100°C, -125°C, -150°C, or colder (e.g., in the vapor phase of a liquid nitrogen freezer or dewar).
  • quality control testing is performed on a master cell bank or cell stock, for example, by evaluating one or more of culture purity, presence of lytic bacteriophage, presence of lysogenic bacteriophage, host cell identity, viability, plasmid retention, restriction map analysis, plasmid copy number, and/or DNA sequencing.
  • a vial from a master cell bank or cell stock may be thawed to inoculate a culture (e.g., a working cell bank).
  • working cell bank culture growth may be stopped at a particular predetermined threshold.
  • a cryoprotectant is added.
  • a working cell bank is aliquoted, stored, and/or evaluated for quality (e.g., as described for a master cell bank).
  • master cell bank and working cell banks are monitored for quality over time (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years or more after release) until the cell bank is depleted and/or no longer used.
  • a pDNA can be amplified by first thawing and subsequent fermentation of the genetically engineered bacterial cells (e.g., E. coli cells from a cell bank), followed by purification of the pDNA (e.g., by filtration, chromatography, etc.), linearization (e.g., by an endonuclease), and optionally a polishing step as appropriate, thereby generating a linearized pDNA.
  • the genetically engineered bacterial cells e.g., E. coli cells from a cell bank
  • purification of the pDNA e.g., by filtration, chromatography, etc.
  • linearization e.g., by an endonuclease
  • polishing step as appropriate
  • a resulting linearized pDNA is assessed for a set of relevant specifications, including, for example, DNA concentration, purity, appearance, residual host cell DNA and/or RNA, residual selection drug, residual protein, pH, PolyA tail integrity and/or identity, linearization efficiency (e.g., least 75%, 80%, 85%, 90%, 95%, or more), identity of transcribed region, bioburden, and/or endotoxins.
  • linear DNA template is stored in water (e.g., high purity water).
  • linear DNA template is stored in buffer (e.g., HEPES, pH 7-9).
  • Ribonucleotides for use in in vitro transcription may include at least two or more (e.g., at least three or more, at least four or more, at least five or more, at least six or more) different types of ribonucleotides, each type having a different nucleoside.
  • Ribonucleotides for use in in vitro transcription can include unmodified and/or modified ribonucleotides.
  • Unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). In some embodiments, all four types of unmodified ribonucleotides may be used for in vitro transcription.
  • At least one type of ribonucleotide included in in vitro transcription is a modified ribonucleotide.
  • Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g.
  • a modified ribonucleotide may have at least one nucleoside ("base") modification or substitution.
  • base nucleoside
  • nucleoside modifications or substitutions are known in the art; one of skill in the art will appreciate that modified nucleosides include, for example, but not limited to synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2- (amino)adenine, 2-(aminoalkyll)adenine, 2- (aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6- (methyl)adenine, 7- (deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine
  • a modified nucleotide utilized in IVT systems and/or methods described herein may disrupt binding of an RNA to one or more mammalian (e.g., human) endogenous RNA sensors (e.g., innate immune RNA sensors), including, e.g., but not limited to toll-like receptor (TLR)3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), protein kinase R (PKR), 2 ’-5’ oligoadenylate synthetase (OAS), and laboratory of genetics and physiology 2 (LGP2), and combinations thereof.
  • mammalian e.g., human
  • endogenous RNA sensors e.g., innate immune RNA sensors
  • RLR toll-like receptor
  • MDA5 melanoma differentiation-associated gene 5
  • PSR protein kinase R
  • OAS oligoadenylate
  • modified ribonucleotides may include modifications as described in US 9,334,328, the contents of which are incorporated herein by reference in their entireties for the purposes described herein.
  • Modified nucleosides are typically desirable to be translatable in a host cell (e.g., presence of a modified nucleoside does not prevent translation of an RNA sequence into a respective protein sequence). Effects of modified nucleotides on translation can be assayed, by one of ordinary skill in the art using, for example, a rabbit reticulocyte lysate translation assay.
  • a modified ribonucleotide may include a modified intemucleoside linkage.
  • modified intemucleoside linkages are known in the art; one of skill in the art will appreciate that non-limiting examples of modified intemucleoside linkages that may be used in technologies provided herein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2 -5' linked analogs of these, and those) having inverted polarity where
  • Modified intemucleoside linkages that do not include a phosphorus atom therein may have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
  • a modified ribonucleotide may include one or more substituted sugar moieties.
  • modified sugar moieties are known in the art; one of skill in the art will appreciate that, in some embodiments, a sugar moiety of a ribonucleotide may include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted.
  • a sugar moiety of a ribonucleotide may include a 2' methoxyethoxy (2'-O- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2 -MOE), 2'- dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2 -DMAOE, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'0-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e.
  • 2' methoxyethoxy 2'-O- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2 -MOE
  • 2'- dimethylaminooxyethoxy i.e., a O(CH2)2ON(CH3)2 group, also known as 2 -DMAOE
  • a mixture of ribonucleotides that are useful for an in vitro transcription reaction may comprise ATP, CTP, GTP, and Nl-methylpseudouridine-5’ triphosphate (m 1 TTP).
  • the ratio of ATP, CTP, GTP, and m l TTP for an in vitro transcription reaction is 1 : 1 : 1 : 1.
  • the ratio of ATP, CTP, GTP, and mlTTP for an in vitro transcription is optimized such that relative proportions of nucleotides correspond to fractions of the respective nucleotides in an mRNA molecule, e.g., as described in the International Patent Publication No. WO 2015188933.
  • an individual reaction component or components are thawed prior to their addition to an in vitro transcription reaction mixture.
  • an in vitro transcription reaction mixture typically includes a DNA template (e.g., as described herein), ribonucleotides (e.g., as described herein), a RNA polymerase (e.g., DNA dependent RNA polymerases), and an appropriate reaction buffer for a selected RNA polymerase.
  • an in vitro transcription reaction mixture may further comprise an RNase inhibitor.
  • an in vitro transcription reaction mixture may further comprise a pyrophosphatase (e.g., an inorganic pyrophosphatase).
  • an in vitro transcription reaction mixture may further comprise one or more salts (e.g., monovalent salts and/or divalent salts), a reducing agent (e.g. , dithithreitol, 2-mercaptoethanoI, etc.), spermidine, or combinations thereof.
  • certain reaction components are added in a specific order (e.g., pyrophosphatase and polymerase added last).
  • agitation rate is increased following the addition of specific reaction components (e.g., pyrophosphatase, polymerase).
  • RNA polymerases that are suitable for in vitro transcription are known in the art, including, e.g., but not limited to DNA dependent RNA polymerases (e.g. , a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, or a variant or functional domain thereof).
  • DNA dependent RNA polymerases e.g. , a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, or a variant or functional domain thereof.
  • an RNA polymerase utilized herein may be a recombinant RNA polymerase, and/or a purified RNA polymerase, z.e., not as part of a cell extract, which contains other components in addition to the RNA polymerases.
  • an RNA polymerase that is useful for commercial-scale in vitro transcription is a T7 RNA polymerase.
  • an inorganic pyrphosphatase may be added to improve the yield of in vitro transcription reaction (e.g. , in some embodiments catalyzed by T7 RNA polymerase).
  • Transcription buffer is typically optimized for a selected RNA polymerase.
  • a transcription buffer may comprise Tris-HCl, HEPES, or other appropriate buffer.
  • a transcription buffer can comprise 20-60 mM HEPES, 20-60 mM divalent salt (e.g., magnesium salts, such as magnesium chloride, magnesium acetate, etc.), 5-15 mM reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, etc.) and 0.5 - 3 mM spermidine.
  • a transcription buffer has apH of 7-9 (e.g., about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0).
  • an RNA produced by technologies described herein may comprise a cap at its 5’ end.
  • RNA e.g., mRNA
  • addition of a 5' cap to an RNA can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency.
  • a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life.
  • Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g.
  • a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system).
  • a capping agent may be introduced into an in vitro transcription reaction mixture (e.g. , ones as described herein), along with a plurality of ribonucleotides such that a cap is incorporated into an RNA during transcription (also known as co-transcriptional capping). While it may be desirable to include, in some embodiments, a 5' cap in an RNA, an RNA, in some embodiments, may not have a 5’ cap.
  • a 5’ capping agent can be added to an in vitro transcription reaction mixture.
  • a 5’ capping agent may comprise a modified nucleotide, for example, a modified guanine nucleotide.
  • a 5’ capping agent may comprise, for example, a methyl group or groups, glyceryl, inverted deoxy abasic moiety, 4’5’ methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4’ thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-di
  • a 5’ capping agent may be or comprise a dinucleotide cap analog (including, e.g., a m7GpppG cap analog or an N7-methyl, 2’-O- methyl -GpppG anti-reverse cap analog (ARCA) cap analog or an N7-methyl, 3'-O-methyl-GpppG ARCA cap analog).
  • a 5’ capping agent comprises a 5' N7-Methyl-3'-O-Methylguanosine structure, e.g., CleanCap® Reagents (Trilink BioTechnologies).
  • a 5’-capping agent is added in excess to a particular ribonucleotide or ribonucleotides (e.g., GTP, ATP, UTP, CTP, or modified version thereof) to enable incorporation of the 5 ’-cap as the first addition to the RNA transcript.
  • a particular ribonucleotide or ribonucleotides e.g., GTP, ATP, UTP, CTP, or modified version thereof
  • an in vitro transcription reaction is conducted, e.g., in a bioreactor described herein (selected for a certain in vitro transcription reaction volume, e.g. , as described herein) for a period of time.
  • the period of time is at least 20 minutes, including, e.g., at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 55 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, or at least 180 minutes.
  • the period of time is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes. In some embodiments, the period of time is about 1.5-3 hours. In some embodiments, the period of time is about 25-35 minutes.
  • an in vitro transcription reaction is conducted, e.g., in a bioreactor described herein for a period of time (e.g., as described herein) at a temperature at which a selected RNA polymerase is functionally active.
  • a temperature at which a selected RNA polymerase is functionally active e.g., a selected RNA polymerase is functionally active.
  • typical phage RNA polymerases e.g. , T7 polymerases
  • thermostable RNA polymerases e.g., thermostable variants of T7 RNA polymerases such as ones as described in US10519431, the contents of which are incorporated by reference for purposes described herein
  • in vitro transcription is performed at a temperature of approximately 25°C or higher, including, e.g., 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • in vitro transcription is performed at a temperature of approximately 45 °C or higher, including, e.g., 46°C, 47°C, 48°C , 49°C, 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C or higher.
  • an in vitro transcription is conducted e.g., in a bioreactor described herein at a pH of about 6, 6.5, 7, 7.5, 8, or 9.
  • a suitable pH for an in vitro transcription may be approximately 7.5-8.5.
  • in vitro transcription reactions performed in accordance with the present disclosure e.g., in a bioreactor as described herein
  • one or more nucleotides may be added to an in vitro transcription reaction in a step-wise manner (e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more bolus feeds).
  • an agitation rate is selected such that a particular blend time to enable rapid mixing of bolus additions to ensure optimal availability of modified nucleotide solution and one or more other nucleotide solutions during RNA synthesis is achieved.
  • an in vitro transcription reaction comprises UTP or a functional thereof at a limiting concentration in combination with at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof.
  • a functional analog of UTP is or comprises Nl- methylpseudouridine-5’ triphosphate (ml TTP).
  • UTP or a functional analog thereof is present in an in vitro transcription reaction at a starting concentration that limits the rate of transcription. In some embodiments, UTP or a functional analog thereof is present in an in vitro transcription reaction at a starting concentration that is lower than the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof.
  • the starting concentration of UTP or a functional analog thereof is at least 30% lower (including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower) than the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof.
  • the ratio of the starting concentration of UTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof is about 1:1.3 or lower, including, e.g., 1: 1.4; 1:1.5; 1:2, 1:2.5; 1:3; 1 :3.5; 1:4; 1:4.5; 1:5; 1 :6; 1:7; 1:8, 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; 1:15; 1:16; 1:17; 1:18; 1:19; 1 :20, or lower.
  • the ratio of the starting concentration of UTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof is about 1 : 1.3 to about 1 :20, or about 1 : 1.5 to about 1 : 15, or about 1 :5 to about 1 : 15, or about 1 : 8 to about 1:12.
  • the starting concentration of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof may be the same.
  • an in vitro transcription reaction is supplemented at least once with UTP or a functional analog thereof over the course of the reaction.
  • an in vitro transcription reaction is supplemented multiple times (e.g., at least 2 or more, including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • supplementation of UTP or a functional analog thereof is performed when its concentration in the reaction mixture is near depletion. In some embodiments, supplementation of UTP or a functional analog thereof is performed when its concentration in the reaction mixture is less than 100 uM, 90 uM, 80 uM, 70 uM, 60 uM, 50 uM, 40 uM, 30 uM, 20 uM, 10 uM, 5 uM, 3 uM, 2, uM, 1 uM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 25 nM, or lower.
  • UTP (or a functional analog thereof) supplementation may be performed continuously during the course of the transcription reaction.
  • UTP (or a functional analog thereof) supplementation may be performed in a continuous manner at a rate that is comparable to (e.g., within 10% or lower) of its consumption rate.
  • UTP (or a functional analog thereof) supplementation may be performed at a rate such that after such supplementation, UTP or functional analog thereof is present in the reaction at a concentration lower than that of one or more, and in some embodiments, all of ATP or functional analog thereof, GTP or functional analog thereof, and/or CTP or functional analog thereof.
  • UTP (or a functional analog thereof) supplementation may be performed periodically during the course of the transcription reaction.
  • UTP (or a functional analog thereof) supplementation may be performed in a periodic manner such that after each addition, UTP or functional analog thereof is present in the reaction at a concentration lower than that of one or more, and in some embodiments, all of ATP or functional analog thereof, GTP or functional analog thereof, and/or CTP or functional analog thereof.
  • periodic supplementation may be performed as one or more bolus or batch addition(s), including, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • Such periodic supplementation may be performed by a fed-batch process.
  • the concentration of UTP or a functional analog thereof added during supplementation is same as the starting concentration of UTP or a functional analog thereof. In some embodiments, the concentration of UTP or a functional analog thereof added during supplementation is lower than the starting concentration of UTP or a functional analog thereof, e.g., at least 10% lower (including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower) than the starting concentration of UTP or a functional analog thereof.
  • UTP (or a functional analog thereof) supplementation is performed at a concentration and/or at a rate or manner such that the ratio of the concentration of UTP or a functional analog thereof to the concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof (during the course of the reaction) is maintained substantially the same (e.g., within 10% or less) as the initial ratio of the concentration of UTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof (at the beginning of the reaction).
  • UTP or a functional analog thereof is supplemented until the end of the transcription reaction.
  • UTP or a functional analog thereof is present in an initial transcription reaction at a starting concentration of 0.1 to 2 mM or 0.1 to 1.5 mM, or 0.1 to 1 mM, or 0.5 to 2 mM, or 1 to 2 mM. In some embodiments, UTP or a functional analog thereof is maintained during the course of an in vitro transcription reaction at a concentration of 0.1 to 2 mM or 0.1 to 1.5 mM, or 0.1 to 1 mM, or 0.5 to 2 mM, or 1 to 2 mM.
  • At least one of non-UTP is provided at a limiting concentration (in addition to limited UTP or a functional analog thereof) at the initial in vitro transcription reaction (e.g., the beginning of the in vitro transcription reaction).
  • at least one of ATP or a functional analog thereof, CTP or a functional analog thereof, or GTP or a functional analog thereof is provided at a limiting concentration (in addition to limited UTP or a functional analog thereof) at the initial in vitro transcription reaction (e.g., the beginning of the in vitro transcription reaction).
  • GTP or a functional analog thereof is provided at a limiting concentration (in addition to limited UTP or a functional analog thereof) at the initial in vitro transcription (e.g., the beginning of the in vitro transcription reaction).
  • GTP or a functional analog thereof is present in an in vitro transcription reaction at a starting concentration that limits the rate of transcription. In some embodiments, GTP or a functional analog thereof is present in an in vitro transcription reaction at a starting concentration that is lower than the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof. In some embodiments, the starting concentration of GTP or a functional analog thereof is at least 30% lower (including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower) than the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof.
  • the ratio of the starting concentration of GTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof is about 1 : 1.3 or lower, including, e.g., 1:1.4; 1:1.5; 1 :2, 1:2.5; 1:3; 1:3.5; 1:4; 1:4.5; 1:5; 1:6; 1:7; 1:8, 1:9; 1 : 10; 1:11; 1:12; 1: 13; 1:14; 1:15; 1:16; 1 :17; 1:18; 1:19; 1 :20, or lower.
  • the ratio of the starting concentration of GTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof is about 1 : 1.3 to about 1 :20, or about 1:1.5 to about 1:15, or about 1:5 to about 1:15, or about 1:8 to about 1:12. In some such embodiments, the starting concentration of ATP or a functional analog thereof and/or CTP or a functional analog thereof.
  • an in vitro transcription reaction is supplemented at least once with GTP or a functional analog thereof over the course of the reaction.
  • an in vitro transcription reaction is supplemented multiple times (e.g., at least 2 or more, including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) with GTP or a functional analog thereof over the course of the transcription reaction.
  • supplementation of GTP or a functional analog thereof is performed when its concentration in the reaction mixture is near depletion.
  • supplementation of GTP or a functional analog thereof is performed when its concentration in the reaction mixture is less than 100 uM, 90 uM, 80 uM, 70 uM, 60 uM, 50 uM, 40 uM, 30 uM, 20 uM, 10 uM, 5 uM, 3 uM, 2, uM, 1 uM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 25 nM, or lower.
  • GTP (or a functional analog thereof) supplementation may be performed continuously during the course of the transcription reaction.
  • GTP (or a functional analog thereof) supplementation may be performed in a continuous manner at a rate that is comparable to (e.g., within 10% or lower) of its consumption rate.
  • GTP (or a functional analog thereof) supplementation may be performed at a rate such that after such supplementation, GTP or functional analog thereof is present in the reaction at a concentration lower than that of ATP or functional analog thereof and/or CTP or functional analog thereof.
  • GTP (or a functional analog thereof) supplementation may be performed periodically during the course of the transcription reaction.
