WO2025021946A1

WO2025021946A1 - Method and system for the production of a carrier

Info

Publication number: WO2025021946A1
Application number: PCT/EP2024/071186
Authority: WO
Inventors: Aurélien VANDER STRAETEN; Arnaud DEGEMBE; José CASTILLO
Original assignee: Quantoom Biosciences S.A.
Priority date: 2023-07-25
Filing date: 2024-07-25
Publication date: 2025-01-30

Abstract

The current invention relates to a method for manufacturing one or more lipid-based carriers. Particularly, the invention relates to a method comprising mixing two solutions in two different phases, aqueous and organic phase where the organic 5 phase comprises lipids to form a lipid-based carrier suspension, removing at least part of said organic phase from said suspension by an evaporation process, and collecting the at least part of the suspension comprising formed lipid-based carriers. In a second aspect, said invention also relates to a system for manufacturing such lipid-based carriers, wherein said system comprises an evaporation device for the 10 removal of one or more organic solvents from a formulation comprising one or more lipid-based carriers.

Description

METHOD AND SYSTEM FOR THE PRODUCTION OF A CARRIER

FIELD OF THE INVENTION

This invention relates to a method and a system for the production of a carrier as a therapeutic delivery system.

BACKGROUND

Carriers, such as lipid-based carriers (e.g. lipid nanoparticles (LNPs)) are the most widely used gene delivery systems. For their fabrication, an aqueous phase containing the genetic material is mixed with an organic phase containing a lipid, a polymer or a mix of lipids and/or polymers. This process results in the encapsulation of the genetic material inside a nanoparticle or nanocarrier. After their fabrication, the organic solvent must be removed to meet the regulatory requirements for medicinal products. Solvent extraction and exchange are frequently performed by Tangential Flow Filtration (TFF) with several consecutive cycles of concentration and dilution with an aqueous buffer until the desired residual solvent concentration is in compliance with current regulatory requirements. WO2023018773 for instance describes the processing of LNPs, including steps to purify, pH adjust, buffer exchange, and/or concentrate LNPs using TFF.

However, the TFF operation can result in considerable shear stress with an adverse impact on the quality of the nanocarriers.

Furthermore, TFF is a limiting step in the nanocarrier production process as it requires dilution of the solution and increases the volume of the solution to be processed which leads to increased waste. Such a TFF step must be operated in batch (as opposed to a continuous processing model), with several disadvantages, such as an increased equipment footprint, a longer total processing time and increased human intervention. Finally, traditional TFF operations are difficult to scale up or scale down. For instance, TFF cannot be used for carrier discovery and screening because the minimal volume needed is too large. The process has a strong impact on the carrier. Hence, translation from discovery to pre-clinical volumes is currently unpredictable and challenging.

Therefore, there is a need for a more efficient, less harsh method for separating organic solvent from produced lipid-based carriers, preferably one which is scalable from discovery to GMP commercial production and can be operated in continuous mode.

SUMMARY OF THE INVENTION

The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a method for manufacturing a lipid-based carrier, according to claim 1. Particularly, the invention relates to a method comprising mixing two solutions in two different phases, aqueous and organic phase where the organic phase comprises lipids to form a lipid-based carrier suspension, removing at least part of said organic phase from said suspension by an evaporation process, and collecting the at least part of the suspension comprising formed lipid-based carriers.

Preferred embodiments of the method are shown in any of the claims 2 to 26.

In a second aspect, the present invention relates to a method for manufacturing a lipid nanoparticle (LNP), according to claims 27-38.

In a third aspect, the present invention relates to a method for storing a lipid-based carrier suspension, according to claims 39-53.

In a last aspect, the present invention relates to a system for manufacturing a formulation comprising one or more of lipid-based carriers, according to claim 54.

Summarized, the invention pertains at least to the following embodiments:

1. A method for manufacturing a lipid-based carrier, comprising: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming a lipid-based carrier suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said lipid-based carrier in said suspension; and c. collecting said concentrated lipid-based carrier suspension. 2. The method according to embodiment 1, wherein said concentration of said lipid-based carrier in said concentrated lipid-based carrier suspension is at most 100 times higher than a concentration of said lipid-based carrier in said lipid-based carrier suspension.

3. The method according to embodiment 1 or 2, wherein said concentrated lipid- based carrier suspension maintains its stability.

4. The method according to the previous embodiments, wherein the aqueous phase solution comprises one or more polynucleotides.

5. The method according to the previous embodiments, wherein said lipid-based carrier does not contain said one or more polynucleotides.

6. The method according to the previous embodiments, wherein said aqueous phase solution does not contain said one or more polynucleotides.

7. The method according to the previous embodiments, wherein a second aqueous phase solution is added to said collected concentrated lipid-based carrier suspension before, during or after the evaporation process, wherein said second aqueous phase optionally comprises one or more polynucleotides.

8. The method according to the previous embodiments wherein said second aqueous solution is added during or after the evaporation process.

9. The method according to the previous embodiments, wherein said lipid-based carrier comprises said one or more polynucleotides.

10. The method according to the previous embodiments, wherein a total surface to volume ratio of said organic phase solution and said gas is maximized.

11. The method according to any of the embodiments, wherein the evaporation process is spray evaporation or film evaporation.

12. The method according to the embodiments 11, wherein said spray evaporation is applied by aerosolizing the suspension to generate an aerosol and condensing said aerosol to form said concentrated lipid-based carrier suspension, wherein said one or more organic solvents are evaporated.

13. The method according to the embodiments 11 or 12, further comprising spraying said lipid-carrier based suspension through a nozzle. The method according to the embodiment 13, wherein said nozzle has a diameter of 0.1 to 5 mm. The method according to embodiment 13 or 14, further comprising setting a temperature of said nozzle at between 20 and 60°C, resulting in an outlet temperature of between 4 and 40°C. The method according to any of the embodiments 11 to 15, wherein said spraying of said lipid-carrier based suspension comprises a flow rate ranging from 3 mL/min to 5 L/min. The method according to any of the embodiments 1 to 11, wherein said evaporation is film evaporation, said method further comprises applying a vacuumpressure between 0.0001 to 1 bar. The method according to any of the embodiments 1 to 16, wherein said evaporation process is spray evaporation, said gas pressure is between 2 to 6 bar. The method according to embodiment 11 or 17, wherein said film evaporation is thin film evaporation or falling film evaporation. The method according to embodiments 11, 17, or 19 wherein said film evaporation is carried out at a temperature ranging between 5 to 50°C and at a flow rate of said lipid carrier based suspension ranging from 0.1 to 1000 mL/min. The method according to the embodiments 111, 17, 19 or 20, wherein said film evaporation is carried out at a minimum pressure of 0.001 bar. The method according to any of the previous embodiments, wherein the volume of said collected concentrated lipid-based carrier suspension is at least 0.01 % v/v of the aqueous phase solution of step a. The method according to any of the previous embodiments, wherein said mixing of step a occurs by means of impingement jet mixing and/or microfluidic mixing, wherein said microfluidic mixing is laminar microfluidic mixing or chaotic microfluidic mixing. 24. The method according to any of the previous embodiments, wherein the formed lipid-based carrier suspension is diluted before step b.

25. The method according to any of the previous embodiments, wherein the collected concentrated lipid-based carrier suspension is mixed with a solution containing a buffer, a cryoprotectant, or a mixture thereof, to protect the lipid-based particles from aggregation and degradation during collection and/or to adjust osmolality and/or pH to meet requirements for injectables.

26. The method according to any of the previous embodiments, wherein the collected concentrated lipid-based carrier suspension is mixed with a solution containing a buffer, a cryoprotectant, or a mixture thereof, to make said collected concentrated lipid-based carrier suspension an injectable solution at a physiological pH.

27. The method according to any of the previous embodiments, wherein the collected concentrated lipid-based carrier suspension is formulated in an injectable solution at a physiological pH of between 7.35 and 7.45.

28. The method according to any of the previous embodiments, wherein said method is further followed by a post-treatment step, preferably a sterilizing filtering step.

29. The method according to previous embodiments, wherein the organic solvent in the organic phase solution is selected from the group of ether, chloroform, benzene, acetone, and alcohol, wherein said alcohol is preferably selected from the group consisting of ethanol, methanol, and isopropanol.

30. The method according to previous embodiments, wherein the one or more lipids in the organic phase solution comprise one or more of the following: cholesterol, a phospholipid, a cationic lipid, a PEGylated lipid, an ionizable lipid, or a mixture thereof.

31. The method according to previous embodiments, wherein the one or more polynucleotides in the aqueous phase solution comprise RIMA, DNA, siRNA, miRNA, mRNA, saRNA, circRNA or a mixture thereof. 32. The method according to previous embodiments, wherein the one or more polynucleotides in the aqueous phase solution comprise RIMA wherein said RNA is a modified RNA.

33. The method according to any of the previous embodiments, further comprising reducing shear stress on said one or more carriers.

34. The method according to any of the previous embodiments, further comprising reducing oxidative stress on said one or more carriers by using nitrogen as the carrier gas.

35. The method according to any of the previous embodiments, wherein said evaporation process generates a continuous process of manufacturing said formulation.

36. A system for manufacturing a formulation comprising one or more lipid-based carriers comprising polynucleotides, wherein, said system comprises one or more mixing units, wherein said mixing units are impingement jet mixing units and/or microfluidic mixing units, suited to mix at least one aqueous solution comprising polynucleotides with an organic phase solution comprising one or more lipids and/or one or more polymers thereby forming a formulation comprising one or more carriers, wherein said one or more microfluidic mixing units are fluidly connected to a spray-drying apparatus or a film evaporation apparatus for removal of at least a part of the organic solvents from said formulation.

37. The system according to embodiment 36, further comprises one or more filtering unit, one or more condenser, and/or one or more buffering tank fluidly connected to spray-drying or evaporation apparatus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to a method and a system for manufacturing a formulation comprising one or more lipid-based carriers. In some embodiments, the carriers consist of cargo such as pharmaceutical ingredients (API), particularly polynucleotides like DNA or RNA. An example of such a carrier is a lipid nanoparticle (LNP). The method involves the mixing of two or more liquids to form lipid-based carriers. For instance, an aqueous solution is mixed with one or more solvents containing lipids and/or polymers, resulting in a formulation comprising one or more lipid-based carriers. Subsequently, one or more organic solvents are removed by means of an evaporation step.

After the fabrication of lipid-based carriers, such as nanocarriers, by mixing an aqueous solution and organic solvent, the organic solvent needs to be removed. Traditional methods, including TFF (Tangential Flow Filtration), used for removing organic solvent from a solution containing carriers, can lead to significant shear stress, which negatively affects the quality of the carriers. Additionally, these methods require the use of large buffer volumes to achieve the desired dilution. Moreover, traditional methods typically operate in batches, which brings several other disadvantages, such as an increased equipment footprint, a longer total processing time, increased human intervention, and difficulties in scaling up production. Utilizing TFF for the removal of organic solvents particularly becomes a disadvantage when scaling-up from discovery to Phase 1 and above stages, as the discovery stage requires the use of consumables such as dialysis cassettes or centrifuge tubes and does not allow the use of TFF. A switch to the TFF method can have a significant toll on the carrier.

It is an objective of the present invention to provide a method for manufacturing lipid-based carriers that meet the regulatory requirements for medicinal products.

It is also an objective of the present invention to provide a method for manufacturing lipid-based carriers that offers a suitable solution for the removal of organic solvent from the manufactured carriers, thereby meeting current regulatory requirements.

Another objective of the present invention is to provide a method for manufacturing lipid-based carriers that incorporates an organic solvent removal approach with reduced or no adverse impact on the quality of the manufactured lipid-based carriers.

Furthermore, it is another objective of the present invention to provide a method for manufacturing lipid-based carriers that optimizes the efficient removal of organic solvent from the mixture of aqueous phase and organic solvent, comprising the produced carriers. This can be achieved, for example, by maximizing the total surface to volume ratio of the liquid-gas.

Additionally, it is also an objective of the present invention to provide a continuous manufacturing method for manufacturing lipid-based carriers that is suitable for scaling up. Definitions

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

As used herein, the following terms have the following meanings:

"A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

"About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

"Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

"Carrier", "nanocarrier" is material being used as a transport module for another substance, such as a drug. The term "carrier" is used if the diameter of the material is ranging from 1 nm to 10 pm whereas the term "nanocarrier" is used to define materials with ranging sizes of diameter 1 nm to 1000 nm. Therefore, the term "carrier" as used herein includes the "nanocarriers". The term "carrier" as used herein also refers to nanocarriers, nanoparticles, nanoparticle drug carriers. Commonly used carriers include micelles, polymers, carbon-based materials, liposomes and other substances. Other non-limiting examples of carriers include polymer-based carriers, polymer conjugates, polymeric nanoparticles, lipid-based carriers, polymer lipid hybrid carriers, dendrimers, carbon nanotubes, gold nanoparticles and the silica nanoparticle.

Non limiting examples of lipid-based carriers includes liposomes, solid-lipid nanoparticles, nanostructured lipid carriers, nanoemulsions. In an embodiment, "lipid-based carrier" as used herein refers to a lipid nanoparticle (LNP). In another embodiment, "lipid-based carrier" as used herein refers to lipoplexes. Lipid-based carrier as used herein can further comprise polymers. Non limiting examples of polymer comprising lipid-based carriers includes polymer core-lipid shell nanoparticles, hollow core/shell lipid-polymer-lipid hybrid nanoparticles, lipid bilayer-coated polymeric particle, and mixed lipid polymer nanoparticles.

As used herein, the term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

The term "lipid nanoparticle ("LNP")" refers to a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and comprises a plurality of lipid molecules physically associated with each other by intermolecular forces. An active agent or therapeutic agent, such as a nucleic acid, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells.