  • GTP (or a functional analog thereof) supplementation may be performed in a periodic manner such that after each addition, GTP or functional analog thereof is present in the reaction at a concentration lower than that of one or more, and in some embodiments, all of ATP or functional analog thereof, and/or CTP or functional analog thereof.
  • such periodic supplementation may be performed as one or more bolus or batch addition(s), including, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more bolus or batch addition(s).
  • such periodic supplementation may be performed by a fed-batch process.
  • the concentration of GTP or a functional analog thereof added during supplementation is same as the starting concentration of GTP or a functional analog thereof. In some embodiments, the concentration of GTP or a functional analog thereof added during supplementation is lower than the starting concentration of GTP or a functional analog thereof, e.g., at least 10% lower (including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower) than the starting concentration of GTP or a functional analog thereof.
  • GTP (or a functional analog thereof) supplementation is performed at a concentration and/or at a rate or manner such that the ratio of the concentration of GTP or a functional analog thereof to the concentration of ATP or a functional analog thereof, and/or CTP or a functional analog thereof (during the course of the reaction) is maintained substantially the same (e.g., within 10% or less) as the initial ratio of the concentration of GTP or a functional analog thereof to the starting concentration of ATP or a functional analog thereof and/or CTP or a functional analog thereof (at the beginning of the reaction).
  • GTP or a functional analog thereof is supplemented until the end of the transcription reaction.
  • GTP or a functional analog thereof is present in an initial transcription reaction at a starting concentration of 0.1 to 2 mM or 0.1 to 1.5 mM, or 0.1 to 1 mM, or 0.5 to 2 mM, or 1 to 2 mM. In some embodiments, GTP or a functional analog thereof is maintained during the course of an in vitro transcription reaction at a concentration of 0.1 to 2 mM or 0.1 to 1.5 mM, or 0.1 to 1 mM, or 0.5 to 2 mM, or 1 to 2 mM.
  • non-UTP supplementation does not include supplementation of CTP or functional analog thereof or ATP or functional analog thereof.
  • non-UTP supplementation can be performed concurrently with UTP supplementation over the course of the reaction.
  • non-UTP or functional analog thereof and UTP or functional analog thereof can be added to a reaction mixture as a single composition.
  • non-UTP or functional analog thereof and UTP or functional analog thereof can be added to a reaction mixture as separate compositions, for example, each at the same or different concentrations and/or each introduced at the same or different flow rates to a reaction mixture).
  • non-UTP supplementation and UTP supplementation can be performed by different methods, e.g., one is performed continuously (e.g., as described herein) while another is performed periodically (e.g., as described herein).
  • in vitro transcription in accordance with the present disclosure is carried out, e.g., in a bioreactor as described herein, using a fed-batch process and the present disclosure teaches that such fed-batch process may have certain advantages including, for example, ability to maintain one or more reagents or components within a particular concentration range.
  • a fed-batch process may involve multiple additions of a nucleotide that competes with a cap analog (a “competing nucleotide”) such as, e.g., a GTP, in the course of an in vitro transcription reaction, for example to maintain a low concentration of GTP (e.g., 0.1 to 2 mM or 0.1 to 1.5 mM, or 0.1 to 1 mM, or 0.5 to 2 mM, or 1 to 2 mM) in order to effectively cap a synthesized RNA.
  • a nucleotide such as, e.g., a GTP
  • a fed- batch process may involve supplementation of an in vitro transcription reaction with a competing nucleotide at a ratio between about 1 : 1 and about 1 :50 relative to the concentration of a cap analog in the reaction, e.g., as described in the International Patent Publication No. WO 2006004648.
  • the concentration of a competing nucleotide in an in vitro transcription reaction is maintained at a level that is less than the concentration of a cap analog throughout the reaction but is not a limiting component.
  • a programmable pump may be used.
  • a programmable syringe pump may be used, for example, to automatically perform step- wise addition of one or more reaction components.
  • a monitor e.g., a sensor
  • a monitor may be utilized to detect level(s) of one or more components; in some such embodiments, a monitor may communicate automatically with a pump, for example so that additional feeds may be released upon detection of a reduced amount of such component(s).
  • an in vitro transcription reaction is optimized such that relative proportions of nucleotides correspond to fractions of the respective nucleotides in an mRNA molecule, e.g., as described in the International Patent Publication No. WO 2015188933.
  • a DNA template can be removed or separated from an in vitro transcription RNA composition, for example using methods known in the art, e.g., DNA hydrolysis.
  • DNase e.g., DNase I
  • DNase I may be added to remove or digest or fragment DNA template under appropriate conditions (e.g., in the presence of divalent salt such as a calcium salt and/or incubation at an optimum temperature for DNase).
  • DNA removal is performed for a period of 15-20 minutes, 15-25 minutes, 20-25 minutes, 20-30 minutes, 25-30 minutes, 25-35 minutes, 30-35 minutes, 30-40 minutes, 35-40 minutes, 35-45 minutes, 45-50 minutes, SO- 55 minutes, or 55-60 minutes.
  • DNA removal is performed at a temperature of approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • DNA removal is performed at a temperature of 30-40 °C.
  • agitation rate is maintained during DNA removal (e.g., DNA hydrolysis) from the previous FVT step.
  • an RNase inhibitor may be added during DNA removal or digestion to protect RNA from potential degradation.
  • a chelating agent may be added to a DNase- treated transcription mixtures to complex with divalent ions that may be added during in vitro transcription reaction.
  • An exemplary chelating agent may be or comprise ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the temperature may be shifted at least 1°C (including e.g, at least 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, I0°C or more).
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA removal and/or digestion) can be subjected to a protein digestion or fragmentation process.
  • an exemplary protein digestion or fragmentation may comprise use of a proteinase (e.g., but not limited to proteinase K).
  • protein digestion utilizes a relative amount of enzyme (e.g., proteinase) to starting IVT volume, for example, at least 0.5 rnL/L, at least 0.75 rnL/L, at least 1 mL/L, at least 1.25 mL/L, or more.
  • protein digestion is conducted at a particular temperature (e.g., at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, or higher) for a particular duration of time (e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, or longer).
  • RNA concentration, bioburden, and/or endotoxins are assessed and/or monitored after protein digestion.
  • an in vitro transcription RNA composition following in vitro transcription and optional pre-purification processing may be maintained at 2-8°C for a period of time before further processing (e.g., removal of impurities).
  • the maintained period of time may be at least 6 hours or longer, including, e.g., at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or longer.
  • a RNA preparation may be held in a container, for example, a bag, tube, vial, etc.
  • the container is a polymer-based container (e.g., polyethylene, ethylene vinyl acetate).
  • RNA concentration and/or integrity may be monitored during or after in vitro transcription.
  • RNA concentration may be assessed following purification of an aliquot of a transcription mixture with a commercial kit after in vitro transcription.
  • RNA concentration and/or integrity of a produced RNA solution after in vitro transcription may be assessed before maintaining at 2-8°C for a period of time (e.g., as described herein).
  • an in vitro transcription RNA composition is produced by in vitro transcription
  • one or more components e.g. , added reagents, reaction by products, and/or impurities
  • an in vitro transcription RNA composition can be purified using phenol-chloroform extraction, enzymatic digestions of undesired components (e.g., protein components), precipitation, chromatography, spin column purification, membrane filtration, and/or affinity-based purification (e.g. , in the form of a solid substrate, e.g., but not limited to magnetic beads or particles).
  • an in vitro transcription RNA composition (e.g, in some embodiments after DNA and/or protein removal and/or digestion) can be purified by an affinity-based purification method, chromatography-based purification methods (e.g., size exclusion chromatography (SEC), high-performance liquid chromatography (HPLC), ion exchange chromatography (IEC)), and/or filtration methods (e.g. , centrifugal ultrafiltration, membrane filtration, etc.).
  • SEC size exclusion chromatography
  • HPLC high-performance liquid chromatography
  • IEC ion exchange chromatography
  • filtration methods e.g., centrifugal ultrafiltration, membrane filtration, etc.
  • an in vitro transcription RNA composition (e.g. , in some embodiments after DNA and/or protein removal and/or digestion) can be purified by an affinity-based purification method.
  • an affinity-based purification method may be performed with a solid substrate known in the art. It will be apparent to one skilled in the art that a variety of solid substrates may be used, including, without limitation, membranes; beads; tubes; wells; microtiter plates or wells; slides; discs; columns; beads (including, e.g., polymeric beads, magnetic beads); membranes; films; chips; and composites thereof.
  • a solid substrate e.g., magnetic beads or particles coated with a substance or composition that has a high binding affinity for high-molecular weight nucleic acids
  • RNA will bind to the solid substrate, while any other undesirable components present in an RNA transcription mixture, including, e.g., short hydrolyzed DNA fragments, free nucleotide triphosphates (NTPs), 5’ capping agent, proteins, divalent ions complexed with a chelating agent, will remain in solution.
  • a silicate-coated solid substrate e.g., particles or magnetic beads
  • a silicate-coated solid substrate may be used.
  • a carboxylate-coated solid substrate e.g. , particles or magnetic beads
  • an RNA transcription mixture may be divided into a plurality of (e.g. , at least two, at least three, at least four, or more) portions such that they can be purified in parallel (e.g. , in batch mode).
  • magnetic bead- or particle-based purification (e.g., as described herein) is carried out at room temperature (e.g., about I8°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20- 30°C, or about 23-27°C or about 25°C).
  • magnetic bead- or particle-based purification is performed under a suitable binding condition (e.g., in the presence of salt and organic solvent (e.g., ethanol)).
  • a suitable binding condition e.g., in the presence of salt and organic solvent (e.g., ethanol)
  • magnetic beads or particles e.g., ones described herein
  • the solid substrate can be separated from supernatant.
  • a magnet can be used to retain RNA-bound magnetic beads in a batch reaction vessel, while supernatant is subsequently removed.
  • the RNA is then eluted from the magnetic beads under a suitable eluting condition (e.g, in the presence of a buffer and/or a chelating agent at a suitable pH).
  • a suitable eluting condition e.g, in the presence of a buffer and/or a chelating agent at a suitable pH.
  • such bind-and-elute process may be performed for a number of cycles (e.g., at least two, at least three, at least four or more cycles).
  • an in vitro transcription RNA composition is divided into aliquots.
  • such RNA aliquots are purified in parallel in batch mode with wash steps prior to the elution of purified RNA.
  • one, two, three, four, five, or six wash steps are carried out.
  • different buffers are utilized in different wash steps.
  • the same solution is utilized in initial wash step or steps and a different solution is utilized in a final wash step.
  • RNA bound magnetic beads can be washed in multiple steps (e.g., three consecutive steps) with a first wash buffer comprising an organic solvent (e.g., ethanol) and a salt (e.g., sodium salt).
  • such a first wash buffer may comprise a 20-40% (v/v) ethanol/O.lM- 1M NaCl.
  • RNA bound magnetic beads can be further subjected to a final wash with an organic solvent (e.g., 80% ethanol).
  • RNA that is bound on magnetic beads is subsequently eluted (e.g., after wash steps) by addition of an elution buffer.
  • an elution buffer comprises a chelating agent to complex and thus remove residual divalent ions (e.g. , magnesium and/or calcium ions) that may be added during RNA synthesis process.
  • an elution buffer may comprise EDTA. While a skilled artisan will be able to select an appropriate buffer for elution, in some embodiments, an elution buffer may comprise HEPES buffer. In some embodiments, an elution buffer is a buffer selected for use in a pharmaceutical-grade composition comprising RNA.
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by a chromatography method.
  • a chromatography purification method may be performed with a chromatographic method known in the art (e.g. HPLC, SEC, IEC, etc.), wherein components of a mixture travel through a stationary phase at different speeds, resulting in separation from one another.
  • a variety of solid substrates e.g., beads, particles, microspheres, resins, etc.
  • silica silica, dextran polymers, agarose, polyacrylamide, etc.
  • a solid substrate has properties such that, in accordance with the present disclosure, permits a different retention time for RNA relative to any other undesirable components present in an RNA transcription mixture, including, e.g., short hydrolyzed DNA fragments, free nucleotide triphosphates (NTPs), 5’ capping agent, proteins, divalent ions complexed with a chelating agent.
  • NTPs free nucleotide triphosphates
  • 5’ capping agent proteins, divalent ions complexed with a chelating agent.
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • RNA is purified by HPLC using a column matrix of alkylated non- porous polystyrene-divinvylbenzene copolymer microspheres, e.g., in triethylammonium acetate (TEAA) buffers, e.g., as described in Kariko et al. “Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA” Nucleic Acids Res.
  • TEAA triethylammonium acetate
  • RNA content from desired fractions is concentrated and/or desalted (e.g., in some embodiments, using centrifugal filtration).
  • RNA is recovered by precipitation.
  • RNA is purified by HPLC using a diethylaminoethyl anion exchange column, e.g., as described in Anderson et al. “HPLC purification of RNA for crystallography and NMR” RNA. 1996;2(2): 110-117.
  • buffer comprising salt and sodium acetate is used for RNA elution.
  • RNA from RNA containing fractions is precipitated (e.g. , by ethanol precipitation) and dried to a powder.
  • a dried powder comprising RNA is re-suspended, for example, in water.
  • HPLC is not used to purify an in vitro transcription RNA composition.
  • precipitation is not used to purify an in vitro transcription RNA composition.
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by size exclusion chromatography (SEC).
  • RNA is purified by using a gel filtration matrix, e.g., as described in Lukavsky and Puglisi. “Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides” RNA. 2004;I0(5):889-893. doi:I0.126I/ma.5264804.
  • fractions are collected and/or analyzed by denaturing polyacrylamide gel electrophoresis.
  • RNA-containing fractions are combined.
  • RNA-containing fractions are concentrated, for example, using centrifugal filtration.
  • filtered RNA is washed twice with buffer (e.g., 10 mM sodium phosphate, pH 6.4).
  • buffer e.g. 10 mM sodium phosphate, pH 6.4.
  • RNA is concentrated a second time.
  • RNA concentration process e.g., centrifugal filtration
  • RNA is washed again (e.g., 1 additional wash, 2 additional washes, 3 additional washes, etc.) with buffer (e.g., 10 mM sodium phosphate, pH 6.4).
  • a final concentration step is conducted using centrifugal filtration.
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by ion-exchange chromatography (IEC).
  • RNA is purified by applying a transcription reaction mixture to a pre-equilibrated column and eluted using a linear salt gradient (e.g. , using sodium chloride), e.g., as described in Koubek et al. “Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription” RNA. 2013; 19(10): 1449-1459. doi:10.1261/ma.038117.113) .
  • fractions are collected.
  • RNA is purified by directly applying a transcription reaction mixture to a Sepharose column (e.g. , a diethylaminoethanol (DEAE) Sepharose column).
  • a Sepharose column e.g. , a diethylamino
  • an in vitro transcription RNA composition (e.g. , in some embodiments after DNA and/or protein removal and/or digestion) can be purified by membrane filtration.
  • Membrane filtration is a separation technique widely used in the life science separation/purification. Depending on membrane porosity, it can be classified as a microfiltration or ultrafiltration process.
  • Microfiltration membranes with pore sizes typically between 0.1 pm and 10 pm, are generally used for clarification, sterilization, and/or removal of microparticulates, while ultrafiltration membranes, with much smaller pore sizes between 0.001 and 0.1 pm, can be useful for removing, concentrating and/or desalting dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchanging buffers, and gross fractionation.
  • ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size.
  • MWCO molecular weight cutoff
  • filtration membranes can be of different suitable materials, including, e.g., polymeric, cellulose, ceramic, etc., depending upon the application. In some embodiments, membrane filtration may be more desirable for large-volume purification process.
  • DFF Direct Flow Filtration
  • TMF Tangential Flow Filtration
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA removal) can be purified by membrane filtration may be purified by a process comprising direct flow filtration.
  • an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by a process comprising tangential flow filtration (TFF).
  • a filtration membrane with an appropriate molecular weight cut-off (MWCO) may be selected for TFF. The MWCO of a TFF membrane determines which solutes can pass through the membrane (i.e. into the filtrate) and which are retained (i.e. in the retentate).
  • the MWCO of a TFF membrane used in accordance with the present disclosure is selected such that substantially all of the solutes of interest (e.g, desired synthesized RNA species) remains in the retentate, whereas undesired components (e.g. , excess ribonucleotides, small nucleic acid fragments such as digested or hydrolyzed DNA template, peptide fragments such as digested proteins and/or other impurities) pass into the filtrate.
  • the retentate comprising desired synthesized RNA species may be re-circulated to a feed reservoir to be re-filtered in additional cycles.
  • a TFF membrane may have a MWCO of at least 30 kDa (including, e.g., at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, or more). In some embodiments, a TFF membrane may have a MWCO of at least 100 kDa (including, e.g., at least 150 kDa, at least 200 kDa, at least 250 kDa, at least 300 kDa, at least 350 kDa, at least 400 kDa, or more).
  • a TFF membrane may have a MWCO of about 250-350 kDa.
  • a TFF membrane e.g., a cellulose-based membrane
  • a filtration membrane that is particularly useful for TFF purification in accordance with the present disclosure is or comprises a cellulose-based membrane.
  • a filtration membrane is not a thermoplastic membrane (e.g., polysulfone or polyethersulfone).
  • a filtration membrane is a filter cassette.
  • TFF is performed at a transmembrane pressure that is less than, for example, 2 bar (including, e.g., less than 2 bar, less than 1.9 bar, less than 1.8 bar, less than 1.7 bar, less than 1.6 bar, less than 1.5 bar, less than 1.4 bar, less than 1.3 bar, less than 1.2 bar, less than 1.1 bar, less than 1.0 bar, less than 0.9 bar, less than 0.8 bar, less than 0.7 bar, less than 0.6 bar, or lower). In some embodiments, TFF is performed at a transmembrane pressure in a range of about 0.5 bar to 2 bar. In some embodiments, TFF is performed at a transmembrane pressure of about 1 bar.
  • TFF is performed with a feed flow rate of less than, for example, 400 liters/m 2 /hour (LMH) (including, e.g., less than 400 LMH, less than 350 LMH, less than 300 LMH, less than 250 LMH, less than 200 LMH, less than 150 LMH, less than 100 LMH, or less). In some embodiments, TFF is performed with a feed flow rate of about 75 LMH to about 500 LMH, or about 50 LMH to about 400 LMH.
  • LMH 400 liters/m 2 /hour
  • an in vitro transcription RNA composition following RNA transcription that is subject to TFF purification has not be treated with a protein denaturing agent such as, e.g., urea, guanidinium chloride thiocyanate, salts of alkali metals (e.g. , potassium chloride), sodium dodecyl sulfate, sarcosyl, and combinations thereof.
  • a protein denaturing agent such as, e.g., urea, guanidinium chloride thiocyanate, salts of alkali metals (e.g. , potassium chloride), sodium dodecyl sulfate, sarcosyl, and combinations thereof.
  • a purification buffer may be fed into a TFF process in addition to an RNA preparation comprising an RNA transcription mixture.
  • the choice and composition of the purification buffer may influence the efficiency of RNA purification, levels of protein aggregation, RNA-protein separation, and/or RNA stability.
  • Typical buffers may include Tris buffer and citrate buffers.
  • a purification buffer that may be particularly useful for TFF purification in accordance with the present disclosure may be or comprise HEPES buffer.
  • a purification buffer may further comprise a chelating agent (e.g, as described herein) and/or a salt(s) (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate).
  • a chelating agent e.g, as described herein
  • a salt(s) e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate.
  • TFF purification may be performed without a buffer change.
  • TFF purification is performed in a buffer that has been utilized for in vitro transcription; in some such embodiments, TFF purification may be performed in a HEPES buffer.
  • a TFF purification process may comprise at least two separate steps of tangential flow filtration.
  • a first step of tangential flow filtration and a second step of tangential flow filtration may utilize different buffers.
  • a first buffer used in a first step of tangential flow filtration may comprise salt(s) (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate), while a second buffer used in a second step of tangential flow filtration may not comprise the same salt(s) as used in the first step (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate).
  • a second buffer used in a second step of tangential flow filtration may not comprise a salt.
  • a first step of tangential flow filtration may be performed with a defined number of volume exchanges (e.g., at least one, at least two, at least three, at least four at least five, at least six, at least seven, or more volume exchanges).
  • a second step of tangential flow filtration may be performed with a defined number of volume exchanges (e.g., at least one, at least two, at least three, at least four at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least or more volume exchanges).
  • a first step of tangential flow filtration may be performed with a minimum of 5 volume exchanges and a second step of tangential flow filtration may be performed with a minimum of 10 volume exchanges.
  • an in vitro transcription RNA composition described herein e.g, in some embodiments after DNA and/or protein removal and/or digestion
  • a suitable purification method known to one of ordinary skill in the art.
  • an in vitro transcription RNA composition described herein can be subjected to precipitation followed by membrane filtration (e.g., as described in WO2015164773).
  • an in vitro transcription RNA composition described herein can be subjected to one or more steps of TFF, wherein at least one or more steps of TFF comprises use of a TFF membrane cassette (e.g., as described in WO2016193206).
  • an in vitro transcription RNA composition described herein can be subjected to a high salt condition chromatography (e.g., by hydrophobic interaction chromatography).
  • an in vitro transcription RNA composition described herein can be a crude RNA reaction IVT mixture or high performance liquid chromatography purified RNA which is subsequently subjected to a high salt condition chromatography (e.g., as described in WO2018096179).
  • an in vitro transcription RNA composition described herein can be subjected to filtering centrifugation. In some embodiments, an RNA is precipitated prior to centrifugation (e.g., as described in WO2018157141). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to a stirred cell or agitated Nutsche filtration device. In some embodiments, a high concentration of salt is added to a RNA composition to denature and solubilize contaminating proteins prior to subjection to a stirred cell or agitated Nutsche filtration device (e.g., as described in WO2018157133).
  • an in vitro transcription RNA composition described herein can be subjected to standard flow filtration (e.g., a filtration process in which the material to be purified flows in a direction normal, i.e. perpendicular, to the surface of the filter).
  • RNA is precipitated prior to standard flow filtration (e.g., as described in W02020041793).
  • an in vitro transcription RNA composition described herein can be subjected to precipitation in a buffer comprising high concentration of salts (e.g., guanidinium salts) and a detergent (e.g, as described in W02020097509).
  • an in vitro transcription RNA composition described herein can be subjected to a protein digestion or fragmentation process prior to one or more additional purification methods known in the art (including, e.g., precipitation, affinity-based purification, ion exchange chromatography methods, high performance liquid chromatography, hydrophobic interaction chromatography, size exclusion-based methods such as size exclusion chromatography, filtration methods such as, e.g., centrifugal ultrafiltration and/or membrane filtration (e.g., direct flow filtration or tangential flow filtration), etc., or combinations thereof).
  • additional purification methods known in the art including, e.g., precipitation, affinity-based purification, ion exchange chromatography methods, high performance liquid chromatography, hydrophobic interaction chromatography, size exclusion-based methods such as size exclusion chromatography, filtration methods such as, e.g., centrifugal ultrafiltration and/or membrane filtration (e.g., direct flow filtration or tangential flow filtration), etc.
  • an exemplary protein digestion or fragmentation may comprise use of a proteinase (e.g., but not limited to proteinase K).
  • a proteinase e.g., but not limited to proteinase K
  • an in vitro transcription RNA composition described herein e.g., in some embodiments after DNA removal and/or digestion and/or removal of impurities
  • a method of removing or reducing bioburden e.g., microbial contamination
  • an exemplary method for bioburden removal or reduction may be or comprise filtration.
  • filtration may be or comprise gravity filtration.
  • gravity filtration may be performed using a filter with pore size that is small enough to capture bioburden (e.g., a filter with 0.45 pm pore size or smaller, a filter with 0.2 pm pore size or smaller).
  • filtration may be performed using a 0.45 pm pore filter.
  • filtration may be performed using a 0.2 pm pore filter.
  • filtration may be performed first using a 0.45 pm pore filter and subsequently using a 0.2 pm pore filter.
  • filtration may be performed first using a 0.2 pm pore filter and subsequently using a 0.45 pm pore filter.
  • an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA removal and/or digestion) can be subjected to at least one or more of purification methods described herein, including, e.g., bind-and elute process (e.g., utilizing solid substrate with high RNA affinity such as magnetic bead-based purification, membrane filtration (e.g., tangential flow filtration), and/or filtration (e.g., gravity filtration).
  • an in vitro transcription RNA composition described herein may be purified by magnetic-bead-based purification (e.g, as described herein) followed by bioburden filtration (e.g., as described herein), to produce an RNA transcript preparation.
  • an in vitro transcription RNA composition described herein may be purified by a TFF process that may comprise one or a plurality of (e.g., at least two) TFF steps (e.g., as described herein) followed by bioburden filtration (e.g. , as described herein), to produce an in vitro transcription RNA composition.
  • a TFF process may comprise one or a plurality of (e.g., at least two) TFF steps (e.g., as described herein) followed by bioburden filtration (e.g. , as described herein), to produce an in vitro transcription RNA composition.
  • purification methods described herein can be sufficient to remove or reduce residual host cell proteins by a factor of at least 100, 200, 250, 300, 350, 400, 450, 500, 550, or 600.
  • a starting RNA in vitro transcription mixture contains an amount of host cell proteins of approximately 400 ng/mg RNA
  • subsequent purification of the RNA by a reduction factor of 400 decreases this amount theoretically to 1 ng/mg RNA.
  • Residual host cell proteins e.g., residual bacterial host cell proteins such as E.
  • coli proteins may be present in an in vitro transcription RNA composition as impurity from a DNA template or as a recombinant protein expressed in host cells.
  • recombinant proteins may include recombinant enzymes added during in vitro transcription, including, e.g. , RNA polymerase, pyrophosphatase, DNases, and/or RNase inhibitors.
  • an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA removal and/or digestion) after one or more purification methods described herein can be maintained at 2-8°C for a period of time before further purification/processing.
  • the maintained period of time may be at least 6 hours or longer, including, e.g., at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or longer.
  • RNA concentration and/or integrity of an in vitro transcription RNA composition may be monitored during or after each purification method described herein.
  • RNA concentration and/or integrity of an in vitro transcription RNA composition may be assessed before or after maintaining at 2-8°C for a period of time (e.g., as described herein).
  • impurities such as Fe 2+ can be derived from magnetic beads.
  • residual Fe 2+ ions in an in vitro transcription RNA composition can be analyzed.
  • filter integrity after gravity filtration may be assessed. Filter integrity may be assessed, for example, for extractables and/or leachables.
  • weight of RNA produced after purification may be assessed.
  • an RNA transcript preparation (e.g., as described herein) may comprise RNA at a concentration of at least 1 mg/mL (including, e.g., at least 1.5 mg/mL, at least 2 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, at least 4 mg/mL, at least 4.5 mg/mL, at least 5 mg/mL, at least 6 mg/mL, or higher).
  • an RNA transcript preparation may comprise RNA at a concentration of 1.5 mg/mL to 5 mg/mL or 2 mg/mL to 4 mg/mL.
  • an RNA transcript preparation may comprise an aqueous buffer.
  • An exemplary aqueous buffer may comprise HEPES (e.g., at a concentration of 5 mM- 15 mM) at an RNA-compatible pH (e.g., pH 7.0).
  • an RNA transcript preparation may comprise a chelating agent, e.g. , EDTA.
  • an RNA transcript preparation (e.g., as described herein) may be characterized to determine one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more) product quality attributes of RNA drug substance.
  • product quality attributes include, but are not limited to appearance, RNA length, identity of drug substance as RNA, RNA integrity, RNA sequence, RNA concentration, pH, osmolality, residual DNA template, residual double stranded RNA, bacterial endotoxins, bioburden, degree of capping, presence and composition of poly(A)-tail, nucleotide composition, secondary and tertiary structure, residual salt contaminants, protein contamination, residual solvent contamination, residual bacterial DNA contamination, and combinations thereof.
  • a skilled artisan will understand that various methods known in the art can be used to characterize such product quality attributes, certain examples of which are described below with exemplary release and/or acceptance criteria.
  • an RNA transcript preparation (e.g., as described herein) that has been determined to meet a set of pre-determined acceptance criteria can be maintained for further steps of manufacturing, and/or formulation and/or distribution.
  • a qualified RNA transcript preparation can be dispensed in a bioprocessing bag (e.g., with a bag chamber volume of at least 5 L, including, e.g. , at least 6 L, at least 7 L, at least 8 L, at least 9 L, at least 10 L, at least 15 L, at least 20 L, at least 25 L, or more).
  • a RNA preparation can be dispensed in a bioprocessing processing polymer bag, e.g., comprising ethylene vinyl acetate copolymer, polyethylene copolymer.
  • predetermined specifications are not met (e.g., post-integrity filter testing, integrity of holding vessel post-sterile filtration) and refiltration may be utilized.
  • a RNA preparation may be refiltered (e.g., through a filter).
  • refiltration is performed in the same manner as the initial final filtration. In some embodiments, refiltration is performed in a different manner than the initial final filtration.
  • each RNA transcript preparation can be manufactured, filled, and stored as an independent batch, e.g. RNA from one production run forms one batch of composition comprising RNA. In some embodiments, each batch can be identified by a unique batch number.
  • an RNA transcript preparation can be transported from its manufacturing facility for further characterization and/or processing. In some embodiments, RNA preparation(s) are transported from its manufacturing facility in a container, for example, a bag, tube, vial, etc. In some embodiments, a RNA preparation may be transported in a bioprocessing polymer bag, e.g., comprising ethylene vinyl acetate copolymer.
  • a RNA preparation is held in a container with a volume of at least 4 L, at least 5 L, at least 10 L, at least 15 L, or larger.
  • a RNA preparation may be transported at a refrigerated or frozen temperature, e.g., at least less than or equal to 15°C, 10°C, 5°C, 0°C, -5°C, -10°C, - 15°C, -20°C or less for a period of time (e.g., up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 hours or more).
  • a freezing process may utilize controlled freeze equipment and/or temperature controlled freezers.
  • monitoring one or more of the parameters described herein may improve product output and/or provide increased reproducibility of composition comprising RNA between batches.
  • an exemplary process to produce an RNA transcript preparation involves a three step process comprising cell-free in vitro transcription from a DNA template, purification of in vitro transcription product, and concentration adjustment and filtration as outlined in Figure 4.
  • RNA quality control may be performed and/or monitored at any time during production process of RNAs and/or compositions comprising the same.
  • RNA quality control parameters including one or more of RNA identity (e.g., sequence, length, and/or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of an RNA manufacturing process, e.g., after in vitro transcription, and/or each purification step.
  • RNAs e.g., produced by in vitro transcription
  • compositions comprising RNAs can be assessed under various test storage conditions, for example, at room temperatures vs. refrigerated or sub-zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer).
  • RNAs e.g., ones described herein
  • compositions thereof may be stored stable at a fridge temperature (e.g., about 2°C to about 8°C, or in some embodiments about 2 °C to about 10°C, or about 4°C to about I0°C or about 2°C to about 8°C) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • a fridge temperature e.g., about 2°C to about 8°C, or in some embodiments about 2 °C to about 10°C, or about 4°C to about I0°C or about 2°C to about 8°C
  • a fridge temperature e.g., about 2°C to about 8°C, or in some embodiments about 2 °C to about 10°C, or about 4°C to about I0
  • RNAs e.g., ones described herein
  • compositions thereof may be stored stable at a sub-zero temperature (e.g., - 15 °C or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • a sub-zero temperature e.g., - 15 °C or below
  • RNAs e.g., ones described herein
  • compositions thereof may be stored stable at room temperature (about 18°C- 30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C) for at least 1 month or longer.
  • one or more assessments as described in Example 4 may be utilized during manufacture, or other preparation or use of RNAs (e.g., as a release test).
  • one or more quality control parameters may be assessed to determine whether linear DNA templates described herein meet or exceed acceptance criteria (e.g., for subsequent IVT).
  • quality control parameters may include, but are not limited to, DNA concentration, DNA identity, identity of transcribed region, identity of PolyA tail, plasmid topology, residual host cell RNA, residual host cell DNA, residual selection drug, appearance, coloration, pH, polyA tail integrity, linearization efficiency, residual protein, bioburden, and/or endotoxins.
  • Certain methods for assessing linear DNA template quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for DNA quality assessment. Examples of such analytical tests may include, but are not limited to, gel electrophoresis, sequencing, and/or UV absorption.
  • one or more quality control parameters may be assessed to determine whether RNAs described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution).
  • quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment.
  • Examples of such certain analytical tests may include but are not limited to gel electrophoresis, UV absorption, and/or PCR assay.
  • a batch of RNAs may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of RNAs can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if RNA quality assessment indicates that such a batch of RNAs meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of single stranded RNAs does not meet or exceed the acceptance criteria.
  • a batch of RNAs that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.
  • manufacturing methods described herein may further comprise monitoring one or more features of a RNA preparation including, e.g., appearance, identity (RNA length), identity (as RNA), RNA integrity, RNA sequence, content (RNA concentration), pH, osmolality, residual DNA template, residual double-stranded RNA (dsRNA), bacterial endotoxins, bioburden, degree of capping, presence and composition of poly(A)-tail, nucleotide composition, secondary and tertiary structure, residual salt contamination, protein contamination, bacterial DNA contamination and/or residual solvent contamination.
  • at least one or more features e.g.
  • At least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen) described herein can be characterized and/or monitored for quality control.
  • RNA substance appearance of RNA substance is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, visual inspection is utilized to monitor appearance.
  • an RNA substance is clear ( ⁇ 6 NTU, ⁇ 5 NTU, ⁇ 4 NTU, or ⁇ 3 NTU). In some embodiments, an RNA substance is a colorless liquid. In some embodiments, an RNA substance is a clear ( ⁇ 6 NTU) and colorless liquid.
  • RNA length is determined by denaturing agarose gel electrophoresis in comparison to a standard ladder with RNAs of known lengths. In some embodiments, sizes obtained must be consistent with theoretically expected lengths, e.g., transcripts from the respective DNA template used, and with reference RNAs.
  • the electrophoresis gel is a precast and buffered agarose gel prestained with a nucleic-acid specific dye.
  • RNA identity is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, RNA identity is determined by incubating an RNA sample for a defined period of time with an RNase, separating by gel-electrophoresis, and comparing to an RNA sample that has been incubated under identical conditions except for the addition of RNase. In some embodiments, disappearance of the RNA band upon incubation with RNase verifies the identity as RNA. In some embodiments, gel-electrophoresis is completed on a precast and pre-stained agarose gel. In some embodiments, the RNase is RNase A.
  • RNA identity is determined by reverse transcribing (RT) said RNA into cDNA and amplifying said cDNA (e.g., by PCR) using primers and/or a probe specific to the cDNA sequence.
  • RT-PCR is conducted in a single-step.
  • RNA integrity is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, RNA integrity can be assessed and/or monitored by agarose gel electrophoresis. In some embodiments, RNA integrity can be assessed and/or monitored by capillary gel electrophoresis. In some embodiments, RNA integrity can be quantitatively determined using capillary electrophoresis. In some embodiments, RNA solution must give rise to a single peak at the expected retention time consistent with the expected lengths as compared to the retention times of a standard ladder. In some embodiments, RNA integrity is above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, quantification of the main RNA peak is calculated in relation to signal intensities in the electropherogram where degradation products are detectable.
  • the sequence of RNA is assessed and/or monitored (e.g., determined at one or more points over time).
  • the RNA sequence can be deduced from sequencing the DNA template which serves as a template for in vitro transcription and defines the primary structure of each RNA.
  • identity of the starting material, and thus the identity of the transcribed RNA is controlled by automated sequencing of the RNA encoding region of the template.
  • RNA sequence is determined by reverse transcribing said RNA into cDNA, amplifying (e.g., by PCR), and sequencing the amplified product.
  • RT-PCR is conducted in a single-step.
  • the sequencing method is Sanger sequencing.
  • the sequencing method is next generation sequencing.
  • the sequence of an RNA has 100% identity to the corresponding DNA from which it was generated.
  • RNA sequence is determined by RNA sequencing using Next Generation Sequencing technology ⁇ e.g., Illumina MiSeq).
  • the sequence of RNA can be determined by liquid chromatography tandem mass spectrometry (LC/MS/MS)-oligonucIeotide mapping.