As such, , the phrase "lipid nanoparticle (LNP)" also refers to a nanosized vesicle or carrier comprising one or more lipids (e.g., cationic and/or non-cationic lipids, cholesterol, phospholipid, a PEGylated lipid, an ionizable lipid, a fusogenic lipid, or a mixture thereof). An LNP usually comprises at least an ionizable lipid, a sterol, a helper lipid (usually a zwiterionic phospholipid) and a PEGylated lipid.

Lipoplexes are complexes formed by ion pairing between cationic lipids and negatively charged nucleic acids (DNA or RNA). Lipoplexes are multilamellar vesicles or condensed complexes where nucleic acids are either intercalated within the lipid layers or adsorbed on the surface of the vesicle. As used herein, the term "cationic" means that the respective structure permanently bears a positive charge.

In the context of the present disclosure, the term "ionizable" in the context of a compound or lipid means the presence of any uncharged group in said compound or lipid which is capable of associating with an ion (usually an H⁺ ion) and thus itself becoming positively charged (also referred to as "cationizable"). Alternatively, any uncharged group in said compound or lipid may yield an ion (usually an H⁺ ion) and thus becoming negatively charged. In the context of the present disclosure any type of ionizable lipid can suitably be used.

The term "cationizable" or "ionizable cationic lipid" as used herein means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also, in non-aqueous environments where no pH value can be determined, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the respective cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of cationizable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e. under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In other embodiments, it is preferred that the cationizable compound or moiety is predominantly neutral at physiological pH values, e.g. about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the cationizable compound or moiety is about 5 to about 7.

The term "permanently cationic" as used herein will be recognized and understood by the person of ordinary skill in the art, and means, for example, that the respective compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge results from the presence of a quaternary nitrogen atom. Where a compound carries a plurality of such positive charges, it may be referred to as permanently polycationic, which is a subcategory of permanently cationic.

In the context of the present disclosure, the term "PEGylated lipid" is meant to be any suitable lipid modified with a PEG (polyethylene glycol) group.

In the context of the present disclosure, the term "sterol", also known as steroid alcohol, is a subgroup of steroids that occur naturally in plants, animals and fungi, or can be produced by some bacteria. In the context of the present disclosure, any suitable sterol may be used, such as selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol. As used herein, the term "aqueous" refers to a composition comprising in whole, or in part, water.

As used herein, the term "lipid solution" refers to a composition comprising in whole, or in part, an organic solvent having a lipid.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to genetic material (e.g., DNA or RIMA), and when such terms are used with respect to the lipid nanoparticles, they generally refer to the genetic material encapsulated by such lipid nanoparticles.

As used herein, the terms "API", "active pharmaceutical ingredient", and "active pharmaceutical agent" are used interchangeably to refer to a biologically active compound. API generally refers to the substances in pharmaceuticals that are responsible for the beneficial health effects experienced by consumers. Examples of APIs include, without limitation a nucleic acid, a polynucleotide, such as RNA and DNA, a peptide, a polypeptide, an excipient, a chemical substance and intermediates, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer or any combination thereof.

An "active ingredient" or "active principle" is any component that provides biologically active or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the body of humans or animals.

An "aerosol", "droplets" or "atomized droplets" as used interchangeably here refer to small liquid droplets that are produced by atomization which can be a suspension of fine solid or liquid particles in a gas. It is typically composed of tiny particles that are dispersed and suspended in the air or another gas medium. Atomization can be achieved through various methods, such as spraying, nebulizing, or using specialized devices like atomizers. These droplets are typically very small and have a high surface-to-volume ratio, which allows for efficient evaporation.

As used here in "Spray drying" or "spray evaporation" is a process used to remove part of a liquid from a liquid, suspension or slurry. It involves atomizing the liquid feed material into fine droplets and then rapidly evaporating liquid from those droplets in a heated chamber.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of acomposition.

Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Description

In the first aspect, the invention pertains to a method for manufacturing lipid-based carriers. In an embodiment, said method comprises the following steps: a) Mixing an aqueous phase solution with an organic phase solution that comprises one or more lipids, thus forming a lipid-based carrier suspension. b) Removing at least a portion of the organic phase from the suspension through an evaporation process. c) Collecting the concentrated lipid-based carrier suspension.

In embodiments of step b of the method disclosed herein, the mixture comprising the aqueous phase and the organic phase is contacted with a gas, causing the organic solvent to be removed through evaporation. However, at least a portion of the aqueous solution is also removed during this process. Since the organic solvent is more volatile than the aqueous phase, a larger amount of it is evaporated, but some water is also lost, resulting in a concentrated lipid-based carrier solution.

In an embodiment, the organic phase solution comprises one or more lipids dissolved in one or more organic solvents.

In an embodiment according to the disclosed method, the aqueous phase solution comprises one or more polynucleotides. The delivery of genetic material to the cell cytosol has many applications, including for instance nucleic acid-based prophylactic vaccines.

RNA therapeutics for instance comprise a rapidly expanding category of drugs that will change the standard of care for many diseases and actualize personalized medicine. These drugs are cost effective, relatively simple to manufacture, and can target previously undruggable pathways. However, employing nucleic acids as therapeutics is challenging because they are susceptible to degradation by nucleases, contribute to immune activation and have unfavorable physicochemical characteristics that prevent facile transfection into cells. Safe and effective nucleic acid therapeutics therefore require sophisticated delivery platform technologies.

In an embodiment, said lipid-based carriers are used for the delivery of polynucleotides. As such, in an embodiment, said aqueous phase solution comprises one or more polynucleotides as described above.

The composition of lipid-based carriers may include an ionizable cationic lipid and three neutral lipids: phospholipid, cholesterol, and lipid-anchored polyethylene glycol (PEGylated lipid).

As such, in an embodiment said one or more lipids in the organic phase solution comprise one or more of the following: a sterol (for instance cholesterol), a phospholipid, a cationic lipid, a PEGylated lipid, an ionizable lipid, a fusogenic lipid, or a mixture thereof.

In an embodiment, said one or more lipids in the organic phase solution consist of an ionizable cationic lipid, a sterol, a phospholipid and a PEGylated lipid.

Sterols are known to modulate membrane fluidity and stability, making them important constituents of lipid carriers, more specifically of LNPs. Suitable sterols for the lipid carrier according to disclosure can be selected from the group of cholesterol, sitosterol, sitosterol-amino acid conjugates, stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11-dehydroergosterol, and hydroxycho I esterol. In an embodiment, said sterol is present in the lipid carrier produced by the method of the current invention at a mass fraction of 15-60% (w/w), such as 15%, 16%, 17%,

18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,

32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% (w/w) or any value in between. Phospholipids are essential components of biological membranes and can be used in the lipid carrier to enhance stability and biocompatibility. Suitable phospholipids for the lipid carrier can be selected from the group of phosphatidylcholines, phosphatidylethanolamines, and sphingolipids. In an embodiment, said phospholipid is present in said lipid carrier produced by the method of the current invention at a mass fraction of 5-35% (w/w), or 10-30% (w/w), such as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w/w) or any value in between.

In embodiments, the lipid carrier further comprises a PEGylated lipid, PEG lipid or PEG conjugate to increase the circulation time of said lipid carrier and reduce an unwanted host response.

PEGylated lipids are lipids that have been modified by the attachment of polyethylene glycol (PEG) chains, which can improve the stability and circulation time of the lipid carrier. Suitable PEGylated lipids for the lipid carrier can be selected from the group consisting of PEG-diacylglycerols (PEG-DAG), PEG-dialkyloxypropyls (PEG-DAA), PEG-phospholipids, and PEG-ceramides. PEG-ceramides are chosen from PEG- ceramides having alkyl chain with C16 and PEG with an MW from 500 to 2000 e.g., C16 PEG500, C16 PEG750, C16 PEG 1000, C16 PEG 1250, C16 PEG 1500, C16 PEG 1750, C16 PEG2000 and any ranges and subranges there in between. The concentration of PEGylated lipids in the lipid carrier can be adjusted as needed. In an embodiment, said PEGylated lipid is present in said lipid carrier produced by the method of the current invention at a mass fraction of 1-15% (w/w), or 1-10% (w/w), such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (w/w) or any value in between.

PEG is often used for its stealth functions in nanoparticle formulations because it is a hydrophilic and flexible polymer. The conjugation of PEG to the lipid carrier reduces the interaction of the lipid carrier with plasma proteins. As a result, this prevents plasma proteins from adsorbing to the surface of the lipid carrier and consequent uptake of the lipid carrier by the reticuloendothelial system (RES). The conjugation of PEG or PEGylation allows the lipid carrier to circulate within the body for a longer period of time, extending their circulation half-life and, consequently, increasing the accumulation of the lipid carrier within the target tissues. Suitable cationic lipids can be selected from the group consisting of DOTAP (1,2- dioleoyl-3-trimethylammonium-propane), DC-cholesterol (30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol), DORI (N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(oleoyloxy)propan-l-aminiumbromide), DOSPA (2,3-dioleyloxy-N- (2-(sperminecarboxamido)ethyl)-N,N-dimethyl-l-propanaminium Trifluoroacetate), ICE (imidazole cholesterol ester), DOTMA (1,2-di-O- octadecenyl-3- trimethylammonium propane), or any combination thereof. In an embodiment, said cationic lipid is present in said lipid carrier produced by the method of the current invention at a mass fraction of 0.1-10% (w/w), 10-20% (w/w), 20-30% (ww/w), 30-40% (w/w), 40-50% (w/w) or 50-60% (w/w). In an embodiment, said cationic lipid is present in said lipid carrier produced by the method of the current invention at a mass fraction of 0.1-50% (w/w), 0.1-40%, 0.1-30%, 0.1-20% or 0.1-10% (w/w). In an embodiment, said cationic lipid is present in said lipid carrier produced by the method of the current invention at a mass fraction of 0.2-10% (w/w), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 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% (w/w) or any value in between.

However, in an embodiment, an ionizable cationic lipid can be provided in the organic phase solution. In an embodiment, said ionizable cationic lipid has an apparent pKa between 5 and 7.

The ionizable cationic lipids are complexed with polyanionic RNA through ion pairing interactions to enable its encapsulation by the neutral lipids and facilitate cellular uptake and endosomal escape. The ionizable cationic lipid is considered to be the most important factor for improving encapsulation efficiency and intracellular delivery, as this is the component responsible for complexing with RNA cargo.

In an embodiment, in order for the ionizable cationic lipid to be able to associate with an ion and thus itself become positively charged after mixing of the aqueous phase solution with the organic phase solution, the aqueous phase solution has an acidic pH, such as a pH below 7, or below 6. In an embodiment, the aqueous phase solution has a pH between 3 and 6. In an embodiment, the aqueous phase solution has a pH below the pKa of the ionizable lipid, such as a pH with one unit below the pKa value.

In an embodiment, said organic phase solution further comprises one or more calixarenes, thus forming a lipid-based carrier comprising one or more calixarenes. In an embodiment, an ionizable calixarene is used to replace the traditional ionizable lipid in lipid-based carriers. In an embodiment, the resulting lipid-based carrier can comprise 4 components: an ionizable calixarene, a helper lipid, a sterol, and a PEG lipid.

Calixarenes are platforms which facilitate the synthesis of ionizable compounds with multiple amine heads, meaning that the charge density (number of amines/molecule) could be increased easily. Besides reducing the amount of ionizable component necessary for efficiently encapsulating nucleic acids, this property also facilitates the encapsulation of very long RNA, such as self-amplifying RIMA (saRNA), by increasing the number of amines without changing the mass ratio between the ionizable component and RNA, while this task remains challenging with the current LNP technology having a formulation that does not comprise calixarenes.

In an alternative embodiment, a cationic calixarene is incorporated as a fifth component into lipid-based carriers made of an ionizable lipid, a helper lipid, a sterol, and a PEG lipid. As described above and similar to the ionizable calixarenes, the charge density (number of amines/molecule) could be increased easily in cationic calixarenes, facilitating the encapsulation of nucleic acids, especially that of very long RNA, such as self-amplifying RNA (saRNA).

The calixarene concentration in the resulting lipid-based carrier can range from 0.1- 60 mol%. The calixarene concentration in the resulting lipid-based carrier can range from 0.1-50 mol%, from 0.1-44% mol%, from 0.1-40 mol%, from 0.1-30 mol%, from 0.1-20 mol%, from 0.1-10 mol%, from 0.1-5 mol% or from 1-60 mol%, from 10-60 mol%, from 20-60 mol%, from 30-60 mol%, from 35-60 mol% or any ranges and subranges therein between.

When the organic phase solution comprising an ionizable cationic lipid (or an ionizable cationic calixarene) is mixed with an aqueous phase solution having an acidic pH (below the pKa of the ionizable lipid/calixarene) and comprising one or more negatively charged polynucleotides, the ionizable cationic lipids/calixarenes become positively charged and are able to complex with the polyanionic polynucleotides through ion pairing interactions, thereby enabling the encapsulation of the polynucleotides by the neutral lipids and facilitating their cellular uptake and endosomal escape after delivery to a subject to which the lipid carrier is administered. As the evaporation process does not involve a concentration step using a membrane, the method of the current invention is especially well suited to concentrate lipid- based carriers comprising charged lipids, as said charged lipids could otherwise adhere to the membranes used in membrane-based concentration processes and be lost from the lipid-based carrier suspension, leading to a significant loss in the lipid- based carriers production yield.

In other embodiments of the disclosed method, the aqueous phase solution does not contain said one or more polynucleotides.

In an embodiment, the method further comprises the mixing of a second aqueous solution. The second aqueous solution comprises one or more polynucleotides. According to the disclosure, the second aqueous phase comprising one or more polynucleotides is added to lipid-based carrier suspension. In further embodiments, the second aqueous solution is added before, during, or after the evaporation process to the lipid-based carrier suspension. In further embodiments, a second aqueous phase solution is added to the collected concentrated lipid-based carrier suspension.

According to the disclosure polynucleotides such as RNAs or DNAs can be selected from any modified, unmodified, natural or synthetic polynucleotides.

The terms "DNA" or "DNA molecule" are used herein to generally refer to any type of DNA. Non-limiting example of DNA includes any (single-stranded or doublestranded) DNA, preferably, without being limited thereto, e.g. genomic DNA, singlestranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a DNA oligonucleotide, a short DNA oligonucleotide ((short) oligodesoxyribonucleotides), viral DNA, or a combination thereof.

The terms "RNA" or "RNA molecule" are used herein to generally refer to any type of RNA. Non-limiting example of RNA includes long-chain RNA, coding RNA, noncoding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), Trans amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), transcription start site-associated (TSSa-)RNAs, upstream antisense (ua) RNAs, promoter upstream transcripts (PROMPTS), or a combination thereof.

According to the present disclosure, the term "RNA" includes and preferably relates to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide, a polypeptide, or protein. mRNA typically comprises a 5' untranslated region (5' -UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the disclosure, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

In embodiments, the manufactured lipid-based carrier according to the disclosed method does not contain one or more polynucleotides. In other embodiments, the manufactured lipid-based carrier comprises one or more polynucleotides.

In an embodiment, the encapsulation efficiency of one or more polynucleotides is at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%.

According to the present disclosure, the formed lipid-based carrier suspension by mixing of aqueous and organic phase solvents is formed lipid-based carriers in a mixture liquid of aqueous and organic phase solvents.

In embodiments, the organic phase solvent and aqueous phase solvent are miscible, which means they can mix together at least in specific portions, preferably in all portions. In embodiments, the organic solvent and aqueous solvent are miscible following the mixing step according to the disclosed method. The removal of at least part of the organic solvent or solvents is performed by an evaporation process. In embodiments, the evaporation process comprises separating the organic solvent or solvents from the suspension by transporting the one or more organic solvents from said suspension to a gas. In embodiments, during the evaporation process of said organic phase solvent is in direct contact with the gas, allowing the solvents to evaporate. As a result, the concentration of the lipid-based carrier in the suspension increases. In embodiments, the evaporation step results in the decrease of the concentration of organic solvent in lipid-based carrier suspension.

According to embodiments, once the evaporation process is complete, the volume of the suspension comprising formed lipid-based carriers is reduced, and the remaining suspension contains a higher concentration of the lipid-based carrier and a lower concentration of organic solvent. In embodiments, this concentrated suspension is then collected for further use or processing.

In an embodiment, the lipid-based carriers obtained through the manufacturing method disclosed herein maintain a liquid form even after the removal of one or more solvents. It is important to note that the collected lipid-based carriers are not completely dried out following the evaporation process.

Traditionally, in the pharmaceutical and gene delivery systems field, evaporation drying has been utilized to obtain solid particles, such as powders. However, the present invention differentiates itself from these previous disclosures by selectively removing a portion of the liquid from the manufactured lipid-based carriers, without achieving or allowing complete dryness.

Furthermore, the prior art primarily focuses on eliminating liquid content, regardless of its phase, from active principles. In contrast, in some embodiments, the disclosed invention enables a more targeted removal of organic solvents from a mixed solution comprising aqueous and organic solvents by utilizing a unique combination of specific parameters.

In some embodiments, the concentration of lipid-based carrier in the concentrated lipid-based-carrier suspension collected after the evaporation step is at most 100 times higher than a concentration of lipid-based carrier in the lipid-based carrier suspension prior to the evaporation step. For example, the concentration of lipid- based carrier in concentrated lipid-based suspension is at most 100 times, at most 75 times, at most 50 times, at most 45 times, at most 40 times, at most 35 times, at most 30 times, at most 27 times, at most 25 times, at most 23 times, at most 20 times, at most 18 times, at most 16 times, at most 14 times, at most 12 times, at most 10 times, at most 8 times, at most 6 times, at most 4 times, at most 2 times higher than a concentration of lipid-based carrier in lipid-based carrier suspension prior to evaporation step.

In some embodiments, the concentration of lipid-based carrier in the concentrated lipid-based-carrier suspension collected after the evaporation step is at least 0.01 times higher than the concentration of lipid-based carrier in the lipid-based carrier suspension prior to the evaporation step. For example, at least 0.05 %, at least 0.1 %, at least 0.5 %, at least 1 %, at least 1.5 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 10 %, at least 15%, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 % higher than the concentration prior to evaporation step.

In embodiments, the volume of aqueous phase in collected concentrated lipid-based carrier suspension is at least 0.01 % v/v of the initial aqueous phase solution described in step a of the disclosed method, such as at least 0.05 %, as at least 0.1 %, at least 0.5 %, at least 1 %, at least 1.5 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 10 %, at least 15%, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 % of the initial aqueous phase solution.

In embodiments, lipid-based carriers collected after the evaporation process is not fully dry such as in dry powder form. The manufactured and collected lipid-based carrier at the end of the method as disclosed here are in liquid.

In some embodiments of the disclosed method, the manufactured lipid-based carriers maintain its stability in concentrated suspension. It is important to note that complete drying of lipid-based carriers, such as LNPs (lipid nanoparticles), is not desirable due to the negative impact of the drying process on the quality and stability of the carriers. However, there are certain applications, such as treating pulmonary diseases, where localized delivery to the lungs through inhalation is more advantageous than systemic delivery. In such cases, complete drying may be necessary. On the other hand, for applications where the lipid-based carriers need to be in solution form, the organic phase still must be removed from the liquid to meet regulatory requirements for medicinal products. Traditionally, organic solvent removal is achieved through diafiltration, which involves multiple cycles of concentration and dilution with an aqueous buffer until the desired residual organic solvent concentration is reached. However, this diafiltration process, known as tangential flow filtration (TFF), can exert significant shear stress on the lipid-based carriers, negatively affecting their quality. In the disclosed method, the inventors successfully produced lipid-based carriers in a solution where the organic solvents are removed to meet medicinal product requirements without the adverse effects of TFF or complete drying. Instead, they utilized an evaporation process within the manufacturing method, which overcomes the drawbacks of traditional methods and ensures the desired removal of organic solvents. The fact that the ethanol is removed quickly from the suspension comprising the manufactured lipid-based carriers increases the stability of the manufactured carriers. Even further, utilizing an evaporation procedure with specific parameters as disclosed in this invention where the complete drying of lipid-based carriers is avoided, solves the problem of reduced stability of manufactured lipid-based carries during the solvent removal step.

In an embodiment, said method further comprises reducing shear stress on said one or more lipid-based carriers. In an embodiment, said method further comprises reducing shear stress by at least 10%, preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40%, on said one or more carriers. As described above, "lipid-based carriers" as used herein can comprise LNPs, polymeric, or hybrid carriers.

During the evaporation process according to the disclosed method, the one or more organic solvents are transported from the organic phase solution to a gas and the organic phase solution is in direct contact with said gas. In particular, the evaporation process does not involve a membrane separation process, allowing direct contact between the solvent and the gas to achieve more efficient transport of solvent from a solution to a gas compared to pervaporation. In addition, as the evaporation process does not involve a concentration step using a membrane, the method of the current invention is especially well suited to concentrate lipid carriers comprising charged lipids, as said charged lipids would otherwise adhere to the membranes used in membrane-based concentration processes.

In an embodiment of the method as disclosed herein, the lipid-based carrier manufacturing does not involve tangential filtration with a membrane, such as a TFF membrane. In some embodiments, the total surface size of the interface of said organic phase solution and said gas is maximized to allow an efficient removal of said organic solvent. In some embodiments, the evaporation process is chosen between spray evaporation or film evaporation in order to maximize the interface between the organic phase solution and the gas. This objective is achieved either by atomizing (spray evaporation) the suspension containing the formed lipid carriers, which increases the liquid's surface area in direct contact with the gas, or by flowing the suspension over falling tubes (falling film evaporation) or spreading it over an evaporation surface (horizontal thin film evaporation) to create a thin film. These methods enhance the liquid's surface area in direct contact with the gas, facilitating rapid evaporation.

In one embodiment, the removal of at least part of the one or more organic solvents is accomplished through a spray evaporation process, also known as spray drying. Spray drying is a widely used technique in the production of dry pharmaceuticals, which involves converting a liquid or slurry into a dry powder form through rapid solvent evaporation. The idea of this invention is to spray the LNP-containing mix of water and organic solvent to evaporate at least some of the organic solvent. The most efficient transport of solvent from a solution to a gas is done when the gas is in direct contact with the solution and without having a physical barrier such as a membrane in between them. Another parameter to consider in order to increase the overall mass-transport is the total surface of the interface, which must be maximal. In embodiments, spraying the solution into an aerosol maximized these two aspects by maximizing the surface of the liquid-gas direct interface.

In embodiments of the disclosed method, the suspension is aerosolized to generate an aerosol by atomizing the liquid into small droplets, thereby increasing the liquid's surface area to facilitate faster evaporation. The atomization step is followed by a drying process, in which the droplets are introduced into a drying chamber or tower and exposed to hot air or a gas stream. The heat from the air or gas causes the organic solvent to evaporate rapidly, traditionally resulting in the formation of solid particles.

In embodiments of the disclosed method, at least part of the one or more organic solvents is removed from the generated aerosol or droplets without allowing complete drying of the aerosol containing the formed lipid-based particles. This is different than any known prior art. Unlike the conventional procedure, in further embodiments, the disclosed method additionally involves condensing the aerosol to form a concentrated lipid-based carrier suspension, where the one or more organic solvents are evaporated and concentrated. In some embodiments, the method described here involves utilizing spray drying or spray evaporation to remove organic solvents and obtain a concentrated lipid-based carrier suspension, but without completely drying the aerosol containing the lipid- based particles. Spray drying is a common technique in pharmaceutical production, to produce completely dry particles but not for removing one phase of liquid from a mixture of different phases of liquids. In some embodiments, to a smaller extent, the aqueous phase may also be removed during the spray evaporation. Compared to traditional Tangential Flow Filtration (TFF), the spray evaporation step does not require a diafiltration buffer and therefore reduces process footprint and waste. As such, in an embodiment of the method, said evaporation process does not comprise utilizing a diafiltration buffer. In embodiments, the footprint of the manufacturing process is minimized for example down to 10 m², 9 m², 8 m², 7 m², 6 m², 5 m², 4 m², 3 m², 2 m², 1 m², 0,5 m². It is also lower in shear stress since it uses a liquidgas interface instead of a membrane to remove the liquid.

Further, by rapidly removing the solvent, the disclosed method, comprising spray drying, helps preserve the stability and extend the shelf life of the lipid-based carriers. In embodiments, the stability of manufactured carriers is preserved in concentrated suspension.

In some embodiments, operations such as applying a vacuum to the gas, using a purge gas or dry air, or applying heat to the liquid, the suspension, the gas or any possible combination thereof are used during the spraying process to accelerate the evaporation rate and/or to create a sink condition.

It should be noted that any commercially available spray dryer with optimized parameters can be used to remove organic solvent from suspension. In embodiments, a commercially available spray dryer such as Butchi B-290 is used to perform spray evaporation spay of the disclosed method.

Spray drying is a continuous and scalable process, making it suitable for large-scale production. It offers faster drying times compared to traditional methods and can handle heat-sensitive materials. As such, in embodiments disclosed method, is a continuous and scalable process.

In embodiments, the lipid-carrier-based suspension is aerosolized by using one of the various known techniques, such as pressure nozzles, rotary atomizers, or centrifugal atomizers. In one embodiment, aerosolization is performed by spraying said suspension through a nozzle. In embodiments said nozzle has a diameter of 0.1 mm to 5mm, such as 0.1mm to 4.5 mm, 0.1mm to 4 mm, 0.1 mm to 3.5 mm, 0.1mm to 3 mm, 0.1mm to 2.5 mm, 0.1 mm to 2 mm, 0.1 mm to 1.5 mm, 0.1 mm to 1 mm, 0.1 mm to 0.75 mm, 0.1 mm to 0.5 mm, or 0.5 mm to 5 mm, 0.75 mm to 5 mm, 1 mm to 5 mm, 1.5mm to 5 mm, 2 mm to 5 mm, 2.5 mm to 5 mm, 3 mm to 5 mm, 3.5 mm to 5 mm, 4 mm to 5 mm, 4.5 mm to 5 mm, or 0.1 mm to 4.5 mm, 0.2 mm to 4 mm, 0.3 mm to 3.5 mm, 0.5 mm to 2.5 mm, 0.6 mm to 2 mm, 0.7 mm to 1.5 mm and all the ranges and -sub-ranges therein between. The nozzle diameter is preferably between 0.5 mm and 2.5 mm. The nozzle size affects the size of the formed droplets. In embodiments, the nozzle size is selected based on the droplet size, and/or the interface surface area, and/or based on the desired evaporation rate.

The size of the droplets has a significance in the maximizing interface surface of the solvent and the gas, as the smaller droplets have a larger surface area in proportion to their volume compared to larger droplets. In embodiments, the maximum size of formed droplets is 200 pm. in embodiments, the maximum size of the droplets is 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 115 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, and all the sizes therein between, in preferred embodiments the size of the formed droplets is 80 pm. It should be noted that as the organic solvent such as ethanol evaporates from the droplet, the droplet size will quickly reduce. In embodiments, the droplet size is smaller than 80 pm, particularly after the evaporation process starts. It should be also noted that a larger droplet size will decrease the total mass transfer but also decrease the shear stress. Therefore, it's a non-linear optimum and it will be obvious to a skilled person in the field that any droplet size to balance these two effects can be selected.