  • LC/MS/MS liquid chromatography tandem mass spectrometry
  • an RNA preparation is fragmented (e.g., by RNase) and separated (e.g., by liquid chromatography).
  • major and minor peaks in the oligonucleotide map can be identified, for example, by MS/MS. Observed masses and MS/MS fragmentation patterns of oligonucleotides in each peak can be mapped to expected RNA fragments.
  • oligonucleotide maps can be assigned via software using decoy sequences to confirm correct peak assignments.
  • protein size after expression of a RNA preparation or RNA preparation(s) are evaluated using Western blot. In some embodiments, expressed protein size is compared to that of a known protein standard.
  • RNA concentration is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, RNA concentration is determined using UV absorption spectrophotometry. In some embodiments, RNA concentration is determined according to the method described within Ph. Eur. 2.2.25. In some embodiments, a desirable RNA concentration can vary with the batch scale. For example, a high RNA concentration may be desirable for a large-scale manufacturing process.
  • an RNA concentration may be at least 1 mg/mL (including, e.g., at least 1.5 mg/mL, at least 2 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, at least 4 mg/mL, at least 4.5 mg/mL, at least 5 mg/mL, at least 6 mg/mL, or higher).
  • an RNA concentration may be 1.5 mg/mL to 5 mg/mL or 2 mg/mL to 4 mg/mL.
  • pH value of the RNA solution is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, the pH value is determined according to the method described within Ph. Eur. 2.2.3. In some embodiments, pH value of the RNA solution is 6-8.
  • osmolality of an RNA solution is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, osmolality of an RNA solution is determined according to the method described within Ph. Eur. 2.2.35. In some embodiments, osmolality of an RNA solution is less than 500 mOsmol/kg, 400 mOsmol/kg, 300 mOsmol/kg, 200 mOsmol/kg, 100 mOsmol/kg, or lower. In some embodiments, osmolality of an RNA solution may be less than 200 mOsmol/kg.
  • residual DNA template is assessed and/or monitored (e.g., determined at one or more points over time).
  • residual DNA template content is assessed and/or monitored (e.g., determined at one or more points over time) using, for example, PCR, absorbance, fluorescent dyes, and/or or gel electrophoresis.
  • residual DNA template content is determined using a real-time quantitative PCR (qPCR) test method.
  • qPCR is completed using a pre-mixed Sybr Green master mix according to the manufacturer’s recommendations.
  • the amplification and detection of DNA is performed in a real-time thermocycler.
  • residual DNA template in a sample is quantified in comparison to a standard or reference.
  • a standard is a serial dilution of pDNA.
  • results are reported in ng DNA/mg RNA.
  • the qPCR method comprises one or more of using a pre-mixed Sybr Green master mix according to the manufacturer’s recommendations, amplifying and detecting DNA in a real-time thermocycler, and quantifying residual DNA template in comparison to a standard (serial dilution of plasmid DNA).
  • residual dsRNA can be assessed and/or monitored (e.g., determined at one or more points over time).
  • residual dsRNA level is determined using a test limit. For example, RNA samples and a dsRNA reference (2000 pg dsRNA/pg RNA, 1500 pg dsRNA/pg RNA, 1000 pg dsRNA/pg RNA, 500 pg dsRNA/pg RNA, or lower) representing the upper limit of accepted residual dsRNA content) are immobilized on a positively charged nylon membrane and incubated with a dsRNA-specific monoclonal antibody.
  • a dsRNA reference 2000 pg dsRNA/pg RNA, 1500 pg dsRNA/pg RNA, 1000 pg dsRNA/pg RNA, 500 pg dsRNA/pg RNA, or lower
  • HRP horseradish peroxidase
  • ECL enhanced chemiluminescence
  • HRP-conjugated secondary is an anti-mouse IgG secondary.
  • bacterial endotoxins are assessed and/or monitored (e.g., determined at one or more points over time), for example, using an analytical kinetic turbidimetric limulus amebocyte lysate (LAL) procedure.
  • Gram-negative bacterial endotoxins are assessed and/or monitored (e.g., determined at one or more points over time).
  • Gram-negative bacterial endotoxins are determined to have an acceptable level if the acceptance criteria in regional pharmacopoeia (e.g., Ph. Eur. 2.6.14, USP ⁇ 85>, JP 4.01) are met when the level of Gram-negative bacterial endotoxins is determined according to the method described therein.
  • RNA solutions have ⁇ 12.5 EU/rnL (including, e.g., ⁇ 10 EU/mL, ⁇ 7.5 EU/rnL, or ⁇ 5.0 EU/mL) of bacterial endotoxins.
  • bioburden is assessed and/or monitored (e.g., determined at one or more points over time) using a membrane filtration method.
  • bioburden is determined to have an acceptable level if the acceptance criteria in regional pharmacopoeia (e.g., Ph. Eur. 2.6.12, USP ⁇ 61>, JP 4.05) are met when the bioburden is determined according to the method described therein.
  • bioburden of an RNA solution is ⁇ 1 CFU per 10 mL.
  • bioburden of an RNA solution is ⁇ 100 CFU per 10 mL when assessed prior to, or during, the process of removing impurities.
  • capping of in vitro transcribed RNA is assessed and/or monitored (e.g., determined at one or more points over time).
  • capping of in vitro transcribed RNA can be verified, for example by assessing translation (which typically requires presence of a functional cap).
  • a biological activity test for example that may be performed during process characterization of animal trial materials, is confirmatory that the RNA is translated into a protein of correct size.
  • nonclinical studies are performed to demonstrate capping of various different mRNA batches.
  • percentage of capped RNA can be assessed and/or monitored (e.g., determined at one or more points over time).
  • characterization of percentage of capped RNA is conducted by an RNase based assay.
  • characterization of percentage of capped RNA comprises one or more of the following steps: annealing RNA samples to a probe or probes binding close to the 5’ end of the RNA, digesting the RNA-probe complex with RNase generating a short fragment corresponding to the 5’ part of the RNA, purifying for sample clean-up, subjecting the purified samples with the 5’ part of the RNA to mass spectrometry (MS), capped and non-capped species are identified, and/or the percentage of capped RNA is calculated.
  • MS mass spectrometry
  • percentage of capped RNA is characterized by an RNase H based assay.
  • characterization of percentage of capped RNA comprises one or more of the following steps: annealing RNA samples to customized biotinylated nucleic acid probe binding close to the 5’ end of the RNA, digesting the RNA- probe complex with RNase H generating a short fragment corresponding to the 5’ part of the RNA, purifying the sample for sample clean-up with streptavidin-coated spin columns or magnetic beads, subjecting the purified samples with the 5’ part of the RNA to LC-MS, identifying capped and noncapped species by the observed mass values, and calculating the percentage of capped RNA using MS signals.
  • an RNase is RNase H.
  • a probe is a customizable biotinylated nucleic acid probe.
  • a purification step comprises use of streptavidin coated spin columns.
  • purification comprises use of magnetic beads.
  • the 5’ part of an RNA is subjected to LC-MS.
  • capped and/or noncapped species can be identified by the observed mass values.
  • MS signals are used to calculate percentage of capped RNA.
  • percentage of capped RNA can be assessed and/or monitored by cleaving RNA molecules with a catalytic nucleic acid molecule into a 5’ terminal RNA fragment and at least one 3’ RNA fragment, wherein RNA molecules have a cleavage site for a catalytic nucleic acid molecule, separating RNA fragments, determining a measure for the amount of capped and non-capped 5’ terminal RNA fragments (e.g., by a spectroscopic method, quantitative mass spectrometry, or sequencing), and comparing the measures of capped and non-capped 5’ terminal RNA fragments determined (e.g, as described in EP3090060).
  • percentage of capped RNA can be assessed and/or monitored by contacting a RNA preparation with a DNA oligonucleotide complementary to a sequence in the 5’ untranslated region of a RNA adjacent to the cap or the uncapped penultimate base of RNA under conditions that permit annealing of the DNA oligonucleotide to the sequence, providing one or more nucleases that selectively degrade DNA/RNA hybrid and/or unannealed RNA, resulting in capped and uncapped fragments, separating capped and uncapped fragments by chromatography, and determining relative amount of capped and uncapped fragments (e.g., as described in EP2971102).
  • percentage of polyadenylation (PolyA) attached to the 3’ end of an RNA construct is assessed and/or monitored (e.g., determined at one or more points over time).
  • measurement of percentage of polyadenylation attached to the 3’ end of the RNA construct uses PCR and comprises one or more of: generating cDNA using a reverse transcription and/or quantitating based on normalization to the theoretical input of the test sample.
  • measurement of the percentage of polyadenylation attached to the 3’ end of the RNA construct uses droplet digital PCR (ddPCR) and comprises one or more of the following steps: generating cDNA using a reverse transcription primer that spans the polyA and 3’ sequence of the RNA construct and requires both for binding, and/or quantitating based on normalization to the theoretical input of the test sample as measured by UV absorption at 260 nm.
  • polyadenylation is characterized by liquid chromatography-spectrometry (LC-MS).
  • LC-MS utilizes a particular detector (e.g., an ultraviolet detector).
  • a polyA tail of a RNA is cleaved off (e.g., by a ribonucleases) and isolated, for example, by affinity purification.
  • the higher order structure of an RNA preparation is assessed and/or monitored (e.g., determined at one or more points over time).
  • higher order structure is evaluated using circular dichroism (CD) spectroscopy.
  • a CD spectrum is a measure of differential absorption of the left- and right-circularly polarized light by the test article (e.g., RNA preparation).
  • the ordered structure of RNA yields a CD spectrum that may contain positive and/or negative signals, while absence of CD signal is indicative of a lack of ordered structure.
  • a CD spectrum for a RNA preparation exhibits an expected profile for an RNA molecule (e.g., contains both negative and positive signals between approximately 200 and 300 nm), indicating quality of folding.
  • RNA preparation comprises 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or is substantially free of salt contaminants.
  • protein contamination is assessed and/or monitored.
  • protein content in an RNA preparation is determined using one or more of UV absorbance at 280 nm (due to presence of aromatic amino acids), a Lowry assay, a Bradford assay and/or a Bichinonic Acid assay.
  • an RNA preparation comprises less than a predetermined threshold of protein contamination.
  • residual solvent contamination is assessed and/or monitored.
  • residual solvents are analyzed according to regional pharmacopeia (e.g., Ph. Eur. 2.2.28).
  • bacterial DNA contamination is assessed and/or monitored.
  • residual bacterial DNA may be detected by PCR or quantitative PCR using primers and/or probes specific for bacterial genomic sequences.
  • the present disclosure provides technologies for (large-scale) manufacturing a pharmaceutical-grade composition or preparation comprising LNPs, for example, at a mass batch throughput of at least 5 g (including, e.g., at least 10 g, at least 15 g, at least 20 g, at least 25 g, at least 30 g, at least 35 g, at least 40 g, at least 45 g, at least 50 g, at least 55 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, or more).
  • methods described herein are particularly useful for a mass batch throughput of at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, or more.
  • technologies provided by the present disclosure achieve production of LNP preparations (e.g. , pharmaceutical-grade LNP preparations, including large batch preparations), in particular including nucleic acids, e.g., RNA.
  • the present disclosure includes technologies for manufacturing a pharmaceutical-grade LNP that include, for example, (i) generating a preparation (e.g. , a stable, dispersion preparation) comprising LNPs at a mass batch throughput of about 5 g to 100 g; and (ii) processing the preparation (which in some embodiments may include, e.g., but not limited to purification, concentration adjustment, formulation for storage, aseptic filling, labelling, storage, or combinations thereof).
  • an LNP preparation (e.g., comprising an agent, such as a pharmaceutical agent, for delivery, and particularly comprising a nucleic acid agent such as an RNA agent) is manufactured by controlled mixing of a relevant agent (e.g., a nucleic acid agent, and particularly an RNA agent, often as an aqueous solution) and lipids (e.g., as described herein) in a solvent environment conducive to formation of agent-encapsulating-LNPs (e.g., as described herein).
  • a relevant agent e.g., a nucleic acid agent, and particularly an RNA agent, often as an aqueous solution
  • lipids e.g., as described herein
  • one or more in- process hold (e.g., storage) steps are conducted at 15-25°C unless otherwise specified.
  • Lipid preparations (e.g. for or as the second liquid mentioned further above)
  • lipid(s) to be included in lipid nanoparticles are selected based on at least one or more factors including, but not limited to, minimum encapsulation of RNA, apparent pKa, size, and/or polydispersity of resulting lipid nanoparticles.
  • lipids to be included in lipid nanoparticles comprise at least one helper lipid described herein, at least one cationic lipid described herein, and at least one PEG-conjugated lipid described herein.
  • lipids are selected to a lipid particle composition described herein.
  • frozen lipids are thawed using a temperature-controlled thawing unit.
  • frozen lipids are thawed at controlled room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • controlled room temperature e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C.
  • periodic monitoring is conducted throughout the duration of thawing.
  • a thawed lipid preparation is transferred to a manufacturing vessel.
  • one or more lipid components e.g., cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids
  • lipid components e.g., cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids
  • ethanol e.g., 100% ethanol
  • pre-determined molar ratio e.g., ones described herein
  • lipids are combined (e.g. , at relevant molar ratios) at a concentration above that at which they are combined with an agent to be encapsulated (e.g., a nucleic acid agent such as an RNA), and may be diluted prior to combination with such agent. In some embodiments, lipids are combined at an appropriate concentration for combination with a relevant agent without further dilution.
  • an agent to be encapsulated e.g., a nucleic acid agent such as an RNA
  • lipids are combined at an appropriate concentration for combination with a relevant agent without further dilution.
  • a lipid solution (e.g., a stock or final concentration solution) can be prepared either by directly weighing lipid components in target proportions (e.g., as described herein) to a single container and dissolving in an appropriate solvent, or by volumetrically combining high concentration (e.g., 10-40 mg/mL) solutions of individual lipid components to achieve the same target proportions (e.g., as described herein) and final total lipid concentrations.
  • a lipid solution for combination with an RNA solution may comprise at least one helper lipid described herein, at least one cationic lipid described herein, and at least one PEG-conjugated lipid described herein.
  • a lipid solution for combination with an aqueous solution can have a total lipid concentration of at least 10 mg/mL (including, e.g., at least 15 mg/mL, at least 20 mg/mL, at least 25 mg/mL, at least 30 mg/mL, at least 35 mg/mL, at least 40 mg/mL, or higher).
  • a lipid solution for combination with an aqueous solution can have a total lipid concentration of about 10-50 mg/mL, or about 10-40 mg/mL, or about 15 to 35 mg/mL/.
  • a solvent is selected such that it can support dissolution of all lipid components in a selected combination and has a minimal toxicity risk for any residual solvent remaining after completion of manufacturing.
  • a solvent can be or comprise ethanol.
  • a lipid solution is warmed, for example to improve or achieve lipid dissolution.
  • a lipid solution may be warmed for a period of time, for example within a range of minutes to hours; in some embodiments, such period may be within a range of 10 mins to 6 hours, 30 mins to 4 hours, or 1 to 3 hours.
  • a lipid solution may be warmed for 10 minutes to 2 hours, 1 to 3 hours, 2 to 4 hours, or 3 to 5 hours.
  • a lipid solution is warmed to and/or maintained at a temperature above approximately 25°C; in some embodiments, a lipid solution can be warmed to and/or maintained at a temperature of about 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, a lipid solution can be warmed to and/or maintained at a temperature of about 30-40°C, or about 33-37°C.
  • a lipid solution is subsequently allowed to cool, e.g., to a reduced temperature, e.g., at or near room temperature (e.g, about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • a lipid solution is purified (e.g., before, during, and/or after warming and/or cooling) by methods known in the art.
  • a prepared lipid solution can be purified by gravity filtration (e.g., filtration by passage through a filter with a pore size within a range of about 0.1 to 0.3 pm).
  • a prepared lipid solution can be filtered by passage through a filter with a pore size of about 0.2 pm or smaller.
  • a lipid solution (e.g., before and/or after purification) is stored and/or maintained at an appropriate temperature for a period time. In some embodiments, a lipid solution is stored and/or maintained at a temperature of about -25°C to about 40°C or about - 10°C to about 40°C or about 0°C to about 30°C, or about 10°C to about 25°C, or about 20°C to about 25°C.
  • a lipid solution is stored and/or maintained at room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • a monitor e.g., a sensor
  • a monitor may be utilized to maintain lipid temperature within a particular range (e.g., as described herein); in some such embodiments, a monitor may communicate automatically with a temperature controller, for example so that appropriate warming or cooling may be provided upon detection of a temperature that falls outside of the particular range.
  • a lipid solution (e.g., before and/or after purification) is stored and/or maintained at a selected temperature (e.g., as described herein) for a period of hours, days or weeks.
  • a selected temperature e.g., as described herein
  • such period of time may be at least 1 hour, at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks or longer.
  • such period of time may be within a range of 1 hours to 48 hours or 12 hours to 24 hours.
  • a lipid solution (e.g., before and/or after purification) is stored at room temperature (e.g., about I8°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C) for up to 24 hours.
  • room temperature e.g., about I8°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C
  • a lipid solution prepared for combination with an RNA transcript as described herein is referred to as a lipid stock solution.
  • Nucleic acid preparations (e.g. for or as the first liquid mentioned further above)
  • technologies provided herein are particularly useful for preparation and/or use of LNP preparations that encapsulate nucleic acids, though those skilled in the art will appreciate that various teachings are not limited thereto.
  • a preparation of a therapeutic nucleic acid (e.g., of an RNA such as an RNA transcript preparation or a dilution thereof) is combined with a lipid preparation described herein (e.g., a lipid stock solution) to provide a nucleic acid-LNP preparation.
  • LNP manufacture begins with a frozen nucleic acid preparation.
  • such a frozen preparation is thawed, for example using a temperature-controlled thawing unit.
  • a frozen preparation is thawed at controlled room temperature (e.g, about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C- 25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • controlled room temperature e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C- 25°C, or about 20-30°C, or about 23-27°C or about 25°C.
  • periodic monitoring is conducted throughout the duration of thawing.
  • a thawed preparation is transferred to a manufacturing vessel.
  • a nucleic acid preparation may comprise nucleic acid (e.g, RNA) in an aqueous buffer at an appropriate pH (e.g, pH 2 to pH 8, or pH 4 to pH 7).
  • aqueous buffer may include, but are not limited to Tris buffers, phosphate buffers (e.g., PBS), HEPES, citrate buffers, acetate buffers, etc., or combinations thereof.
  • a nucleic acid preparation for combination with a lipid preparation can be prepared by weighing a desired amount of nucleic acid (e.g. , by volume or weight if liquid or weight if solid or powder).
  • relevant nucleic acid can be dissolved or diluted in an appropriate aqueous buffer (e.g, as described herein).
  • an aqueous buffer can be or comprise an acidic buffer, e.g, a buffer below pH 7 (e.g., pH 2-pH 6), such as, e.g., in some embodiments, a buffer at pH 4.
  • an aqueous buffer can be or comprise an acidic buffer at a concentration of 10-100 mM, or 25-75 mM, or 30-60 mM, or 40-60 mM. In some embodiments, an aqueous buffer can be or comprise an acidic buffer at a concentration of 40-60 mM. In some embodiments, an acidic buffer can be or comprise a citrate buffer at pH 4.
  • a nucleic acidpreparation for combination with a lipid preparation is prepared by diluting with an aqueous buffer described above at an appropriate pH (e.g, pH 2 to pH 8, or pH 4 to pH 7).
  • an aqueous buffer is or comprise a citrate buffer at pH 4.
  • such an aqueous buffer is or comprises a citrate buffer at a concentration of 10-100 mM, or 25-75 mM, or 30-60 mM, or 40-60 mM, or 30-50 mM; in particular embodiments, such an aqueous buffer is or comprises a citrate buffer at a concentration of 30-60 mM or 30-50 mM.
  • mixing speed is controlled during dilution of RNA preparation.
  • mixing speed is, for example, at least 10 rpm, 25 rpm, 50 rpm, 75 rpm, 100 rpm, 125 rpm, 150 rpm, 175 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm or more.
  • RNA is diluted to a particular concentration prior to mixing (e.g., 0.1 mg/mL-1 mg/mL).
  • preparation of a nucleic acid preparation for combination with a lipid preparation can be performed at about 2- 25°C.
  • preparation of a nucleic acid preparation for combination with a lipid preparation can be performed at room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • preparation of a nucleic acid preparation for combination with a lipid preparation can be performed at a temperature that is below room temperature, including, e.g., at a temperature of about 2-8°C.
  • a nucleic acid preparation has been stored prior to combination with a lipid preparation. In some embodiments, a nucleic acid preparation has been stored and/or maintained as a frozen preparation. In some embodiments, a nucleic acid preparation has been stored and/or maintained as a liquid preparation. For example, in some embodiments, a nucleic acid preparation has been stored and/or maintained at an appropriate temperature for a period time. In some embodiments, a nucleic acid preparation has been stored and/or maintained at zero or subzero temperatures (e.g., a temperature of about -80°C to 0 °C, or about -80°C to -60°C, or about -80°C to -25°C).
  • zero or subzero temperatures e.g., a temperature of about -80°C to 0 °C, or about -80°C to -60°C, or about -80°C to -25°C.
  • a nucleic acid preparation has been stored and/or maintained at a fridge temperature (e.g., above 0°C, or about 2- 10°C, about 2-8°C or about 4-6°C). In some embodiments, a nucleic acid preparation has been stored and/or maintained at a temperature of about 10 °C to 25°C. In some embodiments, an a nucleic acid preparation has been stored and/or maintained at room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • a monitor e.g., a sensor
  • a monitor may be utilized to maintain temperature within a particular range (e.g., as described herein); in some such embodiments, a monitor may communicate automatically with a temperature controller, for example so that appropriate cooling may be provided upon detection of a temperature that falls outside of the particular range.
  • a nucleic acid has been stored and/or maintained at a selected temperature (e.g., as described herein) for a period of time, which may vary from hours to days to weeks to months, depending on the selected temperature.
  • a frozen nucleic acid preparation may be stored and/or maintained (e.g., at zero or subzero temperatures) for days to weeks to months or longer, while a liquid nucleic acid preparation may be stored and/or maintained (e.g., at 4°C or above, including room temperature) for a shorter period of time.
  • a frozen nucleic acid preparation may be stored (e.g., at zero or subzero temperatures) for at least 2 weeks, including, e.g., at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or longer.
  • a liquid nucleic acid preparation may be stored and/or maintained (e.g., at 4°C or above, including room temperature) for at least 30 mins, including, e.g., at least 60 mins, at least 90 mins, at least 120 mins, at least 150 mins, at least 180 mins, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, or longer.
  • a liquid nucleic acid preparation may be stored and/or maintained at 4-6°C for a period of 30 mins to 3 hours.
  • a liquid nucleic acid preparation may be stored and/or maintained at room temperature for a period of 30 mins to 3 hours.
  • a charge-based interaction between the phosphate backbone of the nucleic acid and the amine moiety of a cationic lipid can facilitate efficient encapsulation of nucleic acid payload upon mixing.
  • columbic interaction may be achieved and/or supported, for example, by controlling pH of a mixing solution (e.g., a solution comprising a nucleic acid, such as an RNA, as described herein and lipid components) for example with an appropriate buffer, within a range or otherwise at a pH that maintains ionization of both a nucleic acid backbone and a cationic lipid.
  • a mixing solution e.g., a solution comprising a nucleic acid, such as an RNA, as described herein and lipid components
  • a desired pH may be between the pKa of a nucleic acid backbone (which is approximately 2 in some embodiments) and the pKa of a selected cation lipid (e.g. , with a pKa of approximately 6 in some embodiments can be found at pH 4).
  • lipid components may be prepared in an organic solvent
  • nucleic acid may be prepared in a buffer (e.g., an aqueous buffer, such as for example a citrate buffer) at an appropriate pH (e.g., at a pH between about 2 and about 6, for example at a pH of about 4.0).
  • a buffer e.g., an aqueous buffer, such as for example a citrate buffer
  • an appropriate pH e.g., at a pH between about 2 and about 6, for example at a pH of about 4.0.
  • an aqueous buffer e.g., such as may be used to dissolve nucleic acid may have a buffer strength of at least 10 mM, including, e.g., at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, or higher, provided that excess buffering capacity does not significantly impact the size and/or polydispersity of resulting nucleic acid-lipid nanoparticles after mixing and/or encapsulation efficiency.
  • an aqueous buffer that may be useful to prepare a nucleic acid stock solution may have a buffer strength of 10 mM-50 mM.
  • nucleic acid is maintained at acidic pH for only a minimal time prior to combination with a lipid preparation as described herein.
  • a nucleic acid solution is prepared at an pH between about 2 and about 6, for example at a pH of about 4.0, (e.g., in a citrate buffer), and is promptly combined with a lipid preparation (e.g., in an organic solvent).
  • a nucleic acid solution is combined within a time period of not more than several hours of exposure to such acidic pH.
  • such time period is not more than 4, 3, 2, or 1 hour(s); in some embodiments, such time period is less than 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute(s). In some embodiments, such time period is less than about 5 hours, preferably less than about 4 hours or about 3 hours. In some embodiments, such time period is as short as is reasonably feasible.
  • concentrations of stock solutions for mixing can be determined based on a target ratio of cationic lipid to nucleic acid (e.g., RNA), a target ratio of organic to aqueous component in a mixing output, and/or a target output lipid concentration.
  • the lipid concentration is controlled in terms of the mole ratio of cationic lipid (N) to nucleotide groups (P) in the nucleic acid RNA (e.g., RNA); with the other lipid components calculated according the target molar lipid proportions (e.g., as described herein) relative to the cationic lipid.
  • a target ratio of cationic lipid to nucleic acid can be represented by an N/P ratio where N represents an ionized or ionizable amine in a cationic lipid and P represents a phosphate associated with each nucleotide in a nucleic acid (e.g., RNA).
  • N an ionized or ionizable amine in a cationic lipid
  • P a phosphate associated with each nucleotide in a nucleic acid (e.g., RNA).
  • efficient encapsulation can be achieved when there is sufficient cationic lipid (N) to interact with the entire phosphodiester backbone (P) and/or there is a molar excess of cationic lipid relative to the nucleotides.
  • such a target N/P ratio can be selected by determining effects of various N/P ratios on size and/or polydispersity of resulting LNP preparations and/or encapsulation efficiency (EE).
  • a target N/P ratio is selected that such that size of LNPs is less than 80 nm, polydispersity of LNPs is less than or equal to 0.3, and encapsulation is at least 80%.
  • a target N/P ratio may be in a range of approximately 3 to 35, approximately 3 to 30, approximately 4 to 25, approximately 4 to 20, approximately 4 to 15, approximately 3 to 10.
  • a target N/P ratio may be approximately 4 to 7.
  • a nucleic acid preparation for combination with a lipid preparation can comprise nucleic acid described herein at a concentration of 0.1-0.6 mg/mL, 0.1 -0.5 mg/mL, 0.2-0.4 mg/mL, or 0.3-0.5 mg/mL.
  • a lipid preparation for combination with a nucleic acid preparation can comprise lipids at a total concentration of about 10-50 mg/mL, or about 10-40 mg/mL, or about 15 to 35 mg/mL.
  • a nucleic acid (e.g., RNA) preparation for combination with a lipid preparation can comprise nucleic acid (e.g., RNA) described herein at a concentration of 0.1-0.6 mg/mL, and the lipid preparation can comprise lipids at a total concentration of about 10-40 mg/mL.
  • LNP preparations can be produced by rapid mixing of an aqueous solution described herein (e.g., comprising a nucleic acid, e.g., an RNA and/or in an acidic buffer) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in form of LNPs.
  • an aqueous solution described herein e.g., comprising a nucleic acid, e.g., an RNA and/or in an acidic buffer
  • a lipid preparation described herein comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent
  • properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g, RNA).
  • a nucleic acid e.g, RNA
  • particles are formed around the nucleic acid, which, for example in some embodiments can result in much higher encapsulation efficiency (EE) than it is achieved in the absence of interactions between nucleic acids and at least one of the lipid components.
  • nucleic acid preparation for producing an LNP preparation encapsulating the nucleic acid can be combined with an acidic buffer.
  • An exemplary such buffer is or comprises a citrate buffer.
  • flow rate ratio of an acidic buffer (e.g., a citrate buffer) to transcript nucleic acid preparation (e.g. , an RNA preparation) can be 2:1, 3:1, 4:1, or 5:1.
  • a ratio of organic to aqueous component can be controlled such that LNPs effectively form as a precipitation of lipid components upon rapid change of the solubility characteristics of the lipids in a solution when the aqueous component is introduced.
  • proportion of organic component in a combined solution is sufficiently low to induce precipitation with kinetics that are fast enough to support nanoscale particles.
  • a combining volumetric ratio of a lipid preparation and a nucleic acid (e.g., RNA) preparation is about 1:5, 1:4, 1:3, 1 :2, or 1 : 1.
  • a combining volumetric ratio of a lipid preparation and a nucleic acid (e.g., RNA) preparation is about 1:3.
  • the final concentration of organic solvent in a combined solution may be approximately 25% (v/v).
  • a lipid preparation described herein and a nucleic acid (e.g., RNA) preparation described herein can be introduced into a mixing unit or assembly.
  • a mixing unit or assembly may comprise one or more fluidic components or devices.
  • a mixing unit or assembly may comprise one or more components/parts of a high- performance liquid chromatography (HPLC) and/or other fluidic devices.
  • HPLC high- performance liquid chromatography
  • a mixing unit or assembly may comprise a T mixer comprising an inner diameter suitable for selected flow rate.
  • mixing dynamics for example, are controlled by orifices at the outlet of each stream and by the internal diameters of the tubing.
  • lipid and aqueous (e.g., nucleic acid, e.g, RNA) preparations can be mixed at room temperature by pumping each solution independently at controlled flow rates into a mixing unit or assembly, for example, using piston pumps.
  • the volumetric flow rates of a lipid preparation and an aqueous (e.g., nucleic acid, e.g., RNA) preparation into a mixing unit or assembly are maintained at a ratio of 1 :5, 1:4, 1:3, 1 :2, or 1 : 1.
  • volumetric flow rates of a lipid preparation and an aqueous (e.g., nucleic acid, e.g., RNA) preparation into a mixing unit or assembly are maintained at a ratio of 1 :3.
  • volumetric flow rate of a combined preparation is 100-800, 200-800, 200-700, 200-600, 200-500, 100-600, 100-500, or 150-500 mL/min, which in some embodiments may be particularly useful for large-scale production.
  • volumetric flow rate of a combined preparation is 300- 600 mL/min.
  • an aqueous (e.g., nucleic acid, e.g., RNA) preparation is introduced into a mixing unit or assembly such that a mass flow rate is 10-200, 20-180, 20-160, 20-150, 30-160, 40-160, 50-200, 70-200, or 100-200 mg/min.
  • an aqueous (e.g., nucleic acid, e.g., RNA) preparation is introduced into a mixing unit or assembly such that a mass flow rate is 100-300 mg/min.
  • volumetric flow rate of a lipid preparation is 15-50, 25- 75, 50-100, 75-125, 100-150, 100-200, 50-200, or 15-200 mL/min. In some embodiments, volumetric flow rate of a lipid preparation (e.g., in an organic phases) is about 80-160 mL/min.
  • volumetric flow rate of an aqueous (e.g., nucleic acid, e.g., RNA) preparation (e.g., in an aqueous phase) is 15-500, 30-500, 30-400, 50-500, 40-500, 100-400, 100-500, or 200-500 mL/min, which in some embodiments may be particularly useful for large-scale production.
  • volumetric flow rate of an aqueous (e.g., nucleic acid, e.g., RNA) preparation is 240-480 mL/min.
  • a flow rate of 360:120 mL/min (total 480 mL/min) is utilized.
  • a flow rate (aqueous preparation: lipid preparation) of 360: 120 mL/min (total 480 mL/min) is utilized.
  • an aqueous (e.g., nucleic acid, e.g., RNA) preparation and/or a lipid preparation may be introduced into a mixing unit or assembly (e.g., as described herein) at room temperature.
  • an aqueous (e.g., nucleic acid, e.g., RNA) preparation and/or a lipid preparation may be introduced into a mixing unit or assembly (e.g., as described herein) at a temperature of about 13-28°C, or 15-26°C, or 15-25°C, or 16-26°C.
  • homogenous LNP formation with appropriate sizes may require fast and efficient mixing of aqueous and organic components.
  • the present disclosure recognizes that at lower flow rates particles have larger sizes and higher polydispersity characteristics with variable encapsulation efficiency (EE).
  • flow rates of organic and aqueous components are controlled independently by two pumps.
  • the two pumps are two separate pumps.
  • the pump speeds are related to each other.
  • the pump speeds are related to each other, for example, by the target final organic component concentration (e.g., described herein) and by the same ratio as the stock solution volumes (e.g., described herein) to continuously provide the same dilution at the mixing interface throughout the mixing process.
  • total output flow rates (combining volumetric flow rates of aqueous and lipid preparations) of 10 to 30, 15-35, 18-40, 25-45, or 30-50 mL/minute are utilized.
  • total output flow rates (combining volumetric flow rates of aqueous and lipid preparations) of 65-700, 130-550, 130-400, 130-275, 250-400, 200-700, 260-700, or 260-550 mL/min are utilized, which in some embodiments may be particularly useful for large-scale production.
  • nucleic acid (e.g., RNA) concentration post-mixing is 0.05-0.5, 0.1-0.4, 0.1-0.35, or 0.15-0.30 mg/mL.
  • lipid concentration post-mixing is 2-10, 2-8, or 3-9 mg/mL.
  • the concentration of organic solvent in a combined solution may be approximately 25% (v/v).
  • a preparation comprising nucleic acid (e.g., RNA)-LNPs is diluted with an aqueous buffer at an appropriate pH (e.g. , pH 2 to pH 6, or pH 4 to pH 6), for example, to decrease the concentration of organic solvent present in the lipid preparation and/or to maintain physiochemical stability of LNPs.
  • aqueous buffer may include, but are not limited to, citrate buffers, acetate buffers, etc., or combinations thereof.
  • an aqueous buffer for dilution of such a preparation can be or comprise an acidic buffer, e.g. , a buffer below pH 7 (e.g. , pH 2-pH 6), such as, e. g. , in some embodiments, a buffer at pH 4.
  • an aqueous buffer can be or comprise an acidic buffer at a concentration of 10-100 mM, or 25-75 mM, or 30-60 mM, or 40-60 mM, or 10-50 mM.
  • such an aqueous buffer can be or comprise an acidic buffer at a concentration of 40-60 mM; in some embodiments, such an aqueous buffer can be or comprise an acidic buffer (e.g., a citrate buffer) at a concentration of 50 mM. In some embodiments, an acidic buffer can be or comprise a citrate buffer at pH 4.
  • dilution of an LNP preparation with an aqueous buffer described herein can be performed in-line immediately after LNP formation in a mixing unit or assembly, e.g., as a continuous process.
  • flow rate ratio of a preparation comprising LNPs to an acidic buffer can be 1:1 or 3:2 or 2:1.
  • dilution of a preparation comprising LNPs with an aqueous buffer can be performed at room temperature (e.g., about 18°C-30°C, e.g., about 18°C- 25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • an LNP preparation contains approximately 15-20% ethanol.
  • flow rate of such an aqueous buffer described herein can be 50-300, 60-275, 70- 250, 80-240, or 150-400 mL/minute.
  • nucleic acid concentration of an LNP preparation after such dilution can be 0.01-1, 0.05-0.5, 0.075-0.03, 0.05-0.25, or 0.09-0.21 mg/mL.
  • lipid concentration of an LNP preparation after such dilution can be 1-10, 1-7, 2-6, or 2.5-5.5 mg/mL.
  • the concentration of organic solvent, if present can be further reduced to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24% after dilution.
  • an LNP suspension is collected in a vessel which is cooled to approximately 2- 8°C.
  • an LNP preparation may be collected at a higher temperature, e.g. , at a temperature of above 8°C, including, e.g., 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, or higher.
  • an LNP preparation may be collected at room temperature (e.g., as described herein).
  • an LNP preparation may be collected at a temperature of 2-28°C, 2-25°C, 5-28°C, 10-28°C, 15-28°C, or 16-26°C.