In embodiments, the temperature of said nozzle is at between 10 and 80°C such as between 15°C and 80°C, 10°C and 80°C, 25°C and 80°C, 30°C and 80°C, 35°C and 80°C, 40°C and 80°C, 45°C and 80°C, 10°C and 75°C, 10°C and 70°C, 10°C and 65°C, 10°C and 60°C, 10°C and 55°C, 10°C and 50°C, 10°C and 45°C, 10°C and 40°C, 10°C and 35°C, 15°C and 75°C, 20°C and 70°C, 20°C and 65°C, 20°C and 50°C, 20°C and 60°C, 25°C and 65°C, 30°C and 60°C, 30°C and 50°C, 35°C and 45°C, and all the ranges and subranges there in between. The nozzle temperature is preferably between 20°C and 60°C, even more preferably between 20°C and 50°C. The nozzle temperature also referred to as inlet temperature impacts the outlet temperature. In embodiments, the outlet temperature is between 4°C and 50°C such as between 5°C to 50°C, 10°C to 50°C, 15°C to 50°C, 20°C to 50°C, 25°C to 50°C, 30°C to 50°C, 35°C to 50°C, 40°C to 50°C, 45°C to 50°C, 4°C to 50°C , 4°C to 45°C, 4°C to 40°C, 4°C to 35°C, 4°C to 30°C,4°C to 25°C, 4°C to 20°C, 4°C to 15°C, 4°C to 10°C, 4°C to 5°C, , 5°C to 45°C, 10°C to 40°C, 15°C to 35°C, or 20°C to 30°C, and all the ranges and subranges therein between. The nozzle temperature is preferably between 4°C and 40°C.

According to the disclosed method, spraying of said lipid-carrier-based suspension comprises a flow rate ranging from 0.1 mL/min to 10 L/min, preferably from 3 mL/min to 5 L/min. For example, the flow rate is ranging from 0.3 mL/min to lOL/min, from 0.5 mL/min to lOL/min, from 1 mL/min to 9 L/min, from 2 mL/min to 8L/min, from 3 mL/min to 7 L/min, from 3 mL/min to 6 L/min, from 3 mL/min to 5 L/min, from 3 mL/min to 4.5 L/min, from 3 mL/min to 4 L/min, from 3 mL/min to

3.5 L/min, from 3 mL/min to 3 L/min, from 3 mL/min to 2.5 L/min, from 3 mL/min to 2 L/min, from 3 mL/min to 1.5 L/min, from 3 mL/min to 1 L/min, from 3 mL/min to 0.5 L/min, from 3 mL/min to 400 mL/min, from 3 mL/min to 300 mL/min, from 3 mL/min to 200 mL/min, from 3 mL/min to 100 mL/min, from 3 mL/min to 80 mL/min, from 3 mL/min to 60 mL/min, from 3 mL/min to 50 mL/min, from 3 mL/min to 40 mL/min, from 3 mL/min to 30 mL/min, from 3 mL/min to 20 mL/min, from 3 mL/min to 10 mL/min, from 3 mL/min to 9 mL/min, from 3 mL/min to 8 mL/min, from 3 mL/min to 7 mL/min, from 3 mL/min to 6 mL/min, from 3 mL/min to 5 mL/min, from 3 mL/min to 4 mL/min, or from 5 mL/min to 10 L/min, from 10 mL/min to 10 L/min, from 20 mL/min to 10 L/min, from 30 mL/min to 10 L/min, from 40 mL/min to 10 L/min, from 100 mL/min to 10 L/min, from 200 mL/min to 10 L/min, from 300 mL/min to 10 L/min, from 400 mL/min to 10 L/min, from 500 mL/min to 10 L/min, from 600 mL/min to 10 L/min, from700 mL/min to 10 L/min, from 800 mL/min to 10 L/min, from 900 mL/min to 10 L/min, from 1 L/min to 10 L/min, from

1.5 L/min to 10 L/min, from 2 L/min to 10 L/min, from 2.5 L/min to 10 L/min, from 3 L/min to 10 L/min, from 3.5 L/min to 10 L/min, from 1 L/min to 4 L/min, from 4.5 L/min to 5 L/min, from 4 L/min to 5 L/min, or from 5 mL/min to 4.5 L/min, from 10 mL/min to 4 L/min, from 20 mL/min to 3.5 L/min, from 50 mL/min to 3 L/min, from 100 mL/min to 3 L/min, from 500 mL/min to 3 L/min, from 1 L/min to 3 L/min, from

1.5 L/min to 3 L/min, from 2 L/min to 3 L/min, from 2.5 L/min to 3 L/min, from 2 L/min to 2.5 L/min, and all the ranges and subranges therein between.

It would be clear to a skilled person, that the flow rate can change based on various factors in order to minimize shear and thermal stresses. In certain embodiments, when a suspension is aerosolized using a small nozzle, a lower flow rate is employed compared to when a large nozzle is used, as this helps to decrease shear stress. According to the disclosed method, the spray evaporation process of said lipid- carrier-based suspension comprises applying a gas pressure. In embodiments, the carrier gas pressure is between 2 to 6 bar such as 2 bar to 3 bar, 3 bar to 4 bar, 4 bar to 5 bar, 5 bar to 6 bar, 2 bar to 4 bar, 2 bar to 5 bar, 3 to 6 bar, 4 to 6 bar and all the ranges and subranges therein between.

It would be clear to a skilled person, that the nozzle size, liquid flow rate, air flow rate, and pressure may alter in relation to one another to ensure reduced shear and thermal stresses.

In other embodiments of the disclosed method, a film evaporation technique is used to remove at least part of said organic phase solvent from lipid-based carrier suspension. Film evaporation refers to the process of evaporation that occurs on the surface of a liquid film. Several types of film evaporation methods are known to the skilled person and any of such known techniques can be applied to remove organic solvent from the suspension according to the disclosed method. Some non-limiting examples of film evaporation are thin film evaporation, falling film evaporation, rising film evaporation, falling film evaporation, rotating film evaporation, and forced film evaporation. In preferred embodiments, said film evaporation is selected from thin film evaporation or falling film evaporation. As such, in embodiments, the evaporator used to perform the evaporation is a thin film evaporator or a falling film evaporator. In some embodiments, the film evaporation is carried out at a temperature ranging between 5 to 50°C, for example between 5 to 45°C, 5 to 40°C, 5 to 35°C, 5 to 30°C, 5 to 25°C, 5 to 20°C, 5 to 15°C , 5 to 10°C or 10 to 50°C, 15 to 50°C, 20 to 50°C, 25 to 50°C, 30 to 50°C, 35 to 50°C, 40 to 50°C, 45 to 50°C, or 10 to 45°C, 15 to 40°C, 20 to 35°C, 20 to 30°C, and all the ranges and subranges therein between. Preferably, the film evaporation is carried out at a temperature ranging between 15 and 40°C.

In embodiments the film evaporation is carried out at a flow rate of said lipid-based carrier suspension ranging from 0.1 to 10000 mL/min, preferably from 1 to 150 mL/min, for example between 1-145 mL/min, 1-140 mL/min, 1-130 mL/min, 1-120 mL/min, 1-110 mL/min, 1-100 mL/min, 1-90 mL/min, 1-80 mL/min, 1-70 mL/min, 1-60 mL/min, 1-50 mL/min, 1-40 mL/min, 1-30 mL/min, 1-20 mL/min, 1-10 mL/min, 1-150 mL/min, 10-150 mL/min, 20-150 mL/min, 30-150 mL/min, 40-150 mL/min, 50-150 mL/min, 60-150 mL/min, 70-150 mL/min, 80-150 mL/min, 90-150 mL/min, 100-150 mL/min, 110-150 mL/min, 120-150 mL/min, 130-150 mL/min, 140-150 mL/min, and all the ranges and subranges therein between. In embodiments of the disclosed method, a vacuum is applied during the film evaporation process. In some embodiments, said vacuum pressure is between 0.0001 and 1 bar such as 0.0005 bar to 1 bar, 0.001 bar to 1 bar, 0.005 bar to 1 bar, 0.01 bar to 1 bar, 0.05 bar to 1 bar, 0.1 bar to 1 bar, 0.2 to 1 bar, 0.3 to 1 bar, 0.4 to 1 bar, 0.5 to 1 bar, 0.6 to 1 bar, 0.7 to 1 bar, 0.8 to 1 bar, or 0.9 to 1 bar and all the ranges and subranges therein between.

Alternatively, the vacuum pressure is between 0.0001 bar and 0.9 bar, 0.0001 bar and 0.8 bar, 0.0001 bar and 0.7 bar, 0.0001 bar and 0.6 bar, 0.0001 bar and 0.5 bar, 0.0001 bar and 0.4 bar, 0.0001 bar and 0.3 bar, 0.0001 bar and 0.2 bar, 0.0001 bar and 0.1 bar, 0.0001 bar and 0.05 bar, 0.0001 bar and 0.01 bar, 0.0001 bar and 0.005 bar, 0.0001 bar and 0.001 bar, or 0.0001 bar and 0.0005 bar and all the ranges and subranges therein between.

In some embodiments, the vacuum pressure is between 0.0001 to 2 bar, preferably of 0.1 to 2 bar. For example, said pressure is between the ranges of 0.005 bar to 2 bar, 0.01 bar to 2 bar, 0.05 bar to 2 bar, 0.1 bar to 2 bar, 0.2 bar to 2 bar, 0.3 bar to 2 bar, 0.4 bar to 2 bar, 0.5 bar to 2 bar, 0.6 bar to 2 bar, 0.7 bar to 2 bar, 0.8 bar to 2 bar, 0.9 bar to 2 bar, 1.0 bar to 2 bar, 1.1 bar to 2 bar, 1.2 bar to 2 bar, 1.3 bar to 2 bar, 1.4 bar to 2 bar, 1.5 bar to 2 bar, 1.6 bar to 2 bar, 1.7 bar to 2 bar, 1.8 bar to 2 bar, 1.9 bar to 2 bar, 0.001 bar to 0.1 bar, 0.001 bar to 1 bar, 0.1 bar to 0.2 bar, 0.1 bar to 0.3 bar, 0.1 bar to 0.4 bar, 0.1 bar to 0.5 bar, 0.1 bar to 0.6 bar, 0.1 bar to 0.7 bar, 0.1 bar to 0.8 bar, 0.1 bar to 0.9 bar, 0.1 bar to 1.0 bar, 0.1 bar to 1.1 bar, 0.1 bar to 1.2 bar, 0.1 bar to 1.3 bar, 0.1 bar to 1.4 bar, 0.1 bar to 1.5 bar, 0.1 bar to 1.6 bar, 0.1 bar to 1.7 bar, 0.1 bar to 1.8 bar, 0.1 bar to 1.9 bar, 0.1 bar to 2.0 bar, 0.3 bar to 1.7 bar, 0.5 bar to 1.5 bar, 0.7 bar to 1.5 bar, 0.9 bar to 1.3 bar, 1 bar to 1.5 bar, and all the ranges and subranges therein between.

In embodiments, the film evaporation is carried out at a minimum vacuum pressure of 0.0001 bar.

It is preferred that the vacuum pressure is lower than the atmospheric pressure. The decrease in pressure in the vacuum environment, as vapour molecules gain velocity and move away from the liquid surface, contributes to the continuous process of liquid evaporation, as explained by the so-called Bernoulli effect.

In embodiments of the disclosed method, the evaporation process is designed such that when the aerosol is condensed and/or when the evaporation step is completed, the solvent concentration has reached its target value. This provides a plurality of advantages over other techniques used in the field. The advantages include that the manufacturing of carriers according to the disclosed method can be operated in a continuous mode, unlike the commonly used TFF technique. Even if single-pass TFF (SP-TFF) is operated in continuous mode, it is very difficult to implement it for the removal of organic solvent from lipid-based carriers, particularly removing ethanol from LNP. Reasons include the large volume of the buffer that must be used for such application, the process likely to lead to the clogging of membranes and other challenges associated with the chaining of two or more SP-TFFs.

Unlike SP-TFF or pervaporation, the current invention, by utilizing evaporation, solves such problems, allowing the removal of organic solvents such as ethanol in one step. Moreover, continuous processing is possible, as there is no need for additional buffer dilutions and as there is no membrane, there is no risk of clogging. The system can further be coupled with upstream and downstream operations.

In embodiments, said method comprises the mixing of two or more liquids. In embodiments, the method comprises mixing an aqueous solution with one or more lipids and/or polymers and one or more organic solvents, thereby forming a formulation comprising one or more carriers, and subsequently removing at least part of said one or more organic solvents by a pervaporation step.

In some embodiments, said method further comprises mixing. In particular embodiments, said method comprises mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents. In an embodiment, to prepare lipid-based carriers such as, LNPs, the lipid components are first dissolved in an organic solvent such as ethanol and then mixed with an aqueous phase solution to allow particle formation by nanoprecipitation. In some embodiments, aqueous solution comprises pharmaceutical ingredients such as polynucleotides.

In an embodiment, the formulation of carriers involves the rapid microfluidic mixing of an organic phase comprising one or more lipids with an aqueous phase solution thereby forming particles, such as nanoparticles. In some embodiments, these particles organize into a dense structure, wherein the core contains the active pharmaceutical ingredients electrostatically complexed with one or more lipids.

In an embodiment, said mixing occurs by means of microfluidic mixing. In further embodiments, the microfluidic mixing is laminar microfluidic mixing. In another embodiment, chaotic microfluidic mixing is used for mixing. In another or further embodiment, said mixing occurs by means of impingement jet mixing. During impingement jet mixing, two streams (the aqueous phase solution and the organic phase solution) collide at high velocity and pressure in a jet mixing chamber. The mixing at high velocity rapidly reduces the solubility of the lipids so that homogenous nanoparticles are formed.