  • in process-controls and/or monitoring of one or more of preparation of lipid and/or nucleic acid preparations and LNP formation can be conducted.
  • filter integrity after lipid stock filtration and/or dilution of a nucleic acid preparation with an acidic buffer can be assessed.
  • LNP size and/or polydispersity, lipid and/or nucleic acid (e.g., RNA) content (e.g., concentration), and/or encapsulation can be assessed and/or monitored during and/or after LNP formation.
  • an LNP preparation (e.g., a dispersion preparation) described herein can be processed by one or more of buffer exchange, concentration adjustment, purification, formulation for storage, aseptic filling, labelling, storage, or combinations thereof.
  • an LNP preparation e.g., a dispersion preparation
  • an LNP preparation in some embodiments after dilution with an acidic buffer described herein, can be subjected to one or more steps of ultrafiltration and/or diafiltration processes, or combinations thereof.
  • an LNP preparation e.g., a dispersion preparation
  • an acidic buffer described herein can be subjected to at least two, three, four, five, six, seven, or eight steps of ultrafiltration and/or diafiltration processes, or combinations thereof.
  • 3-5 steps of ultrafiltration and/or diafiltration processes, or combinations thereof can be performed.
  • an LNP preparation e.g., a dispersion preparation
  • Ultrafiltration is a membrane filtration process during which external forces, e.g., pressure or concentration gradients lead to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate. Ultrafiltration membranes typically have pore sizes between 0.001 and 0.1 pm and/or MWCO between 10-300 kDa, and can be applied in cross-flow or dead-end mode.
  • Diafiltration can be performed either discontinuously or alternatively, continuously.
  • a diafiltration solution can be added to a sample feed reservoir at the same rate as filtrate is generated.
  • small molecules e.g. salts, solvents, etc.,
  • each additional diafiltration volume reduces the solvent concentration further.
  • a solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of small molecules (e.g. salts, solvents, etc.) remaining in the reservoir is reached.
  • Each additional diafiltration volume (DV) reduces the small molecule (e.g., solvent) concentration further.
  • Continuous diafiltration typically requires a minimum volume for a given reduction of molecules to be filtered.
  • Discontinuous diafiltration permits fast changes of the retentate condition, such as pH, salt content and the like.
  • an LNP preparation is subjected to a diafiltration process.
  • a diafiltration process with a defined number of volume exchanges (e.g. , at least one, at least two, at least three, or more volume exchanges) using an aqueous buffer described herein (e.g., a dilution buffer described herein used in dilution of an LNP preparation such as, e.g., an acidic buffer at pH 4) is performed.
  • a diafiltration process comprises multiple volume exchanges (e.g., 1-10 volume exchanges, 3-10 volume exchanges, 5-9 volume exchanges, 6-10 volume exchanges) to perform buffer exchange, e.g. , in some embodiments replacing the supernatant in a first aqueous buffer with a different aqueous buffer described herein (e.g., a formulation buffer).
  • a formulation may have a pH 6-8 (e.g., pH 7.4).
  • a formulation buffer may comprise one or more salts (e.g., sodium salts, potassium salts, phosphate salts, etc.).
  • a formulation buffer may be or comprise phosphate ions.
  • multivalent anions e.g., from buffer components such as, e.g., citrate, and/or chelating agents (e.g., EDTA) added during processing
  • buffer components such as, e.g., citrate, and/or chelating agents (e.g., EDTA) added during processing
  • chelating agents e.g., EDTA
  • displacement of such multivalent anions by phosphate may further reduce the amount of bound ions in a bulk LNP product.
  • a formulation buffer may be or comprise PBS.
  • a first combination of diafiltration and ultrafiltration is employed using a first buffer, wherein said first buffer is identical to the LNP preparation, so that this first combination of diafiltration and ultrafiltration is removing the solvent and concentrating the LNP preparation and this first combination is followed by a second combination of diafiltration and ultrafiltration steps using a second buffer so that the buffer type and pH are adjusted.
  • the first ultrafiltration is concentrating the LNP preparation to a concentration of between 0.1 and 2mg/mL, preferably between 0.2 and Img/mL and/or the second diafiltration is using phosphate buffer in an amount to change the pH of the LNP preparation to pH7.0 or higher and the second ultrafiltration is concentrating the LNP preparation to a concentration of between 0.5 and 4mg/mL, preferably between 0.66 and 2mg/mL.
  • the required amount of phosphate buffer of the second diafiltration depends on the buffering capacity of the same.
  • the second buffer may needto overcompensate (i) the buffering capacity of the cationic lipid making up the LNP phase and/or (ii) a certain portion of the buffering capacity of the first buffer.
  • the second buffer may needto overcompensate (i) the buffering capacity of the cationic lipid making up the LNP phase and/or (ii) a certain portion of the buffering capacity of the first buffer.
  • between 5 and 15 volumes of a phosphate buffer having a strength of about lOmM phosphate are used for the second diafiltration and the second ultrafiltration is not started before the pH of the LNP preparation is 6.5 or higher.
  • an LNP preparation can be subjected to an ultrafiltration process.
  • an ultrafiltration process can be performed after a diafiltration, e.g., for concentration.
  • an LNP preparation can be concentrated by ultrafiltration by a factor of at least 2 (including, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more).
  • an LNP preparation can be concentrated by ultrafiltration by a factor within a range of about 2 to about 6-fold.
  • an LNP preparation can be subject to a process comprising at least two cycles (including, e.g., at least three, at least four, or more) of diafiltration followed by ultrafiltration.
  • Each cycle can comprise diafiltration using a different diafiltration volume and/or buffer and ultrafiltration with a different concentration factor.
  • the concentration of an LNP preparation following a process comprising diafiltration and/or ultrafiltration can be in the range of 0.1-1, 0.2-0.8, or 0.4-0.6 mg/mL. In some embodiments, such concentration can be 0.5 mg RNA-LNPs/mL.
  • diafiltration and/or ultrafiltration can be performed at room temperature (e.g., about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C).
  • diafiltration and/or ultrafiltration described herein can be performed in a tangential flow filtration (TFF) set-up.
  • a TFF system comprises hollow fiber membranes, which can be polymeric, ceramic, or cellulose.
  • a TFF system used for diafiltration and/or ultrafiltration comprises hollow fiber polymeric membranes, e.g., thermoplastic membranes (e.g., polysulfone or polyethersulfone). In other embodiments, planar membranes are used. In some embodiments, membranes can be stacked. TFF membranes can be several square meters in size depending on the actual scale of the preparation and in some embodiments, a TFF membrane has a filter area requirement of below Im 2 per gram nucleic acid (e.g., RNA). In some embodiments, a TFF membrane has a filter area requirement of between 0.1 and 0.8 m 2 /g or between 0.25 and 0.5m 2 /g.
  • hollow fiber polymeric membranes e.g., thermoplastic membranes (e.g., polysulfone or polyethersulfone).
  • planar membranes are used.
  • membranes can be stacked. TFF membranes can be several square meters in size depending on the actual scale of the preparation and in some embodiments,
  • transmembrane pressure is less than, for example, 500 mbar (including, e.g., less than 450 mbar, less than 400 mbar, less than 350 mbar, less than 300 mbar, less than 250 mbar, less than 200 mbar, less than 150 mbar, less than 100 mbar, less than 50 mbar, or less).
  • shear rate is less than 10,000/s, less than 9,000/s, less than 8,000 per second, less than 7,000 per second, less than 6,000/s, less than 5,000/s, less than 4,000/s, less than 3,000/s, less than 2,000/s, less than 1,000/s or lower.
  • an LNP preparatoin e.g., after ultrafiltration/diafiltration described herein
  • gravity filtration e.g., filtration by passage through a filter with a pore size within a range of about 0.1 to 0.3 pm
  • an LNP preparation e.g., after ultrafiltration/diafiltration described herein
  • filtration is completed over a period of less than 5 hours (including, e.g., less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour).
  • filtration is conducted at a particular pressure. In some embodiments, filtration pressure is less than 30 psig, less than 25 psig, less than 20 psig, less than 15 psig, less than 10 psig, or less than 5 psig.
  • a purified LNP preparation can be adjusted with a formulation buffer to a desired concentration (e.g., nucleic acid concentration).
  • a formulation buffer may have a pH 6-8 (e.g., pH 7).
  • a formulation buffer may comprise one or more salts (e.g, sodium salts, potassium salts, phosphate salts, etc.).
  • a formulation buffer may be or comprise PBS.
  • a formulation buffer may comprise a cryoprotectant.
  • a cryoprotectant may be present in a formulation buffer at a concentration of about 100-500 mM, or 200-400 mM, or 250-350 rnM.
  • cryoprotectants include a sugar (e.g., sucrose, trehalose), glycerin, ethylene glycol, or combinations thereof.
  • a cryoprotectant included in a formulation buffer includes a sugar (e.g., sucrose, trehalose, etc.). In some embodiments, cryoprotected is added with mixing.
  • mixing occurs for a particular duration of time, for example, at least 10 minutes (including e.g., at least 15 minutes, at least 20 minutes at least 25 minutes, at least 30 minutes, or more). In some embodiments, mixing occurs at a particular speed or range of speeds, for example, at least 10 rpm, at least 25 rpm, at least 50 rpm, at least 75 rpm, at least 100 rpm, at least 125 rpm, at least 150 rpm, at least 175 rpm, at least 200 rpm, at least 250 rpm, at least 300 rpm, at least 350 rpm, at least 400 rpm, at least 450 rpm, at least 500 rpm or more).
  • a purified LNP preparation can be adjusted with a formulation buffer described herein such that nucelic acid (e.g., RNA) concentration is 0.1-1 mg/mL, or 0.2-0.8 mg/mL, or 0.3-0.7 mg/mL, or 0.4-0.6 mg/mL.
  • nucelic acid e.g., RNA
  • a bulk LNP product may be stored and/or maintained at an appropriate temperature for a period of time before transportation and/or aseptic filling.
  • a bulk LNP product described herein may be stored and/or maintained as a frozen or liquid composition.
  • a bulk LNP product described herein may be stored as a frozen composition at a subzero temperature, e.g., -10°C or lower, including, e.g., -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, - 80°C or -90°C.
  • a frozen composition comprising a bulk LNP product may be stored for at least 2 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or longer.
  • a bulk LNP product described herein may be stored as a liquid composition at a refrigerated temperature, e.g., 2-10°C or 2-8°C.
  • a liquid composition comprising a bulk LNP product may be stored for at least 5 days, at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or longer.
  • a liquid composition comprising a bulk LNP product may be stored for about 1 week.
  • a bulk LNP product described herein may be stored as a liquid composition at room temperature or lower (e.g., 10-25°C).
  • a liquid composition comprising a bulk LNP product may be stored for at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.
  • one or more product attributes including, e.g., but not limited to LNP size/polydispersity, lipid/nucleic acid content (e.g., concentration), nucleic acid encapsulation, nucleic acid integrity, pH, osmolality, and combinations thereof, can be assessed and/or monitored during and/or after LNP formulation and/or storage.
  • Aseptic filling and/or labeling- a bulk LNP product described herein is aseptically filled into a sterile container, which in some embodiments may be a plastic or glass vessel. In some embodiments, a container may be suitable for a single-dose administration.
  • a container may be suitable for a multi-dose administration (e.g., at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, or more).
  • a container include, but are not limited to a bag, a pouch, a vial, etc.
  • a container may have a volume within a range of less than 30 mL, less than 25 mL, less than 20 mL, less than 15 mL, less than 10 mL, less than 5 mL, less than 4 mL, less than 3 mL, less than 2 mL, less than 1.5 mL, less than 1 mL, or smaller. In some embodiments, a container may have a volume within a range of 4-26 mL.
  • a container may be or comprise glass.
  • a container may be or comprise borosilicate glass, which in some embodiments may be or comprise type I borosilicate glass that meets requirements of applicable ISO standards and pharmacopeias (USP and Ph.Eur.).
  • a container may be a Schott glass vial.
  • a container may be a Gerresheimer glass vial.
  • a container may include a closure, which can be, e.g., but not limited to, a cap, a stopper, or a lid, etc.
  • a closure may be or comprise a flip off cap.
  • a closure may be or comprise a rubber stopper (e.g., a latex-free bromobutyl rubber stopper).
  • a closure may be or comprise a Datwyler stopper (e.g., Datwyler FM457 V9471, Datwyler FM547 V9145, etc.).
  • a container includes an overseal (e.g. an aluminum overseal).
  • crimping speed may occur at, for example, at least 100 units/minute, at least 200 units/minute, at least 300 units/minute, at least 400 units/minute, at least 500 units/minute, at least 600 units/minute, at least 700 units/minute, or more.
  • crimping pressure occurs at, for example, at least 100 N, at least 200 N, at least 300 N, at least 400 N, at least 500 N or more.
  • a single-dose amount or a multi-dose amount of a bulk RNA-LNP product described herein is aseptically filled in a container (e.g., described herein).
  • about 0.1-1 mL e.g, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mL
  • 0.2-0.7 mL or 0.4-0.5 mL of a bulk LNP product described herein is aseptically filled in a container (e.g., described herein).
  • a drug product is provided as a concentrate for suspension; in some such embodiments, about 0.1, about 0.2, or about 0.3 mg of concentrate (e.g., about 0.21, 0.22, 0.23, 0.24, or 0.25 mg, such as about 0.225 mg).
  • Aseptic filling can be manual or automated. In some embodiments, aseptic filling can be operated at a throughput of at least 1000 vials/day, at least 2000 vials/day, at least 3000 vials/day, at least 4000 vials/day, at least 5000 vials, or more.
  • aseptic filling can be operated at a throughput of at least 5000 vials or more, including, e.g., at least 7500 vials/day, at least 10,000 vials/day, at least 20,000 vials/day, at least 30,000 vials/day, at least 40,000 vials/day, at least 50,000 vials/day, at least 60,000 vials/day, at least 70,000 vials/day or more.
  • a lot number is labeled (e.g., printed) on a container and/or a lid.
  • vials prior to storage, vials are visually inspected for visible particles and/or vial weight is assessed (e.g., before and/or after filling).
  • a processes of sterile filtration, aseptic filing, and/or capping are performed under constant environmental monitoring.
  • all personnel involved in clean room activities are microbially monitored.
  • particle monitoring and microbial air monitoring is performed.
  • a bulk LNP product described herein can be sterile filtered, e.g., through a sterilization grade filter with a pore size of 0.1-0.3 pm. In some embodiments, a sterilization grade filter with a pore size of 0.2 pm can be used.
  • a sterile filter may have a filter surface area of at least 200 cm 2 , at least 300 cm 2 , at least 400 cm 2 , at least 500 cm 2 , at least 600 cm 2 , at least 700 cm 2 , at least 800 cm 2 , at least 900 cm 2 , at least 1000 cm 2 , at least 1250 cm 2 , at least 1500 cm 2 , at least 1750 cm 2 , at least 2000 cm 2 , at least 5000 cm 2 , at least 10,000 cm 2 , at least 15,000 cm 2 , at least 20,000 cm 2 or larger.
  • a sterile filtration is performed directly before filling a bulk RNA-LNP product described into containers described herein.
  • bioburden of a bulk LNP product described herein can be assessed prior to sterile filtration.
  • filter integrity can be assessed prior to and/or after sterile filtration.
  • one or more product attributes including, e.g., but not limited to LNP size/polydispersity, nucleic acid content (e.g, concentration), nucleic acid encapsulation, nucleic acid integrity, nucleic acid identity, pH, osmolality, and combinations thereof, can be assessed and/or monitored at the beginning and/or during the filling.
  • sterility e.g., bioburden, endotoxin, etc.
  • a bulk LNP product described herein can be transported to a different location for filling and/or labeling.
  • a bulk LNP product may be transferred to a container, e.g., with flexible wall(s), which, e.g., may be a flexible bag.
  • a container may have a volume of between 2 L and 200 L, in some embodiments between 5 and 50 L.
  • a bulk LNP product may be transported, e.g., in a disposable bioprocessing polymer bag, e.g.
  • such period of time may be less than 90 days, less than 60 days, less than 30 days, less than 14 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day or shorter.
  • a bulk LNP product may be transported at a refrigerated temperature (e.g., 2 to 10 °C or 2 to 8°C) for less than 14 days, less than 20 days, less than 7 days, less than 6 days, or shorter.
  • a bulk LNP product may be transported at a frozen temperature (e.g., -90 to -60°C or -60 to -35°C) for at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months or longer.
  • one or more product attributes including, e.g., but not limited to LNP size/polydispersity, nucleic acid content (e.g., concentration), RNA encapsulation, nucleic acid integrity, nucleic acid identity, pH, osmolality, and combinations thereof, can be assessed. Additionally or alternatively, sterility (e.g., bioburden, endotoxin, etc.) of a defined number of vials, can be assessed after transport to a different location for aseptic filling and/or labeling and prior to aseptic filling.
  • filling is completed using pumps (e.g., piston pumps or rotary piston pumps).
  • multiple containers e.g., multiple vials such as single use or multi-use vials
  • LNP product in which LNP product is disposed
  • a common tray or rack multiple such trays or racks are stacked in a carton that is surrounded by a temperature adjusting material (e.g., dry ice) in a thermal (e.g., insulated) shipper (packaging designed to maintain crucial conditions).
  • a temperature adjusting material e.g., dry ice
  • thermal shipper e.g., insulated
  • a thermal shipper keeps product at ultra-low temperature (e.g., less than -60°C, less than - 70°C, less than -80°C, less than -90°C or lower) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days or longer, e.g., if the thermal shipper is maintained at 15°C to 25°C.
  • product is shipped and/or stored in a thermal shipper for a period of time less than about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.
  • a thermal shipper comprises a thermal sensor.
  • a thermal shipper comprises a global positioning satellite (GPS) monitor.
  • GPS global positioning satellite
  • a thermal shipper comprises a system for communicating location and/or temperature via GPS to another site and/or device (e.g., tower).
  • a thermal shipper comprises a GPS-enabled thermal sensor, for example, with a control site and/or device (e.g., tower) that will track the location and/or temperature of each product shipment across their pre-set routes.
  • a thermal shipper (e.g, as described herein) is utilized to maintain crucial conditions (e.g, temperature) throughout a distribution process and/or during storage.