In the embodiments, the mixing of an organic phase comprising one or more lipids with an aqueous phase solution, preferably containing active pharmaceutical ingredients, results in a single-phase liquid mixture comprising the formed carriers, aqueous phase solution and one or more organic phase solvent.

In conventional methods of producing lipid-based carriers like liposomes, it is crucial to remove the organic solvent from an emulsion in the aqueous phase to ensure the proper formation of lipid vesicles.

The organic solvent is used to dissolve the lipids and create a homogeneous lipid film. However, lipids are hydrophobic molecules and tend to aggregate in organic solvents rather than disperse evenly in water, leading to a multi-phase liquid mixture resembling an emulsion with droplets of lipids in the aqueous phase. To form liposomes, it is essential to remove the organic solvent from the droplets in the emulsion and replace it with an aqueous phase. This can be achieved through evaporation, for example, using a membrane pervaporation unit. The evaporation step allows the lipids to concentrate in the first liquid, forming a thin film and coming into contact with the second liquid. As a result, the lipids spontaneously selfassemble to form liposomes.

WO9610393 for instance describes the formation of multi-lamellar vesicles (MLV) which are prepared by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs. Sizing of the liposomes is subsequently performed by the extrusion method. Such an extrusion method relates to extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to reduce liposome sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes to achieve gradual reduction in liposome size. In WO9610393, said method for instance allows to produce liposomes (MLVs) having a mean size of 570 nm. After the extrusion step, the liposome suspension in WO9610393 is concentrated using a microfiltration system by means of a pump. The lipids forming the deposit in WO9610393 are selected from synthetic or natural, saturated and unsaturated phospholipids and may further contain substances selected from dicetylphosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, a-tocopherol, stearic acid, stearyl amine and mixtures thereof. As such, the organic phase solution in WO9610393 does not comprise an ionizable lipid, a cationic lipid or a PEGylated lipid. WO9610393 is focused on the encapsulation of antibacterial compounds (proteins) and contrast agents. WO9610393 does not disclose the encapsulation of polynucleotides.

KR20100024050 and WO2011127456 describe methods for multivesicular liposomes (MVL).

KR20100024050 describes such a multivesicular liposome having a large size (1 to 1000 pm). In contrast to the current invention, the organic phase solution comprises a weakly acidic amphipathic substance (for instance cholesteryl hemisuccinate, oleic acid, dipamitoylsuccinylglycerol and dioleoylsuccinyl glycerol), which undergoes ionization at specific base conditions, resulting in the surface of the lipid carrier to become anionic. As such, in KR20100024050, in order for the surface of the lipid carrier to become anionic, a pH of about pH 7.0 to 10.0, or about pH 9.0 to 10.0 is preferred. This is in contrast to the current invention, where a cationic ionizable lipid complexes with the negatively charged polynucleotides comprised in the aqueous phase solution which preferentially has an acidic pH.

WO2011127456 relates to pharmaceutical formulations containing large-diameter synthetic membrane vesicles, such as multivesicular liposomes (MVL) and methods for preparing such formulations. The organic phase in WO2011127456 comprises at least one amphipathic lipid (such as a phospholipid) and at least one neutral lipid (for instance a sterol ester or a squalene). The organic phase solution in WO2011127456 does not comprise an ionizable lipid, a cationic lipid or a PEGylated lipid.

Lipid nanoparticles are the leading technology for nonviral nucleic acid delivery. Naked RNA is quickly degraded after administration by cellular ribonucleases (RNases). LNPs slow down the degradation process to ensure RNA stability while also promoting cellular internalization via endocytosis and allowing the intracellular release of RIMA into the cytoplasm for translation by cellular machinery.

As such, in an embodiment, said lipid-based carrier is an LNP and the invention relates to a method for manufacturing an LNP. More specifically, in an embodiment, the invention relates to a method for manufacturing a lipid nanoparticle (LNP), comprising: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming an LNP suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said LNP in said suspension; and c. collecting said concentrated LNP suspension.

In embodiments of step b of the method disclosed herein, the mixture comprising the aqueous phase and the organic phase is contacted with a gas, causing the organic solvent to be removed through evaporation. However, at least a portion of the aqueous solution is also removed during this process. Since the organic solvent is more volatile than the aqueous phase, a larger amount of it is evaporated, but some water is also lost, resulting in a concentrated LNP solution.

The aqueous phase solution comprises one or more polynucleotides.

As described above, the term "lipid nanoparticle" refers to a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.

Various research articles describe the effect of the LNP size on the biodistribution profile and, in the case of vaccine formulations, the immune response obtained by said LNPs. In an embodiment, the LNPs fabricated by the method of the current invention have a mean particle diameter below 1000 nm, below 500 nm, below 200 nm, or 50-150 nm. The "size" of the LNPs relates to the diameter of the LNPs and both terms are used interchangeably herein.

Said (mean) LNP diameter (or (mean) particle diameter) can be quantified by any means known from the state of the art, such as quasi-electric light scattering (QELS), dynamic light scattering (DLS), Nanoparticle Tracking Analysis (NTA) and by imaging methods (such as, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and cryo-(TEM)). In an embodiment, DLS allows to determine the average particle size and polydispersity index (PDI, a measure of particle size distribution), while NTA allows to determine the mean, mode, and span of the particle population. In an embodiment, the (concentrated) LNP suspension comprises a monodispersed population. As such, in an embodiment, the (concentrated) LNP suspension has a PDI below 0.25, or below 0.2. In an embodiment, advanced mathematical analyses (e.g. CUMULANT analysis) can be used to estimate the mean size and PDI of the LNP suspension fabricated by the method of the current invention.

The LNP size is influenced by their fabrication method, more specifically by the parameters related to the mixing of the aqueous phase solution with the organic phase solution.

In some embodiments of the method, where the lipid-based carrier is an LNP, said organic phase solution further comprises one or more calixarenes.

In some embodiments of the method, where the lipid-based carrier is and LNP, the one or more lipids in the organic phase solution consist of a sterol, a phospholipid, a PEGylated lipid and an ionizable cationic lipid.

In some embodiments of the method, where the lipid-based carrier is and LNP, said ionizable cationic lipid has a pKa between 5 and 7.

In an embodiment, in order for the ionizable cationic lipid to be able to associate with an ion and thus itself become positively charged after mixing of the aqueous phase solution with the organic phase solution, the aqueous phase solution has an acidic pH, such as a pH below 7, or below 6. In an embodiment, the aqueous phase solution has a pH between 3 and 6. In an embodiment, the aqueous phase solution has a pH below the pKa of the ionizable lipid, such as a pH with one unit below the pKa value. In embodiments of the present disclosure, the method focuses on the formation of lipid nanoparticles (LNPs). LNPs have a different structure than liposomes (including multivesicular liposomes (MVL), unilamellar liposomes and multilamellar liposomes described above) and their production does not rely on forming droplets of the first liquid in a mixture of two non-miscible liquids or by depositing a selected lipid on the inside wall of a suitable container and by subsequently adding an aqueous phase to the vessel). Instead, LNPs are suspended in a single-phase mixture where the individual molecules of the organic solvent and aqueous solution disperse evenly. For instance, in specific embodiments, ethanol and water are used as the organic solvent and aqueous solution, respectively, with ethanol dispersed in the mixture through vigorous mixing or sonication. LNPs which are not soluble in such a single-phase mixture comprising organic and aqueous solvents will be formed. The common practice to remove the solvent in this step of LNP production is Tangential Flow Filtration (TFF), involving several cycles of concentration and dilution with an aqueous buffer until the residual solvent concentration meets regulatory requirements. However, TFF can impose shear stress on the nanocarriers, negatively impacting their quality. The inventors provide a novel solution by proposing the use of evaporation such as film or spray evaporation to remove the solvent from the single-phase solution comprising the aqueous phase solution and the one or more organic solvent such as ethanol to obtain purified LNPs. This alternative method avoids the adverse effects of shear stress associated with TFF, ensuring the quality and integrity of the nanocarriers. The method also avoids using a membrane as in pervaporation hence avoiding problems due to membrane clogging and allowing a faster solvent removal method from manufactured LNPs.

As such, in an embodiment, the method further comprises yielding a single-phase liquid mixture comprising said lipid nanoparticles or hybrid carriers resulting from the mixing step. In embodiments, the organic solvent and the aqueous solution form a single-phase mixture liquid.

As described above, in an embodiment, the formulation of carriers involves the rapid microfluidic mixing of an organic phase comprising one or more lipids with an aqueous phase solution thereby forming particles, such as nanoparticles.

The microfluidic approach, which does not involve appreciable input of energy to disrupt previously formed structures, is considerably gentler and, in contrast to sonication, the production of limit size lipid carriers such as LNPs can be readily scaled using the microfluidic approach by assembling a number of mixers in parallel. To produce suitably sized LNPs, a microfluidic-based approach has been demonstrated as convenient for forming monodispersed LNPs. Polydispersity index (PDI) is a normalized value that indicates nanoparticle size range in a sample, and is a useful indicator of sample quality. In samples with high dispersity, larger particles in the distribution will tend to aggregate and sediment, which for instance leads to a diminished effective RIMA concentration and inconsistent dosing. Typically, LNP formulations developed for biological application should have a PDI below 0.2, which indicates the colloid is acceptably monodisperse. Monodispersity of nanoparticle drugs is crucial to ensure the consistent behavior of the intended drug, as size influences how particles interact with the body.

As described above, the lipid carrier of the invention is an LNP, referring to a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and comprising a plurality of lipid molecules physically associated with each other by intermolecular forces.

In an embodiment of the method of the current invention, step a (mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming a lipid-based carrier suspension) is performed by using a microfluidics approach.

Microfluidics uses intersecting microchannels for the highly controlled mixing of two or more miscible solvents (the aqueous phase solution and the organic phase solution comprising one or more lipids in one or more organic solvents). During the mixing, the change in polarity promotes nanoprecipitation and the formation of lipid- based nanoparticles. As with other manufacturing techniques, optimization of critical process parameters (total flow rate (TFR), flow rate ratio (FRR), temperature) and material parameters (aqueous buffer selection and composition, solvent) is essential as they impact the properties of the final product. The theory of vesicle formation assumes that LNP formation is based on disk-like bi-layered fragments whose edges are stabilized by an organic solvent such as ethanol. When diluting the organic solvent in water, these planar fragments grow and fuse to even bigger rafts. At low organic solvent concentrations, the destabilized structures bend to form closed LNPs. The faster the increase in the polarity of the organic solvent solution, the smaller the fragments will be before closing into vesicles, resulting in overall smaller LNPs. Two important factors that directly influence the rate at which the polarity of the organic solvent solution changes are the total flow rate (TFR) and the flow rate ratio (FRR).. TFR, as used herein is defined as the total speed in mL/min at which both fluid streams are being pumped through the two separate inlets of the microfluidic cartridge. The FRR, as used herein, is defined as the volumetric ratio of the aqueous phase over the organic phases (FRR). The mixing rate, for example, influences both the size and the homogeneity of the LNPs. The properties of individual LNPs strongly depend on local, microscopic mixing rates, where diffusive transport effects can lead to LNPs with variable compositions. Therefore, rapid mixing of the ethanol-lipid phase with excess water is key for the synthesis of small, uniform LNPs. Higher mixing rates are achieved with staggered herringbone micromixers.

In embodiments of the method disclosed herein, where the lipid-based carrier is and

LNP, the total flow rate (TFR) is comprised between 1 ml/min and 500 ml/min, between 100 ml/min and 500 ml/min between 150 ml/min and 500 ml/min between 200 ml/min and 500 ml/min between 250 ml/min and 500 ml/min between 300 ml/min and 500 ml/min between 350 ml/min and 500 ml/min between 400 ml/min and 500 ml/min, or between 450 ml/min and 500 ml/min.

Alternatively, the TFR is between 1 ml/min and 450 ml/min, between 1 ml/min and 400 ml/min, between 1 ml/min and 350 ml/min, between 1 ml/min and 300 ml/min, between 1 ml/min and 250 ml/min, between 1 ml/min and 200 ml/min, between 1 ml/min and 150 ml/min, between 1 ml/min and 100 ml/min or, between 1 ml/min and 50 ml/min.

In yet another alternative embodiment, the TFR is between 50 ml/ min and 450 ml/min, between 100 ml/min and 400 ml/min, between 150 ml/min and 350 ml/min, between 200 ml/min and 300 ml/min, or between 250 ml/min and 300 ml/min.

In embodiments of the method disclosed herein, where the lipid-based carrier is and LNP, the flow rate ratio (FFR) is between 5/1 and ¹/2, between 5/1 and 4/1, between 4/1 and 3/1, between 3/1 and 2/1, between 2/1 and 1/1, or between 1/1 and 1/2. In some embodiments, said flow rate ratio (FFR) is greater than 1 : 1.

In an embodiment, step a is performed by using a microfluidics approach using one or more herringbone structures.

A series of herringbone structures induce a rotational chaotic flow, essentially wrapping the fluids into one another. This phenomenon is also termed turbulent flow. In this way, the microfluidic device enables extremely rapid mixing of two fluids, with an associated fast increase in the polarity of the lipid solution. The time required for mixing in the staggered herringbone micro mixer, tmix, decreases with the flow velocity, U, as follows: tmix ~ A/[U ln(UI/D)], where A and I are parameters determined by the geometry of the microfluidic device and D is the diffusion coefficient. At low flow rates, mixing rates are also low, leading to larger LNPs as previously described. The adoption of microfluidics as part of production process offers the advantages of robust particle size control and high reproducibility across production scales and hence the ability to support scale-independent and/or continuous operation.

In an embodiment, step a is performed by using microstructures that induce lamination of the flow-stream.

As described above, the size of the LNP diameter depends on, among other factors, the rate of changing the polarity of the solution containing the lipid particle-forming materials (e.g., rapid mixing of two streams with different polarities). In certain embodiments, the rapid mixing is achieved by flow control; control of the ratio of the first flow rate comprising the aqueous phase to the second flow rate comprising the organic phase. In certain embodiments, the ratio of the first flow rate to the second flow rate is greater than 1 : 1 (e.g., 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, including intermediate ratios). In other embodiments, the rapid mixing is achieved by controlling the composition of the streams. Rapid change in solvent polarity past a critical point results in formation of LNPs with a small diameter.

In an embodiment, the mean LNP diameter is decreased by increasing the total volumetric flow rate during mixing. Increasing the total volumetric flow rate during formulation increases mixing efficiency and decreases the time scale of particle formation, resulting in smaller particles. In an embodiment, the total flow rate is decreased from 12 to 0.5 mL/min while keeping the ethanol percentage constant (25%) to obtain LNPs with a larger diameter.

In an embodiment, the mean LNP diameter is decreased by increasing the aqueous phase solution-to-organic phase solution stream ratio. Conversely, decreasing the aqueous phase solution-to-organic phase solution stream ratio increases the organic solvent concentration at the mixing interface, thus increasing lipid solubility and lengthening particle formation time, allowing for more particle growth. In an embodiment, the flow rate ratio is comprised between 5/1 and ¹/2, or between 3/1 and 1/1. In an embodiment, the organic solvent concentration is varied from 25 to 50% during mixing (v/v). No organic solvent concentrations above 50% are used because this generally leads to low polynucleotide entrapment and increased particle heterogeneity. Modifying such mixing parameters changes the dynamics of the nanoprecipitation reaction and solubility of the lipid components. In an embodiment, the formulation of lipid-based carriers involves impingement jet mixing of an organic phase comprising one or more lipids with an aqueous phase solution, thereby forming particles such as nanoparticles. The impingement jet approach, which involves the collision of high-velocity jets of the two phases, is effective for achieving rapid and efficient mixing without extensive energy input to disrupt previously formed structures. However, unlike microfluidic methods, impingement jet mixers do not scale in the same manner and cannot be easily parallelized for high-throughput production. Instead, the capacity of impingement jet mixing is generally limited by the size of the mixer and the associated energy input. In embodiments, the organic solvent and water are miscible, which means they can mix together at least in specific portions, preferably in all portions. In embodiments, the organic solvent and water are miscible following the mixing step according to the disclosed method.

In embodiments, the individual molecules of one or more organic solvents and aqueous solution disperse evenly or at least partially evenly.

In embodiments, the formed lipid-based carrier suspension is diluted before removing the one or more organic solvents as described in step b of the disclosed method. In further embodiments, said suspension is diluted with an aqueous buffer such as water, PBS, TE, or TRIS buffer. In embodiments, the suspension is diluted at most 200 times. For example, in embodiments, the suspension is diluted at most 175 times, 150 times, 100 times, 75 times, 50 times, 25 times, or 10 times.

In embodiments, said method is further followed by one or more downstream steps, preferably with a filtering step, to further remove any remaining traces of the organic solvent from the lipid-based carrier suspension and/or with a formulation step.

In embodiments, the formulation step further comprises diluting the collected concentrated lipid-based carrier suspension, adjusting the pH and/or filtering.

In an embodiment, said stored concentrated lipid-based carrier suspension is buffer- exchanged or diluted with a buffer to make said stored suspension an injectable solution at physiological pH of between 7 and 9, or between 7 and 8. For instance, said stored concentrated lipid-based carrier suspension can be diluted (for instance between 2-200 times) in a 20 mM TRIS buffer having a pH of 7.4 or in a 40 mM TRIS buffer having a pH of 8.4. In embodiments, following the evaporation step, the collected concentrated lipid-based carrier suspension is mixed with a solution. In embodiments said solution contains a buffer, a cryoprotectant, or a mixture thereof, to protect the lipid-based particles from aggregation and/or degradation during collection.

In certain embodiments, the concentrated lipid-based carrier suspension is collected in a liquid holder, such as a liquid collector, tube, or bottle. In embodiments, said holder comprises a solution before it receives the concentrated lipid-based carrier suspension.

In embodiments, following the evaporation step, the collected concentrated lipid- based carrier suspension is mixed with a solution to make said collected concentrated lipid-based carrier suspension an injectable solution at a physiological pH of between 7.35 and 7.45.

In embodiments, the concentrated lipid-based carrier suspension is formulated in an injectable solution at a physiological pH of between 7.35 and 7.45, preferably pH 7.40.

However, when the pH is increased from acidic to pH 7.4, the lipid-based carrier can transition to a less stable state when an ionizable lipid is present, as the latter becomes uncharged and the system must stabilize the negative charges stemming from the RIMA without the positive charges that were provided by the ionizable lipid in acidic conditions. This results in lipid-based carriers being unstable as the highly hydrophilic RNA molecules are encapsulated into a lipid-rich phase without any lipid bearing a net positive charge.

As such, in an embodiment, the organic phase is removed by an evaporation process after fabrication of the lipid-based carrier to increase the stability of the lipid-based carrier, but the pH is not brought to 7.4 or close to 7.4. Instead, the pH is maintained acidic so that the ionizable lipid is positively charged and able to complex and stabilize the RNA inside the lipid-rich phase.

As such, in an alternative embodiment, the collected concentrated lipid-based carrier suspension is not mixed with a solution to make said collected concentrated lipid- based carrier suspension an injectable solution at a physiological pH, but is kept at the same pH and/or salt concentration as obtained after the mixing step. In an embodiment, the aqueous phase solution in step a comprises a buffer having a pH below 7 and/or a low salt concentration. Such conditions, acidic pH and/or low salt concentration, are optimal for storage of the collected concentrated lipid-based carrier suspension. In an embodiment, the pH of the aqueous phase is chosen depending on the pKa of the ionizable lipid, so that a significant fraction of the chemical functions is protonated and complexed with the RIMA. At lower pH, the lipid- based carriers are also more positively charged. Hence, the colloidal stability is improved via electrostatic repulsion.

In an embodiment, said collected concentrated lipid-based carrier suspension is stored in a storage buffer. In a further embodiment, said storage buffer comprises the same pH and/or salt concentration as obtained after the mixing in step a. In an embodiment, said lipid-based carrier comprises an ionizable lipid and said storage buffer has a pH less than the pKa of the ionizable lipid.

In an embodiment, the pH of the storage buffer is below 7, below 6, or below 5. In an embodiment, the pH of the storage buffer is between 2 and 7, or between 3 and 6, such as 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 or any value in between. In an embodiment, said storage buffer has a pH between 3-4.

In an embodiment, said storage buffer has an ionic strength less than about 50 mM, less than about 40 mM, less than about 30 mM, less than about 20 mM, less than about 10 mM, or less than 5 mM, such as 4 mM, 3 mM, 2 mM, ImM, 0.05 mM or any value in between.

In an embodiment, said storage buffer is a 1 mM citrate buffer having a pH of 3.8.

In an embodiment, said storage buffer comprises a monovalent salt. Such a buffer allows to maximize electrostatic repulsion and minimize van der Waals attractive forces.

In an embodiment, said stored concentrated lipid-based carrier suspension is buffer- exchanged or diluted with a buffer to make said stored suspension an injectable solution at physiological pH of between 7 and 9, preferably 7 and 8. For instance, said stored concentrated lipid-based carrier suspension can be diluted (for instance between 2-200 times) in a 20 mM TRIS buffer having a pH of 7.4 or a 40 mM TRIS buffer having a pH of 8.4.

In an embodiment, said one or more organic solvents are chosen from the group of alcohol, ether, chloroform, benzene, and acetone, preferably from alcohols. Non- limiting examples of said alcohols can be ethanol, methanol, propanol, isopropanol, butanol, or mixtures thereof. In another embodiment one organic solvent is acetone.

In an embodiment said one or more lipids in the organic phase solution comprise one or more of the following : cholesterol, a phospholipid, a cationic lipid, a PEGylated lipid, an ionizable lipid, a fusogenic lipid, or a mixture thereof.

In embodiments, said lipid-based carriers can comprise LNPs or hybrid carriers. In preferred embodiments, lipid-based carrier is a lipid nanoparticle (LNP).

In embodiments, the first or second aqueous phase comprises active pharmaceutical ingredients such as polynucleotide, a chemical compound, a polypeptide, a small molecule and any combination thereof. In preferred embodiments said active pharmaceutical ingredient is polynucleotides. A "polynucleotide" as defined herein is a combination of nucleotide monomers which are connected to each other through covalent bonds. In further embodiments, the one or more polynucleotides in the aqueous phase solution comprise RIMA, DNA, siRNA, miRNA, mRNA, saRNA, circRNA or a mixture thereof.

In embodiments, said evaporation process generates a continuous process of manufacturing said formulation.

In some embodiments, the method disclosed herein can be used for the preparation of small-scale nucleic acid vaccines, such as nucleic acid-based personalized vaccines. A non-limiting example of the application of such vaccines is the development of personalized cancer vaccines and other nucleotide-based medicines for rare diseases. These vaccines are produced on a small scale of up to 100 mg RNA per batch.

However, it would be obvious to a skilled person that, in some embodiments, the method disclosed herein can be scaled up. This is particularly practical for the rapid development of vaccines in response to pandemics.

Using evaporation for the concentration of lipid-based carriers in the method described herein provides several advantages, including gentle processing conditions, better and easier scalability, and higher efficiency in solvent removal. These benefits contribute to producing stable, uniform, and effective lipid-based carriers, making evaporation a preferred choice in the manufacturing process. Additionally, the disclosed method of concentration does not rely on membranes, eliminating issues related to membrane fouling, clogging, and compatibility with different solvents.

In a further aspect, the invention relates to a method for storing a lipid-based carrier suspension, said method comprising storing said lipid-based carrier suspension in a storage buffer having a pH below 7 and/or a low salt concentration.

Acidification lowers the pH, leading to the protonation of ionizable lipids, which stabilizes the lipid-based carriers by reducing their surface charge and preventing aggregation. This enhances the stability of lipid-based carriers during storage, especially at elevated temperatures.

It was surprisingly observed that the combination of evaporation and acidification offers a synergistic approach to concentrating and stabilizing lipid-based carriers. The gentle removal of solvents by evaporation maintains the structural integrity of the LNPs, while acidification provides enhanced stability by reducing aggregation and degradation risks. This synergy is particularly advantageous over TFF, which faces significant challenges such as membrane fouling, pH sensitivity, and mechanical stress. Furthermore, the acidic conditions necessary for thermostabilization are incompatible with TFF, as they can damage the filtration membranes and reduce their effectiveness. Therefore, the method of the current invention is especially well- suited for concentrating lipid carriers that comprise charged lipids, as these would otherwise adhere to the membranes used in membrane-based concentration processes, compromising efficiency and consistency.

In an embodiment, the pH of the storage buffer is below 7, below 6 or below 5. In an embodiment, the pH of the storage buffer is between 2 and 7, or between 3 and 6, such as 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 or any value in between. In an embodiment, said storage buffer has a pH between 3-4.

In an embodiment, said storage buffer comprises a monovalent salt. Such a buffer allows to maximize electrostatic repulsion and minimize van der Waals attractive forces. In an embodiment, said storage buffer is a 1 mM citrate buffer having a pH of 3.8. In an embodiment, said stored concentrated lipid-based carrier suspension is buffer- exchanged or diluted with a buffer to make said stored suspension an injectable solution at a physiological pH of between 7 and 9, preferably between 7 and 8. For instance, said stored concentrated lipid-based carrier suspension can be diluted (for instance between 20-200 times) in a 20 mM TRIS buffer having a pH of 7.4.

In an embodiment, said lipid-based carrier is a lipid nanoparticle (LNP), wherein said LNP comprises an ionizable lipid. In a further embodiment, said storage buffer has a pH less than the pKa of the ionizable lipid.

In an embodiment, the lipid-based carrier suspension is manufactured by: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming a lipid-based carrier suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said lipid- based carrier in said suspension; and c. collecting said concentrated lipid-based carrier suspension.

In an embodiment, said lipid-based carrier suspension is able to produce the desired effect when stored at a temperature above 4°C for at least 48 hours.

In an embodiment, said lipid-based carrier suspension is able to produce the desired effect when stored at a temperature above 37°C for at least 2 weeks.

The integrity of the encapsulated component (for instance the polynucleotides comprised in the aqueous solution), when stored according to the method of the current invention, is sufficient to produce the desired effect, e.g., to induce an immune response. For example, the RNA integrity may be at least 50%, such as at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, or at least 60%. In some embodiments, the integrity of saRNA may be lower than 50% and still produce an immune response. Additionally or alternatively, the size (Z_aVerage) (and/or size distribution and/or polydispersity index (PDI)) of the LNPs stored according to the method of the current invention is sufficient to produce the desired effect, e.g., to induce an immune response. In a last aspect, the invention is also directed to a system for manufacturing one or more lipid-based carriers, wherein said system comprises one or more mixing units fluidly connected to an evaporation device such as a spray evaporator or film evaporator.

In embodiments, the system for manufacturing a formulation comprising one or more lipid-based carriers, wherein, said system comprises one or more mixing units suited to mix at least one aqueous solution comprising polynucleotides with an organic phase solution comprising one or more lipids and/or one or more polymers thereby forming a formulation comprising one or more carriers, wherein said one or more microfluidic mixing units are fluidly connected to one or more evaporation device for removal of at least a part of the organic solvents from said formulation.

In embodiments, said evaporation device is a spray-drying apparatus or a film evaporation apparatus. In embodiments, said spray-drying apparatus can be chosen from any commercially available spray-drying devices, such as Butchi B-290.