  • a thermal shipper (e.g. , as described herein) is used to ship product from a manufacturing site to a distribution center and/or point of care, e.g., by air and/or ground transportation.
  • a thermal shipper (e.g., as described herein) is useful for long-distance shipping(e.g., at least 100 kilometers, 200 kilometers, 300 kilometers, 400 kilometers, 500 kilometers, 1,000 kilometers, 2,000 kilometers, 3,000 kilometers, 4,000 kilometers, 5,000 kilometers, 6,000 kilometers, 7,000 kilometers, 8,000 kilometers, 9,000 kilometers, 10,000 kilometers or more).
  • filled products can be stored stable at sub-zero temperatures (e.g., less than -20°C, less than -30°C, less than -40°C, less than -50°C, less than -60°C, less than -70°C, less than -80°C, or lower) over a period of time (e.g., at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer).
  • filled products can be stored stable as a frozen liquid at a temperature of -60°C to -80°C or lower for a period of at least 6 months.
  • a freezing process may utilize controlled freeze equipment and/or temperature controlled freezers.
  • filled products can be stored stable at a refrigerated temperature (e.g., about 2°C to about 10°C or about 2°C to about 8°C) for at least 3 days, at least 5 days, at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or longer.
  • filled products can be stored stable at a temperature of about 2°C to about 8°C for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or longer.
  • filled products can be stored stable at room temperature or lower (e.g., 10-25°C) for at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or longer.
  • room temperature or lower e.g. 10-25°C
  • filled products can be maintained at ultra-low temperature (e.g., as described herein) in a thermal shipper (e.g. , as described herein) as a temporary storage, for example, for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days or longer when the thermal shipper is maintained at 15°C to 25°C.
  • product is shipped and/or stored in a thermal shipper for a period of time less than about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.
  • duration of time a thermal shipper that keeps product at ultra-low temperature can be extended, for example, by at least several days, by opening the thermal shipper and adding and/or replacing ice or dry ice (e.g., re-icing).
  • temperature and/or location can be monitored during storage.
  • one or more quality control parameters may be assessed to determine whether LNPs in a preparation or a bulk drug product described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution).
  • quality control parameters may include, but are not limited to appearance (e.g., color, dryness, presence and/or size, color, type, etc.
  • lipid identity e.g., absolute and/or relative amount of any lipid
  • nucleic acid e.g., RNA
  • nucleic acid identity e.g., sequence and/or other structure
  • nucleic acid integrity e.g., nucleic acid content (e.g., presence and/or absolute or relative amount)
  • nucleic acid encapsulation LNP size (e.g., average size, size distribution, etc.), LNP polydispersity, pH, osmolality, subvisible particles (e.g., too small to be visible to unaided eye, e.g., particles in the size range of 0.1 pm to 100 pm), presence and/or amount of one or more endotoxins, sterility, etc., may be assessed.
  • Certain methods for assessing quality of an LNP preparation or a bulk drug product are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests (e.g., as described herein) can be used for quality
  • a batch of an LNP preparation or a bulk drug product described herein may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of a preparation or a bulk drug product described herein can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if quality assessment indicates that such a batch of a preparation or a bulk drug product described herein meets or exceeds the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of a preparation or a bulk drug product described herein does not meet or exceed the acceptance criteria.
  • a batch of a preparation or a bulk drug product described herein that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.
  • manufacturing methods described herein may further comprise assessing and/or monitoring (e.g., assessing at one or more time points) one or more features of an LNP preparation or a bulk drug product described herein including, e.g., appearance (e.g., color, dryness, presence and/or size, color, type, etc.
  • lipid identity e.g., absolute and/or relative amount of any lipid
  • nucleic acid e.g., RNA
  • identity e.g., sequence and/or other structure
  • nucleic acid integrity e.g., nucleic acid content (e.g., presence and/or absolute or relative amount)
  • nucleic acid encapsulation LNP size (e.g., average size, size distribution, etc.), LNP polydispersity, pH, osmolality, subvisible particles, presence and/or amount of one or more endotoxins, sterility, etc.
  • at least one or more features e.g.
  • At least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve at least thirteen, at least fourteen) described herein can be characterized and/or monitored for quality control.
  • an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, visual inspection is utilized to monitor appearance. In some embodiments, an LNP preparation or bulk product described herein is a white to off-white suspension.
  • visible particles present in an LNP preparation or bulk product described herein are assessed and/or monitored (e.g., determined at one or more points over time).
  • visible particle testing is performed according to Ph. Eur. 2.9.20.
  • visible particle testing is performed according to Ph. Eur. 2.9.20 with minor adaptions.
  • LNPs are free or essentially free from observable particles (e.g., visible to unaided eye).
  • lipid identity and/or lipid content of lipids, lipid stock solutions, and/or LNPs is assessed and/or monitored (e.g., determined at one or more points over time).
  • an HPLC-CAD assay determines the identity and concentration of lipids in the tested sample (e.g. LNPs).
  • individual lipid identities and/or content is determined by comparison of retention times with those of the reference standards.
  • lipid identities and content determined comprise monitoring or particular lipids.
  • particular lipids comprise cationic lipid, PEG-lipid, helper lipid (e.g., DSPC, and/or cholesterol).
  • concentration of each individual lipid is determined by sample area response against the respective five-point calibration curve generated from the reference standards, with peak detection performed use a CAD.
  • results for lipid identity and lipid content are reported as relative retention time compared to reference standard and as mg/rnL, respectively.
  • a predetermined acceptance criterion is met for release for lipids, lipid stock solutions, and/or LNPs.
  • nucleic acid identity is assessed and/or monitored (e.g., determined at one or more points over time).
  • nucleic acid identity is determined by capillary electrophoresis.
  • LNPs are treated with Tween20 are applied to a gel matrix contained in a capillary.
  • nucleic acid (e.g., RNA) and its derivatives, degradants, and impurities are separated according to their sizes.
  • the gel matrix contains a fluorescence dye which binds specifically to nucleic acid (e.g., in some embodiments specifically to RNA) components which allows detection by a laser-induced fluorescence (LIF) detector.
  • the excitation wavelength is 495 nm.
  • the emission wavelength is 537 nm.
  • RNA identity is verified by comparing with the reference standard.
  • RNA identity is determined by reverse transcribing said RNA into cDNA and amplifying said cDNA (e.g., by PCR) with a target specific probe and/or primers.
  • sequence of anRNA is determined by reverse transcribing said RNA into cDNA, amplifying (e.g., by PCR), and sequencing the amplified product.
  • nucleic acid length is determined by denaturing agarose gel electrophoresis in comparison to a standard ladder with nucleic acid s of known lengths. In some embodiments, sizes obtained must be consistent with theoretically expected lengths.
  • the electrophoresis gel is a precast and buffered agarose gel pre-stained with a nucleic-acid specific dye.
  • nucleic acid integrity is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, nucleic acid integrity is determined use agarose gel electrophoresis. In some embodiments, nucleic acid integrity is determined by capillary electrophoresis. In some embodiments, nucleic acid integrity can be quantitatively determined using capillary electrophoresis.
  • determination of nucleic acid integrity comprises one or more of LNP treatment with nonionic surfactant, application of nonionic surfactant treated LNP to a gel matrix contained in a capillary, separating nucleic acid and its derivatives, degradants, and impurities according to their sizes, detection of intact nucleic acid and its derivatives, degradants, and impurities, and/or determining the integrity of the RNA.
  • a non-ionic surfactant is Tween20.
  • a gel matrix comprises a fluorescent dye which binds specifically to nucleic acid (e.g., in some embodiments specifically to RNA) components.
  • detection is conducted using a laser-induced fluorescent (LIF) detector.
  • LIF laser-induced fluorescent
  • an excitation wavelength is 495 nm. In some embodiments, an emission wavelength is 537 nm.
  • a nucleic acid (e.g., RNA) solution must give rise to a single peak at the expected retention time consistent with the expected lengths as compared to the retention times of a standard ladder.
  • quantification of a main nucleic acid (e.g., RNA) peak is calculated in relation to signal intensities in the electropherogram where degradation products are detectable. In some embodiments, > 30.0, 40.0 50.0, 60.0, 70.0, 80.0 or 90.0 % in the peak corresponds to intact nucleic acid (e.g., RNA).
  • nucleic acid (e.g., RNA) encapsulation is assessed and/or monitored (e.g., determined at one or more points over time).
  • encapsulation is monitored using a nucleic acid-binding (e.g., an RNA-binding) dye.
  • an RNA-binding dye is Ribogreen (Invitrogen, Eugene, OR, USA).
  • nucleic acid encapsulation is calculated by comparing signals (e.g., fluorescent signals) of LNP samples in the absence (free nucleic acid) and presence (total nucleic acid) of detergent.
  • the detergent is TritonX-100.
  • > 60, 70, 80, or 90% of nucleic acid (e.g., RNA) is encapsulated.
  • nucleic acid (.e.g, RNA) content is assessed and/or monitored (e.g., determined at one or more points over time).
  • nucleic acid (.e.g, RNA) content is determined using UV absorption spectrophotometry.
  • nucleic acid (.e.g, RNA) content is measured using a nucleic acid-binding (e.., an RNA-binding) dye.
  • an RNA- binding dye is Ribogreen (Invitrogen, Eugene, OR, USA).
  • nucleic acid (.e.g, RNA) content is determined by disrupting LNPs with detergent and measuring the total nucleic acid (.e.g, RNA) content based on a signal.
  • the detergent is Triton X-100.
  • the total nucleic acid (.e.g, RNA) content signal is measured using a spectrofluorophotometer.
  • nucleic acid (.e.g, RNA) content is 0.1-1 mg/rnL or 0.3- 0.7 mg/mL, or 0.4-0.6 mg/mL.
  • LNP size and/or polydispersity is assessed and/or monitored (e.g., determined at one or more points over time).
  • evaluation of mean particle size and size distribution of LNP in a sample involves use of dynamic light scattering.
  • results are reported as the Z-average size of the particles and the polydispersity index.
  • polydispersity values are used to describe the width of fitted log-normal distribution around the measured Z-average size and are generated using proprietary mathematical calculations within a particle sizing software.
  • dynamic light scattering methods comprise use of a particle sizer that uses back-scatter at 173°.
  • LNP size is ⁇ 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 nm.
  • LNP polydispersity is ⁇ 0.1, 0.2, 0.3, 0.4, or 0.5.
  • pH value of an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time).
  • the pH value is determined according to regional pharmacopeia (e.g., Ph. Eur. 2.2.3, USP ⁇ 791>).
  • pH value is 6-8, or 7-8, or 6.8-7.9.
  • osmolality of an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, osmolality of LNPs is determined according to regional pharmacopeia (e.g., Ph. Eur. 2.2.35, USP ⁇ 785>). In some embodiments, osmolality of LNPs is 400-650 mOsmol/kg, 425-625 mOsmol/kg, or 450-600 mOsmol/kg, or 475-550 mOsmol/kg.
  • subvisible particles of an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, detection of subvisible particles is determined according to Ph. Eur. 2.9.19 / USP ⁇ 787> (method 2, microscopic particle count).
  • an LNP preparation or bulk product described herein can comprise particles with a size of > 25 pm is no more than 600 particles/container. In some embodiments, an LNP preparation or bulk product described herein can comprise particles with a size of > 10 pm is no more than 6000 particles/container.
  • presence and/or level of bacterial endotoxins in an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time), for example, using an analytical kinetic turbidimetric limulus amebocyte lysate (LAL) procedure.
  • LAL analytical kinetic turbidimetric limulus amebocyte lysate
  • Gram-negative bacterial endotoxins are assessed and/or monitored (e.g., determined at one or more points over time).
  • Gram-negative bacterial endotoxins are determined to have an acceptable level if the acceptance criteria in regional pharmacopoeia (e.g., Ph. Eur.
  • LNPs have ⁇ 12.5 EU/mL of bacterial endotoxins. In some embodiments, for example, prior to filtration, LNPs may have ⁇ 46 EU/mL.
  • bioburden is assessed and/or monitored (e.g., determined at one or more points over time) using a membrane filtration method.
  • bioburden is determined to have an acceptable level if the acceptance criteria in regional pharmacopoeia (e.g., Ph. Eur. 2.6.12, USP ⁇ 61>, JP 4.05) are met when the bioburden is determined according to the method described therein (e.g., less than or equal 10 1 CFU per 10 mL). In some embodiments, for example, prior to filtration, bioburden may be less than or equal to 20 CFU per 20 mL.
  • sterility of an LNP preparation or bulk product described herein is assessed and/or monitored (e.g., determined at one or more points over time). In some embodiments, sterility testing is performed according to regional pharmacopoeia (e.g., Ph. Eur. 2.6.1, USP ⁇ 71>, JP 4.06). In some embodiments, LNPs are sterile. In some embodiments, sterility is assessed and/or monitored by determining the presence or absence of detectable growth. In some embodiments, sterility is assessed and/or monitored, for example, by subjecting LNP samples to luciferase which catalyzes a reaction with microbial ATP. Light emitted during the reaction can be measured, for example, using a luminometer. In some embodiments, additional characterization may be carried out in addition to, or in combination with, any other characterization and/or quality control method.
  • regional pharmacopoeia e.g., Ph. Eur. 2.6.1, USP ⁇ 71>, JP
  • protein expression from nucleic acids encapsulated in LNPs can be assessed.
  • protein expression is measured using a process comprising one or more of the following steps: adding LNPs to mammalian cells and/or measuring protein expression.
  • mammalian cells are HEK-293T cells.
  • n LNP dose is added to mammalian cells.
  • protein expression is measured using an antibody directed against an expressed protein or a portion thereof.
  • cells are labeled with a live/dead dye.
  • live/dead dye labeled cells are separated by flow cytometry.
  • the percent of live cells expressing relevant protein is enumerated.
  • nucleic acid (e.g., RNA) substance is transfected as a control to confirm protein expression.
  • a control substance transfection comprises use of electroporation.
  • a control transfection comprises use of calcium carbonate transfection.
  • expression is measured by quantifying the number of cells that have positive signal for bound antibody directed against the expressed protein or portion thereof. In some embodiments, expression is measured by quantifying the number of cells that have positive signal for bound antibody directed to a target protein.
  • protein expression of is measured using a process comprising one or more of the following steps: adding LNPs to mammalian cells, e.g., HEK-293T cells, at a pre-determined dose level, labeling cells with a live/dead dye and separating by flow cytometry, enumerating the percent of live cells expressing relevant protein, transfecting a control compositon with lipofectamine to confirm protein expression, and/or measuring expression by quantifying the number of cells that have positive signal for bound antibody directed to a target protein.
  • mammalian cells e.g., HEK-293T cells
  • characterization of LNPs is performed. In some embodiments, characterization comprises use of one or more of electron microscopy, CD spectroscopy, small angel X-ray scattering (SAXS), in vitro expression, and/ or mouse immunogenicity and comparing to a reference standard and/or control LNPs.
  • SAXS small angel X-ray scattering
  • excipients present in an LNP preparation or bulk product are assessed and/or monitored (e.g., determined at one or more points over time).
  • excipients that may be assessed and/or monitored include, but are not limited to cholesterol, cryoprotectant, solvent (e.g., water and/or organic solvent), and/or salts.
  • excipients are tested according to a quality standard set forth in Ph. Eur.
  • the impurity profile of LNPs is based primarily on the impurity profile of the materials used for its manufacture.
  • possible process-related impurities include residual solvent (e.g., ethanol), buffer components (e.g., citrate, HEPES), and/or chelating agent (e.g., EDTA).
  • residual solvent (e.g., ethanol) content present in an LNP preparation or a bulk product described herein is less than 10,000 ppm, 7,500 ppm, 5,000 ppm, 2,500 ppm, 1,000 ppm, or lower.
  • all buffers and solutions held at least 24 hours are assessed and/or monitored for microbial content.
  • the container and/or closure of the container is assessed and/or monitored.
  • the closure system comprises, for example, a vial and/or a vial stopper.
  • container closure integrity is assessed when exposed to low temperatures (e.g., less than - 50°C, less than -60°C, less than -70°C, less than -80°C, less than -90°C) and/or to assess and/or monitor the impact of crimping force on container closure integrity.
  • vial quality testing is performed according to regional pharmacopoeia (e.g., Ph. Eur. 3.2.1, USP ⁇ 660>, JP 7.01).
  • vial stopper quality testing is performed according to regional pharmacopoeia (e.g., Ph.
  • closure of the container is assessed and/or monitored by incursion of a dye.
  • container closure integrity (e.g., before and/or after exposure to low temperature) can be assessed and/or monitored using laser-based headspace carbon dioxide and oxygen detection analysis (HSA).
  • HSA headspace carbon dioxide and oxygen detection analysis
  • headspace analyzers e.g., Lighthouse Instruments Oxygen and FMS-Carbon dioxide headspace analyzers.
  • analyzers are calibrated using traceable standards (e.g., NIST-traceable standards).
  • traceable standards e.g., NIST-traceable standards.
  • an increase in the percent of oxygen measured of about 0.5%, 1%, 1.5% or 2% is considered to be a failure and/or loss of container closure integrity.
  • RSF residual seal force
  • a container closure system is assessed and/or monitored.
  • RSF is stress an elastomeric closure will continue to exert against the glass vial finish and the overseal after capping is complete.
  • RSF is measured prior to and/or after sample exposure to low temperature.
  • samples are warmed to room temperature and RSF is measured.
  • an initial RSF alert limit of no less than 10 Ibf, 9 Ibf, 8 Ibf, 7 Ibf, 6 Ibf, 5 Ibf, 4 Ibf, or 3 Ibf is utilized for monitoring RSF.
  • physiochemical properties e.g., density, viscosity, size distribution and shape, surface charge, and/or surface PEG
  • thermal transitions of LNPs are assessed and/or monitored, for example, using differential scanning calorimetry.
  • provided technologies include one or more quality assessment steps.
  • one or more of aqueous (e.g., nucleic acid, e.g., RNA) solution, lipid solution, and/or LNP preparation is subjected to one or more quality control steps, assessments, and/or characterizations during and/or after its production and/or use as described herein.
  • an assessed material is subjected to repeat or alternative assessment. In some embodiments, if an assessment indicates a defect or failure, an assessed material is discarded.
  • an assessed material continues along a predetermined workflow.
  • a reference standard for a particular quality control assessment can be any quality control standard, including, e.g., a historical reference, a set specification. As will be understood by a skilled artisan, in some embodiments, a direct comparison is not required.
  • a reference standard is an acceptance criterion based on, for example, assessment and/or characterization of features described herein, including, e.g., physical appearance, lipid identity and/or content, LNP size, LNP polydispersity, nucleic acid (e.g., RNA) encapsulation, nucleic acid (e.g., RNA) length, nucleic acid (e.g., RNA) identity (e.g., as RNA), integrity, sequence, and/or concentration, pH, osmolality, potency, bacterial endotoxins, bioburden, residual organic solvent, osmolality, pH, and combinations thereof.
  • a quality control assessment involves an assessment of presence of air and/or of one or more manifestations (e.g., loss of polydispersity, disruption of nanoparticle structure and/or of colloidal structure of an LNP composition, etc.) of air having been present.
  • one or more manifestations e.g., loss of polydispersity, disruption of nanoparticle structure and/or of colloidal structure of an LNP composition, etc.
  • Example I Overview of exemplary manufacturing process for a pharmaceutical-grade composition comprising RNA
  • the present Example depicts an exemplary manufacturing process for pharmaceutical-grade RNA comprising an in vitro RNA transcription followed by removal of components utilized or formed in the course of production by a purification process, and filtration to reduce bioburden (e.g., as illustrated in Figure 4).
  • Optional in-process controls may also be completed depending on whether a hold step is performed.
  • Example 2 Overview of exemplary manufacturing process for pharmaceutical-grade RNA-LNPs
  • the present Example demonstrates an exemplary manufacturing process for pharmaceutical-grade RNA- LNPs comprising six steps and one optional step (Figure 5).
  • a lipid and RNA stock is prepared (the lipid stock corresponds to the second liquid mentioned further above, the RNA stock corresponds to the first liquid mentioned above).
  • LNPs are formulated and stabilized by dilution followed by concentration, buffer exchange, and filtration. Subsequently, the concentration is adjusted and cryoprotectant is added.
  • RNA-LNPs are transported to an external fill and finish site.
  • RNA-LNPs undergo sterile filtration and aseptic filling and storage.
  • Example 3 Overview of exemplary DNA template manufacture via a PCR-based process.
  • the present Example describes an exemplary manufacturing process of a DNA template via a PCR-based process ( Figure 6).
  • a master mix preparation was made.
  • forward primer and vector were added.
  • the PCR-mix is transferred into a reagent reservoir and a PCR plate was filled.
  • a PCR is completed comprising an initial denaturation, a denaturation step, an annealing step, a final extension step for 20-30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) cycles and a hold step.
  • the PCR products can be pooled and purified. Subsequently, the purified, pooled PCR product was filtered and quality control tested.
  • Example 4 Exemplary characterization of pharmaceutical-grade RNA
  • the present Example describes exemplary characterization of pharmaceutical-grade RNA compositions.
  • degree of coloration was tested based on Ph. Eur. 2.2.2. In some embodiments, degree of opalescence was determined based on Ph. Eur. 2.2.1. Results were reported as the clarity and color of the product solution.
  • Gram-negative bacterial endotoxins were detected with a chromogenic-kinetic method according to regional pharmacopeia (e.g. Ph. Eur. 2.6.14, USP ⁇ 85>, JP 4.01). Results were reported as EU/mL of product solution.
  • bioburden tests determined the total aerobic microbial count (TAMC) and the total combined yeast/molds counts (TYMC) using a membrane filtration method according to regional pharmacopeia (e.g., Ph. Eur. 2.6.12, USP ⁇ 61>, JP4.05).
  • the test solution was filtered and the membrane filter was transferred to the surface of a suitable nutrient agar medium. Results were reported as CFU/mL of composition comprising RNA.
  • RNA concentration was determined photometrically according to Eur. 2.2.25 at a wavelength of 260 nm utilizing an extinction coefficient of 0.025 *pg-l*cm-l. Results were reported as mg/mL of product solution.
  • RNA samples were incubated for a defined time period with RNase A, certified to be free of DNases and proteases and then separated by gel-electrophoresis on a precast and pre-stained agarose gel and compared to an RNA sample that had been incubated under identical conditions except for the addition of RNase A.
  • disappearance of the RNA band upon incubation with RNase A verified the identity as RNA. Results were reported as the presence or absence of an RNA band by gel electrophoresis.
  • RNA samples were separated by denaturing gel electrophoresis on precast and buffered agarose gel pre-stained with a nucleic acid specific dye.
  • the gel was photographed using a gel documentation system and the length of the RNA band was compared to an RNA of known size (length standard [RNA ladder]).
  • RNAs were separated by capillary electrophoresis using a system which gives an electropherogram as a result and a quantitative evaluation was performed.
  • the conformance of lengths of RNA (and thus indirectly the molar masses) with theoretical values were verified by denaturing gel electrophoresis in comparison to a standard ladder with RNAs of known lengths.
  • RNAs i.e., transcripts from the respective DNA template used.
  • capillary electrophoresis was applied for quantitative analysis of RNA integrity.
  • RNAs gave rise to a single peak at the expected retention time consistent with expected lengths as compared to the retention times of a standard ladder.
  • quantification of the main RNA peak was calculated in relation to the signal intensities in regions of the electropherogram, where degradation products were detectable.
  • osmolality of a RNA solution was determined according to regional pharmacopeia (e.g., Ph. Eur. 2.2.35, USP ⁇ 785>). Results were reported as mOsmol/kg of water. pH
  • a pH value was potentiometrically determined according to regional pharmacopeia (e.g, Ph. Eur. 2.2.3, USP ⁇ 791>) using a microelectrode with an embedded temperature sensor for automatic correction of the measured values.
  • regional pharmacopeia e.g, Ph. Eur. 2.2.3, USP ⁇ 791>
  • residual DNA template content derived from the respective linear DNA template was determined using a real-time quantitative PCR test method. For example, in some embodiments, for the PCR a pre-mixed Sybr Green master mix was used according to manufacturer’s recommendations. In some embodiments, amplification and detection of DNA was performed in a real-time thermocycler. In some embodiments, residual DNA template in the sample was quantified in comparison to a standard (serial dilution of plasmid DNA). The results were reported in ng DNA/mg RNA.
  • residual dsRNA level was determined using a limit test.
  • RNA samples and a dsRNA reference 2000 pg dsRNA/pg RNA, 1500 pg dsRNA/pg RNA, 1000 pg dsRNA/pg RNA, 500 pg dsRNA/pg RNA, or lower representing the upper limit of accepted residual dsRNA content
  • a dsRNA-specific monoclonal antibody mouse IgG (immuno globulin G), clone J2
  • ECL Enhanced chemiluminescence
  • signal intensities were quantified by densitometry, and the values of the RNA samples compared to the signal intensity of the dsRNA reference. Results were reported as complies with the specified upper limit.
  • RNA sequence was deduced from sequencing the DNA template, which served as template for in vitro transcription and defines the primary structure of each RNA.
  • identity of the starting material and thus identity of the transcribed RNA was controlled by automated sequencing of the RNA encoding region of the template.
  • results were reported as compliments to the target sequence.
  • a cap-analog was included in the in vitro transcription reaction mixture, which, upon incorporation at the 5’ end during transcription led to RNA with a so-called capl structure.
  • the percentage of capped RNA for the exemplary batches were characterized by an RNase H based assay.
  • RNA samples were annealed to a customized biotinylated nucleic acid probe binding close to the 5’ end of the RNA, and RNase H was used to digest the mRNA-probe complex, generating a short fragment corresponding to the 5’ part of the RNA.
  • streptavidin-coated spin columns or magnetic beads were used for sample clean-up.
  • capped and non-capped species were identified by the observed mass values and their MS signals were used to calculate the percentage of capped RNA. Multiple such batches displayed a percentage of capped RNA between 40-70%.
  • percentage of polyadenylation (PolyA) attached to the 3’ end of the RNA construct was measured for exemplary batches using droplet digital PCR (ddPCR).
  • ddPCR droplet digital PCR
  • cDNA was generated using a reverse transcription primer that spanned the PolyA and 3’ sequences of the RNA construct and required both for binding.
  • positive signals indicated polyadenylated RNA and were detected using primers and probes located close to the 3’ end of the RNA construct.
  • quantitation was based on normalization to the theoretical input of the test sample (UV A260 nm concentration).
  • RNAs e.g., of BNT162b2 RNA
  • Multiple such batches displayed identity (RNA length) as a single, distinct band migrating at the expected location as compared to a length standard (RNA ladder).
  • Multiple such batches displayed RNA integrity above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • Multiple such batches displayed content (RNA concentration) of 1.53-1.87 mg/mL.
  • RNA concentration 1.53-1.87 mg/mL.
  • Multiple such batches displayed pH between 6.0-8.0.
  • Multiple batches have been produced, including multiple batches that produced at least 30 g. Multiple such batches displayed an appearance which was a clear (less than or equal to 6 NTU), colorless liquid. Multiple such batches displayed identity (RNA length) as a single, distinct band migrating at the expected location as compared to a length standard (RNA ladder). Multiple such batches displayed RNA identity as RNA by lack of an RNase-resistant band detectable by gel electrophoresis. Multiple such batches displayed RNA integrity above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Multiple such batches displayed a desired RNA sequence based on testing (e.g., sequencing) of DNA starting material.
  • testing e.g., sequencing
  • RNA concentration 1.53-1.87 mg/mL.
  • pH between 6.0-8.0.
  • residual DNA template levels between 0.1-100 ng DNA per mg RNA, wherein residual DNA template level is preferably less than or equal 500 ng DNA per mg RNA, 480 ng DNA per mg RNA, 450 ng DNA per mg RNA, 420 ng DNA per mg RNA, 390 ng DNA per mg RNA, 360 ng DNA per mg RNA, 330 ng DNA per mg RNA 300 ng DNA per mg RNA, 270 ng DNA per mg RNA, 240 ng DNA per mg RNA, 210 ng DNA per mg RNA, or lower.
  • Multiple such batches displayed residual dsRNA less than or equal to 2000 pg dsRNA/pg RNA, 1500 pg dsRNA/pg RNA, 1000 pg dsRNA/pg RNA, 500 pg dsRNA/pg RNA, or lower. Multiple such batches displayed bacterial endotoxins at levels less than or equal to 0.5 EU/mL. Multiple such batches displayed bioburden levels less than or equal to 1 CFU per 1 mL.
  • Example 5 Certain characteristics of exemplary RNA solutions and/or RNA-LNP compositions
  • the present Example describes certain assessments that may be performed of RNA-LNP compositions.
  • RNA length is assessed and, for example, is determined to be a single, distinct band migrating at the expected location as compared to a length standard (RNA ladder).
  • RNA integrity is assessed to be above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • RNA content is assessed to be within a range of about 1.53-1.87 mg/mL.
  • an RNA solution for use in accordance with the present disclosure is characterized by a pH between 6.0-8.0. In some embodiments, an RNA solution is characterized by osmolality of 25- 400 mOsmol/kg, 50-400 mOsmol/kg, wherein osmolality is preferably less than or equal to 200 mOsmol/kg.
  • an RNA solution is characterized by residual DNA template levels between 1-850 ng DNA/mg RNA, wherein residual DNA template level is preferably less than or equal 500 ng DNA per mg RNA, 480 ng DNA per mg RNA, 450 ng DNA per mg RNA, 420 ng DNA per mg RNA, 390 ng DNA per mg RNA, 360 ng DNA per mg RNA, 330 ng DNA per mg RNA 300 ng DNA per mg RNA, 270 ng DNA per mg RNA, 240 ng DNA per mg RNA, 210 ng DNA per mg RNA, or lower.
  • an RNA solution is characterized by residual dsDNA levels between 75-125 ng dsRNA/pg RNA.
  • residual dsDNA levels are preferably less than or equal to 2000 pg dsRNA/pg RNA, 1500 pg dsRNA/pg RNA, 1000 pg dsRNA/pg RNA, 500 pg dsRNA/pg RNA, or lower.
  • an RNA solution is characterized by bacterial endotoxins at levels less than or equal to 0.5 EU/mL. In some embodiments, an RNA solution is characterized by bioburden levels less than or equal to 1 CFU per 10 mL.
  • an RNA-LNP preparation is characterized by RNA-LNP size of 60-90 nm. Multiple such batches displayed a polydispersity index (PDI) of 0.05-1.
  • a polydispersity index (PDI) of 0.05-1.
  • an RNA- LNP preparation is characterized by RNA integrity above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • an RNA-LNP preparation is characterized by RNA content between 0.4-1 mg/mL.
  • an RNA-LNP preparation is characterized by percent encapsulation efficiency above 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and specifically above, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%,
  • an RNA-LNP preparation is characterized by cationic lipid levels between 5-7 mg/mL.
  • an RNA-LNP preparation is characterized by polyethylene glycol (PEG)- lipid levels between 0.5-1 mg/mL.
  • an RNA-LNP preparation is characterized by phospholipid levels between 1-2 mg/mL.
  • an RNA-LNP preparation is characterized by sterol levels between 2-3 mg/mL.
  • an RNA-LNP preparation is characterized by pH between 6.0-8.0. In some embodiments, an RNA-LNP preparation is characterized by osmolality between 400-600 mOsmol/kg, 450-575 mOsmol/kg, 500-575 mOsmol/kg, or 525-575 mOsmol/kg. In some embodiments, an RNA-LNP preparation is characterized by N/P ratio between 4.5-5.5.
  • RNA-LNPs are assessed and/or monitored. Size distribution and particle shape may be assessed, for example, using asymmetrical flow field-flow fractionation (AF4).
  • AF4 asymmetrical flow field-flow fractionation
  • an RNA-LNP preparation is characterized by a size distribution, as measured by AF4 between 20-55 nm.
  • an RNA-LNP preparation is characterized by a particle shape between 0.6 and 0.8 Rz/Rh.
  • an RNA-LNP preparation is characterized by a surface charge, as assessed by electrophoretic light scattering, between -3.5 and -1.5 mV.
  • RNA-LNPs were added to mammalian cells (e.g, HEK-293T cells) at an RNA dose (.e.g., a dose of 60 ng or 100 ng).
  • protein expression is measured (e.g., by flow cytometry) using an antibody directed against the expressed protein.
  • cells ae labeled with a Live/Dead dye and the percent of live cells (to eliminate background signal) expressing protein is enumerated.
  • a positive control is utilized (e.g., RNA drug substance transfected with lipofectamine) to confirm protein expression.
  • expression is measured by quantifying the number of cells that had a positive signal for bound antibody.
  • percent positive cells range from 5-60%. In some embodiments, percent positive cells is at least 30% or higher.
  • protein expression of an exemplary RN A-LNP product is measured on different days.
  • degree of cell viability be considered when evaluating assay results, and particularly when comparing different sets of assay results. For example, in some embodiments, it may be desirable to compare results achieved with populations of cells whose percent viability is comparable, e.g., does not differ by more than about 5%, 4%, 3%, 2%, 1% or less.
  • protein expression is characterized for exemplary RNA-LNPs, with various doses of RNA. In some embodiments, expression within range of 20-70% positive cells is observed. In some embodiments, for a 60 ng dose, at least20-40% positive cells are observed, for a 100 ng dose, expression between 25-60% positive cells is observed, and/or for a 150 ng dose, expression between 40-70% positive cells is observed. Often, observed expression is above 35% positive cells for multiple (e.g., all) doses.
  • percentage of capped RNA and/or polyadenylated RNA is measured. In some embodiments, percentage of capped RNA is characterized by an RNase H based assay. In some embodiments, capped and non-capped species are identified by the observed mass value and their MS signals are used to calculate the percentage of capped RNA. In some embodiments, a percentage of capped RNA is between 40-70%.
  • percentage of polyadenylation (PolyA) attached to the 3’ end of an RNA is measured, for example using droplet digital PCR (ddPCR).
  • quantitation is based on normalization to the theoretical input of test sample (UV A260 nm concentration).
  • Example 7 Exemplary manufacturing increased batch sizes of RNA-LNPs
  • the present example demonstrates Exemplary manufacturing of RNA-LNPs at a larger batch size.
  • an exemplary composition comprising RNA with a concentration with 2.25 mg/mL can be conditioned with citrate pH 4.0 to arrive at 0.2 or 0.4 mg/mL of composition comprising RNA in 40 mM citrate.
  • lipids are dissolved in absolute ethanol at 35 °C and filtered through a 0.2pm PES filter before use.
  • lipid stocks are prepared at a 15 or 30 mg/mL.
  • three volumes of the conditioned RNA drug substance phase is mixed with one volume of the lipid stock in a continuous flow process.
  • another two volumes of citrate pH 4.0 are added continuously to the LNP stream.
  • resulting material constitutes the primary LNP from which in-process control post-mixing is taken.
  • primary LNP e.g. the liquid composition discussed further above
  • primary LNP are connected to a TFF device and (i) diafiltered with two volumes of citrate pH 4, (ii) concentrated to 0.5 mg/mL, (iii) diafiltered with eight volumes of PBS, and (iv) concentrated to about 1.2 mg/mL.
  • in-process control post-TFF is taken.
  • material is harvested from the TFF device and filtered through a 0.2 pm PES filter.
  • in-process control post 0.2pm filtration is taken.
  • RNA-LNPs are filtered a further time through a 0.2 pm PES filter.
  • in-process data and characterization are highly similar between an upscale and a reference manufacturing processes scales.
  • produced RNA-LNP are characterized by particle size of 50-80 nm. In some embodiments produced RNA-LNP are characterized by a polydispersity index (PDI) of 0.05-0.2.
  • PDI polydispersity index
  • produced RNA-LNP are characterized by RNA integrity above 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments produced RNA-LNP are characterized byRNA content between 500-600 pg.

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Abstract

L'invention concerne des procédés, des systèmes et des utilisations pour fournir une composition de nanoparticules lipidiques ayant des propriétés de nanoparticules avantageuses.
PCT/EP2022/079475 2022-10-21 2022-10-21 Procédés et utilisations associés à des compositions liquides Ceased WO2024083345A1 (fr)

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