In embodiments, said film evaporation apparatus is a thin film evaporation apparatus or falling film evaporation apparatus. In an embodiment, said film evaporation apparatus is chosen from a commercially available film evaporation apparatus.

In embodiments, the system further comprises one or more filtering units, one or more condensers, and/or one or more buffering tanks fluidly connected to a spraydrying or evaporation apparatus.

In embodiments, the system for manufacturing a formulation comprising one or more lipid-based carriers comprises one or more polynucleotides.

In embodiments, the system is designed for manufacturing a formulation comprising one or more LNPs, preferably said LNPs comprising one or more polynucleotides.

In embodiments, said system is suited for both laminar and chaotic mixing.

In embodiments, said one or more mixing units used in said system are microfluidic mixing units. In embodiments, said microfluidic mixing unit comprises an assembly of a plurality of microfluidic mixers in parallel. In embodiments, a microfluidic mixing unit comprises 5 to 20 of said mixers such as 5 to 20, 6 to 19, 7 to 18, 8 to 17, 9 to 16, 10 to 15, 11 to 14 or 12 to 13 microfluidic mixers, preferably 10 microfluidic mixers. In embodiments, said one or more mixing units used in said system are impingement jet mixing units.

In embodiments, said microfluidic mixing unit comprises a microfluidic chip comprising an assembly of a plurality of microfluidic mixers in parallel, preferably 5 to 20 of said mixers. For example, said microfluidic chip comprises 5 to 20, 6 to 19, 7 to 18, 8 to 17, 9 to 16, 10 to 15, 11 to 14 or 12 to 13 microfluidic mixers, preferably 10 microfluidic mixers.

In an embodiment, said evaporation device is in fluid connection to the outlet of one or more mixing units, suited to mix an aqueous solution with one or more organic solvents comprising one or more lipids, thereby forming a formulation comprising one or more lipid-based carriers.

In an embodiment, said evaporation device is connected to one or more vacuum pumps, preferably to vacuum pumps with a condenser.

In embodiments, vacuuming, purge gas and/or heating separately or in any combination therein is optionally applied before and/or during the evaporation process to improve the efficiency of solvent separation.

In further embodiments, said evaporation device comprises and/or is connected to one or more condensers. In embodiments, the condensers are vapour condensers.

In embodiments, said evaporation device is in fluid connection to the inlet of one or more filters for further purification of the solution comprising carriers, wherein said filters preferably have pore sizes ranging from 0.1 pm to 2 pm, more preferably pore sizes from 0.1 pm to 1.5 pm, 0.15 pm to 1 pm, 0.2 pm to 0.8 pm and all ranges and subranges therein between.

In embodiments, said system is thermoregulated to maintain the temperature constant and counterbalance the endothermic evaporation process.

In specific embodiments, said system comprises heating and control means for avoiding temperature decrease, which is caused by the endothermic evaporation of the solvent. In embodiments, said heating is applied to maintain a temperature between 5°C and 55°C, such as between 10°C and 50°C, 15°C and 45°C, 15°C and 40°C, 15°C and 35°C, 20°C and 35°C, 20°C and 30°C, and all the ranges and subranges therebetween. In embodiments, said system comprises a plurality of T junctions wherein two or more tubing are joined and/or split. Said T junctions are configured to receive reagents, excipients and/or buffers required for manufacturing of one or more carriers.

In an embodiment, T junctions may further comprise chambers for an effective addition of reagents, excipients and/or buffers to the system.

In embodiments, said system comprises a plurality of chambers wherein each chamber is configured to receive reagents, excipients and/or buffers required for manufacturing of one or more lipid-based carriers. Said chamber may or may not be located at a T junction.

In embodiments, said chambers and/or T junctions are used to adjust the pH of the solution-containing carriers.

In an embodiment, said chambers and/or T junctions are located before the mixing unit, between the mixing unit and pervaporation device, after the evaporation device, between the evaporation device and one or more filters and/or after the filters.

In embodiments, said system comprises a storage unit for storing one or more reagents, said storage unit can be cooled to a temperature below 10°C, preferably to 4°C.

In embodiments, the system is chained with upstream and downstream operators. These operators include an upstream system for microfluidic fabrication of the gene delivery system and/or a downstream formulation system and/or filtration system.

In embodiments, said system comprises one or more SP-TFF (Single-Pass Tangential Flow Filtration) units. Said one or more SP-TFF units can be placed after the microfluidic mixing unit and/or after the evaporation unit.

In an embodiment, said storage unit is in fluid connection with a pump system, such as a peristaltic pump or a syringe pump.

In an embodiment, said system is provided in a cabinet, preferably a wheeled cabinet. In specific embodiments, said cabined is a laminar flow hood. In an embodiment, said system can be connected to a system for the in vitro transcription of RIMA.

The present invention will be now described in more detail, referring to examples that are not limitative.

EXAMPLES AND/OR DESCRIPTION OF FIGURES

The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

Figure 1 shows the Luciferase activities of LNPs carrying mRNA manufactured according to the disclosed method utilizing spray evaporation.

Figure 2 shows ethanol removal as a function of time from produced LNPs using thin film evaporation.

Figure 3 shows the PDI and size of produced LNPs during the thin film evaporation process.

Figure 4 (A and B) shows the Luciferase activities of LNPs carrying mRNA manufactured according to the disclosed method utilizing thin film evaporation. A) Luminescence after 6H and B) Luminescence after 24H of transfection.

Figure 5 (A and B) show schematic overviews of the laminar microfluidic mixing unit.

Figure 5 (C and D) show schematic overviews of a chaotic microfluidic mixing unit.

Figure 6 shows schematic overviews of possible embodiments of falling film units (A) and thin film units (B).

Figure 7 shows schematic set-ups of possible embodiments of the system configuration comprising a thin film evaporator.

Figure 8 shows a schematic overview of a possible embodiment of a falling film unit spray evaporator. Figure 9 shows schematic set-ups of possible embodiments of the system configuration comprising a spray dryer.

Figure 10 shows schematic overviews of a pervaporation unit combined with a vacuum pump (A), a pervaporation unit combined with a purge gas source (B), and a pervaporation unit combined with a heating unit (C).

Figure 11 The effects of the pH, the buffer concentration, the temperature and the storage time on the LNP hydrodynamic diameter (size) and PDI in the acidic buffer (citrate pH 3 to 6) or in the reconstituted buffer (TRIS pH 7.4). The dashed line shows the responses for the selected factors (i.e., pH 3.8, 1 mM citrate, 20°C and 10 days storage).

Figure 12 (left) The hydrodynamic diameter of SM-102-LNP and CX5-LNP stored at 37°C in TRIS sucrose pH 7.4 buffer, (right) The Hydrodynamic diameter of SM-102- LNP and CX5-LNP produced in citrate 1 or 5 mM at pH 3.8, stored at 37°C in citrate 2 or 10 mM at pH 3.8 and reconstituted in TRIS sucrose pH 7.4.

Example 1. Preparation of LNPs

LNPs encapsulating mRNA (Nl-methylpseudouridine-modified) encoding for the Firefly Luciferase and made of 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102, Organix, USA), distearoylphosphatidylcholin (DSPC, Avanti, USA), cholesterol (Avanti, USA) and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000, Avanti, USA) with a molar ratio 50: 10:38.5: 1.5 were produced by microfluidic mixing using the NanoAssemblr Ignite from Precision NanoSystems. LNPs we produced using acetate buffer 25 mM pH 5.5 for the RNA-containing aqueous phase (0.133 g/L of mRNA) and mixed at a flow rate ratio (FRR) of 3: 1 with the organic phase containing the lipids dissolved in ethanol. The N:P ratio was 5.57. After fabrication, these LNPs were subjected to an evaporation process, either spray or film evaporation to remove the organic phase from the manufactured LNPs in suspension. a. Using a spray dryer in the evaporation step: 10 mL of the LNPs produced were then aerosolized to evaporate the ethanol phase using a Butchi B-290 spray-dryer. Process parameters were optimized and the Butchi B-290 was repurposed so that the aerosol was condensed in the cyclone (collector 2) before being completely dried. The lower vapour pressure of ethanol compared to water ensures that most of the ethanol is evaporated when the aerosol is condensed.

The nozzle temperature was set at 40°C, which resulted in an outlet temperature of 25°C. The pressure was set to 4 bar and medium-sized nozzles were used (1.4 mm diameter). The pump was set to 20% (6 mL/min) and aspiration to 50% (20 m3/h). The collector 2 was pre-filled with 1 mL of TRIS 20 mM sucrose 8% at pH 7.4 to dilute and adjust the pH of the aerosolized LNPs. Before spraying the suspension, encapsulation buffer only was sprayed to wet all surfaces and simulate continuous operation.

Several combinations of parameters were evaluated during pre-screening to avoid LNP degradation including inlet temperature from 30-200°C, outlet temperature 25- 120°C, pressure 2-6 bar, Nozzle size 0,7 mm, 1,4mm or 2mm, suction 20-38 m³/h (50-100%), pump flow rate 0-30 ml/min(0-100%).

Preferred optimized process parameters were set

Temperature inlet: 40 °C

Temperature outlet: 25 °C

Pressure: 4 bar

Nozzle size: 1,4 mm (medium)

Suction: 20 m³/h (50%)

Pump flow rate: 6 ml/min (20%) b. Using film evaporation in the evaporation step:

6.5 ml of LNP suspension in acetate buffer 25 mM, pH 5 and 25% ethanol was left to evaporate under the airflow of a BSC for 6h. The LNPs were evaporated in a petri dish such as they formed a film with a thickness of a few millimeters maximum. The ethanol concentration was measured every hour using an enzymatic-based quantification method. Results shown in Figure 2 show that the ethanol becomes undetectable after 6h. This was further confirmed using NMR quantification. Using dynamic light scattering, we also demonstrated that the polydispersity and the size of the LNPs are not affected by the solvent removal process (Figure 3). After 6h, the LNPs were collected, and mRNA concentration was determined using a modified Ribogreen assay. RNA total concentration was diluted to 100 ng/pL by diluting with TRIS-sucrose. This yields a LNPs suspension that is at pH 7.4 and 8% sucrose w/v. Table 1 shows the total RNA concentration, the RNA encapsulation efficiency, the pH, the polydispersity (PDI), the size and the equivalent vaccine dose (assuming 0.5 mL injection volume) for the LNPs obtained at each step of the process (before the solvent removal and after adjustment to a vaccine dose of 50 pg and 10 pg).

Table 1: NMR quantification of water, ethanol and acetate after 6 hours of evaporation of an LNP suspension.

Table 2: Total RIMA concentration, RNA encapsulation efficiency, pH, polydispersity index (PDI), size and equivalent vaccine dose (assuming 0.5 mL injection volume) for the LNPs obtained at each step of the thin film evaporation process (before the solvent removal and after adjustment to a vaccine dose of 50 pg and 10 pg).

LNPs prepared using the thin film evaporation (at 20 ng/pL) were injected intramuscularly in BALB/C mice and luminescence was measured after 6h and 24h at the injection site (1 pg per mice). The luminescence was compared to that of a standard LNP fabrication method. The standard laboratory procedure for LNP downstream processing consists of a dialysis step in a dialysis cassette and using TRIS 20 mM and 8% w/v sucrose as the diluting buffer. The RNA concentration is further adjusted to 20 ng/pL after RNA concentration and using the same buffer as the dialysis buffer. As shown in Figure 4, the luminescence is not significantly different (a=0.05%) between the two groups (n=4), which demonstrates that both fabrication processes yield LNPs that are potent and undistinguishable.

Example 3: Ethanol content of LNP suspension and physicochemical characteristics of manufactured LNS after removal of ethanol

Table 3: Physicochemical characteristics of manufactured LNPs after spray evaporation. EE%: encapsulation efficiency; PDI: polydispersity index; Control shows LNPs dialyzed using a standard laboratory procedure.

7 mL and 1 mL of TRIS 20 mM, sucrose 8% buffer at pH 7.4 were added to collector 1 and collector 2 respectively. The addition of this buffer to both collectors allows to formulate LNPs into the administration buffer upon condensation of the aerosol in the collectors. LNPs were then sprayed using the preferred parameters and procedure (see example 1). LNPs were collected in both collectors 1 and 2, and filtrated. The ethanol content of LNPs in both collector 1 and collector 2 was measured using gas chromatography (GC). The data revealed that the disclosed method allows for obtaining ethanol concentrations as low as 0.7%. Furthermore, the size, polydispersity index (PDI), and encapsulation efficiency of the LNPs were measured and compared to control LNPs (LNP dialyzed in Tris 20 mM sucrose 8% buffer at pH 7.4) that were not subjected to spray evaporation. The results, as shown in Table 3, indicate that the size remained around 100 nm in diameter in collector 1, while in collector 2, the size was maintained at around 150 nm in diameter. The PDI values remained at 0.2 and below for all conditions in collector 2, whereas in collector 1, the PDI was maintained around 0.5. The RNA encapsulation efficiency ranged between 75% and 90% in collector 2. Overall, these findings demonstrate that spray evaporation with specified parameters, at an outlet temperature of 25°C preserves the size, PDI, and encapsulation efficiency of the LNPs. Therefore, this unit operation does not adversely affect the physicochemical characteristics of the LNPs.

Example 4. Functional Evaluation of LNPs with Luciferase Assays

The LNPs are produced and the organic solvent is removed according to the disclosed method. After spraying the suspension comprising the lipid-based carrier with RNA, the total RIMA concentration was estimated using a modified Ribogreen assay. The RNA concentration was adjusted to 20 ng/pL and LNPs were filtrated on a 0.22 m filter. A TRIS 20 mM, 8% sucrose w/v and pH 7.4 solution was used to dilute LNPs and adjust RNA concentration.

These LNPs were administrated intramuscularly to BALB/c mice at a dose of 1 pg per injection. Luminescence was measured after injection and results are shown in Figure 1. Results show that spray evaporation yields LNPs with similar luminescence, i.e., potency, as LNPs that were processed with a standard downstream laboratory procedure, i.e., dialysis in a dialysis cassette. This indicates that the bioactivity of LNPs is preserved during the evaporation step when applying the appropriate conditions of temperature, flow rate, nozzle size, pressure and suction.

Example 5: effect of the pH and salt concentration on the stability of lipid- based carriers (LNPs) during storage.

LNPs are fabricated by mixing lipids solubilized in an organic phase with an acidic RNA solution. The reason for this is that the ionizable lipid must be positively charged to complex and encapsulate the RNA in the lipid-rich phase. The ionizable lipid is positively charged in acidic pH and uncharged at pH 7.4. Hence, the net charge of LNPs is usually positive at acidic pH and becomes neutral when pH is increased to 7.4. Often, after fabrication of LNPs, ethanol is removed by evaporation and the buffer is changed for a buffer that is suitable for injection (usually TRIS 25 mM at pH 7.4 and 8% w/v sucrose) during storage of the LNPs.

To evaluate the effect of pH and salt concentration on the stability of lipid-based carriers (LNPs) during storage, after fabrication of the LNPs, the resulting suspension was buffer-exchanged in a buffer containing citrate at a concentration ranging from 1 to 100 mM and a pH ranging from pH 3 to pH 6.

SM-102, DSPC, cholesterol and DMG-PEG were dissolved in ethanol at a molar ratio of 50: 10:38.5: 1.5. The RNA was dissolved in the aqueous phase at a concentration of 0.133 g/L. Both phases were mixed at a flow rate ratio (FRR) of 3 to encapsulate the mRNA inside the nanoparticle.

LNPs were stored at 4°C, room temperature or 37°C for up to 2 weeks. LNPs were reconstituted after 24h, 48h, 72h, 1 week and 2 weeks by diluting 100 times in TRIS 20 mM pH 7.4. After reconstitution, a ZetaSizer Ultra Red was used to measure the hydrodynamic radius and the polydispersity index (PDI).

The results (see figure 11) show that at pH 3.8 and 1 mM, the temperature (up to 37°C) and storage time (up to 2 weeks) have no impact on the size and PDI of the reconstituted LNP. Indeed, both size and PDI remain constant at respectively 101 nm and 0.12. This indicates excellent colloidal stability. When pH and/or buffer concentration is increased (see pH and log[concentration(mM)] panels), both size and PDI increase (shown for 20°C and 10 days, as indicated by the dashed lines). This indicates poor colloidal stability.

Example 6: Effect of the storage buffer type on the stability of lipid-based carriers (LNPs) during storage

The effect of the type of storage buffer ( 10 mM citrate solution or the administration buffer Tris Sucrose) was tested on two different LNPs.

The first type of LNP, SM-102-LNP, was obtained by dissolving SM-102, DSPC, cholesterol and DMG-PEG in ethanol. The RNA was dissolved in the aqueous phase at a concentration of 0.133 g/L with 1 mM of citrate buffer at pH 3.8. Both phases were mixed at a flow rate ratio (FRR) of 3 to encapsulate the mRNA inside the SM- 102-LNP.

The second type of LNP, CX5-LNP, was obtained by dissolving CX5, DOPE, cholesterol and DMG-PEG in ethanol at a different molar ratio than SM-102-LNP. The RNA was dissolved in the aqueous phase at a concentration of 0.133 g/L with 5 mM of citrate buffer at pH 3.8. Both phases were mixed at a flow rate ratio (FRR) of 3 to encapsulate the mRNA inside the CX5-LNP.

Both resulting LNP suspensions were then evaporated using a Smart Evaporator (BioChromato) until a concentration factor of 2 was achieved. The resulting suspensions containing less than 0.5% w:v remaining ethanol were then stored at 37°C for up to 2 weeks in the citrate buffer (pH 3.8).

As a control, the same two types of LNPs were encapsulated using acetate at pH 5 as an encapsulation buffer. After encapsulation, the buffer was exchanged by diluting LNPs in TRIS 20 mM sucrose 8% and then reconcentrating them to their initial RNA concentration on Amicon tubes.

The LNPs of all conditions were reconstituted after Ih, 1 week or 2 weeks by 2-fold dilution in TRIS 40 mM, 16% sucrose buffer at pH 7.4. After reconstitution, a ZetaSizer Ultra Red was used to measure the hydrodynamic radius and the polydispersity index (PDI).

The hydrodynamic radius of the LNPs before and during the storage are shown in Figure 12. The results indicate that at pH 3.8 and 2 or 10 mM citrate, the size of both reconstituted LNPs does not change. This indicates excellent colloidal stability. When LNPs are stored in their administration buffer, TRIS-sucrose, the size of both SM-102-LNP and CX5-LNP increases after 2 weeks of storage at 37°C. This indicates poor colloidal stability. All PDIs were found to be below 0.35 (data not shown).

The results confirm that optimal colloidal stability is achieved at low pH and low buffer concentration. It also shows that the optimum is valid for at least two very different types of LNPs.

Example 7 Membrane-based concentration vs concentration by evaporation

Two methods of ethanol removal and LNP concentration were compared. To do this, LNP suspensions were prepared by dissolving SM-102, DSPC, cholesterol and DMG- PEG in ethanol at a molar ratio of 50: 10:38.5: 1.5. The RNA was dissolved in the aqueous phase at a concentration of 0.133 g/L in acetate buffer. Both phases were mixed at a flow rate ratio (FRR) of 3 to encapsulate the mRNA inside the nanoparticle.

Membrane-based concentration: To remove ethanol and from LNPs in their acidic buffer, the suspension was diluted in acetate 25 mM at pH 5 and centrifugated on Amicon tubes.

Concentration by evaporation: To remove ethanol from LNPs in their acidic buffer, the suspension was evaporated to reach a concentration factor of 2 and evaporate ethanol. The LNPs could not be reconcentrated using the membrane-based concentration method because they clogged the membrane, which made it impossible to concentrate and remove ethanol post-encapsulation. On the other hand, evaporation of LNPs was successful in removing ethanol (ethanol concentration <0.5% w:v as measured using an enzymatic quantification kit from Megazyme) and LNPs produced could either be diluted in the administration buffer or stored in the encapsulation buffer. This demonstrates the advantage of the evaporation method over membrane-based buffer exchange for charged LNPs (i.e., LNPs in acidic buffers).

It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example of fabrication without reappraisal of the appended claims.

DESCRIPTION OF FIGURES

Figure 1 shows the Luciferase activity of LNPs manufactured according to the disclosed method (aerosolized) and compared to the standard laboratory procedure (Control). Ethanol removal from the LNPs encapsulating RNA encoding for Firefly Luciferase was performed using the preferred parameters and procedure according to the disclosed method (see examples 1, 3 and 4). LNPs were administrated intramuscularly to BALB/c mice at a dose of 1 pg per injection. Luminescence was measured after 6, 12 and 24 hours. Data are shown for the 6h time point. Control shows LNPs not spray evaporated but dialyzed using a standard laboratory procedure. Error bars represent the 95% confidence interval.

Figure 2 shows the ethanol concentration (in % w/v) in a suspension of LNPs as a function of the duration of thin film evaporation under a laminar airflow. Ethanol concentration was measured using an enzymatic reaction. The LNPs were evaporated in a petri dish such as they form a film with a thickness of a few millimeters maximum. LNPs were evaporated in their encapsulation buffer (1:3, ethanokacetate 25 mM pH 5 buffer).

Figure 3 shows the size and PDI of LNPs during the evaporation under a laminar airflow. Size and PDI were measured using dynamic light scattering. The LNPs were evaporated in a petri dish such as they form a film with a thickness of a few millimeters maximum. LNPs were evaporated in their encapsulation buffer (1:3, ethanokacetate 25 mM pH 5 buffer). Figure 4 shows the Luciferase activity of LNPs manufactured according to the disclosed method (Evaporation) and compared to the standard laboratory procedure (Dialysis). Ethanol removal from the LNPs encapsulating RNA encoding for Firefly Luciferase was performed using thin film evaporation and as described in Example 1. LNPs were administrated intramuscularly to BALB/c mice at a dose of 1 pg per injection. Luminescence was measured after 6 and 24 hours. Dialysis shows LNPs not evaporated but dialyzed using a standard laboratory procedure. Error bars represent the 95% confidence interval.

Figure 5 shows a schematic overview of the microfluidic mixing unit. 5A and 5B show a schematic overview of a laminar microfluidic mixing unit for a slow mixing rate with parallelization of up to 10 channels (A) and with a single channel (B). The mixing unit presented induces lamination of the flow-stream for the production of carriers. The unit includes Part A for receiving a first aqueous solution and preferably polynucleotides herein together called stream 1, Part B for receiving a second stream comprising a second aqueous solvent and/or an organic solvent. Streams 1 and 2 are introduced into Part C flowing under laminar flow conditions where rapid dilution occurs, and then to Part D where the final product, carriers containing polynucleotides/therapeutic agent, exit the unit. The unit comprises several pumps to assist the fluid flow. 5C and 5D show a schematic overview of a chaotic microfluidic mixing unit for rapid mixing rate with parallelization of up to 10 channels (C) and with a single channel (D). Unit includes Part Al for receiving a first aqueous solution and optionally therapeutic agent such as polynucleotides, together called herein as stream 1, Part Bl for receiving a second stream comprising one or more organic solvent with carrier forming materials and/or second aqueous solution.

The mixing of two streams coming from Part Al and Part Bl occurs in the zigzagged pattered central channel (Part Cl) where the fluids injected are chaotically mixed. The chaotic mixing results in the assembly of carriers containing therapeutic agents which then flow into Part DI to exit the unit. The unit comprises several pumps to assist the fluid flow.

Figure 6 shows operation units for film evaporation, in particular, a falling film evaporator (A) and a thin film evaporator (B)

Figure 6A illustrates the structure of a falling film evaporator. The evaporator consists of several components, including a feed inlet, evaporator tubes, a heater, a temperature sensor, a gas inlet, a gas outlet for permeate and a feed outlet for concentrate.

To begin the process, a liquid containing an aqueous phase solution, organic phase solution, and lipid-based carriers is introduced into the evaporator tubes through the feed inlet from the top of the tubes. This liquid is referred to as the influent. The liquid is then distributed onto the surface of the tubes, forming a thin film that runs down the tubes.

In order to facilitate evaporation, heated gas or hot air is introduced into the evaporator. As the liquid film flows down the tubes, heat is exchanged with the liquid, causing the organic phase from the lipid-based carrier suspension to evaporate. This evaporation removes the organic phase from the suspension, leading to the concentration of the remaining suspension.

The concentrated suspension is collected as the concentrate, while the evaporated organic phase exits the system to be condensed or collected separately.

Figure 6B illustrates the structure of a thin film evaporator. The evaporator consists of several components, including a feed inlet, evaporation surfaces, a heater, a temperature sensor, a gas inlet, a gas outlet for permeate and a feed outlet for concentrate.

To begin the process, a liquid containing an aqueous phase solution, organic phase solution, and lipid-based carriers is introduced into the evaporator through the feed inlet. This liquid is referred to as the influent. The liquid is then distributed onto the evaporation surface, forming a thin film that flow horizontally.

In order to facilitate evaporation, heated gas or hot air is introduced into the evaporator. As the liquid film flows on the surface, heat is exchanged between the gas and the liquid, causing the organic phase from the lipid-based carrier suspension to evaporate. This evaporation removes the organic phase from the suspension, leading to the concentration of the remaining suspension.

The concentrated suspension is collected as the concentrate, while the evaporated organic phase exits the system to be condensed or collected separately. Figure 7 illustrates an embodiment of a formulation production system designed to produce lipid-based carriers using a thin film evaporator unit, as described in the current disclosure. The system consists of various components and apparatuses that enable the production process. These include a microfluidic mixing unit (1), a thin film evaporation unit (24), buffering tanks (21, 28), a heater (31), a condenser (26), filter units (12), and handling apparatuses such as pumps (18,13) including volumetric pumps (23, 30), a vacuum pump (27), and sensors (22) like level sensors and thermocouples. Additionally, the system incorporates feed inlets (14, 15, 13), an air or gas inlet (25), outlets for product, permeate, and waste (11, 17), air filters, and T junctions.

The formulation system presented facilitates the formulation of biological compounds, specifically nucleotides, within a lipid-based carrier. The system consists of a microfluidic mixing unit (1) connected to a thin film evaporation unit (24) to enable the formulation process.

In more detail, the system includes input inlets (14, 15) that allow the introduction of first and second aqueous buffers, organic solvents, active ingredients, and/or other compounds into the microfluidic mixing unit for the formulation of the biological compound. The mixing unit (1) can comprise multiple microfluidic mixers operating in parallel, such as 5 to 20 microfluidic mixers. These mixers may include staggered herringbone micromixers, laminar flow mixers, fractional flow mixers, or chaotic mixers.

Once the formulation of biological compounds, such as lipid nanoparticles (LNPs), is achieved in the microfluidic mixing unit (1), the fluid containing the particles (such as LNPs) is transferred to a buffering tank (21) through the tubing. From there, the fluid is pumped to the thin film evaporator (24) using a volumetric pump (23).

The thin film evaporation unit consists of several key components, including a feed inlet (32), evaporation surfaces (33), a heater (31), a gas inlet (25), a thermocouple, and outlets for permeate (11) and concentrate (29). Additionally, the evaporation unit can be connected to a condenser (26) and a vacuum pump (27) via the permeate outlet (11). In operation, the fluid containing the particles is introduced into the thin film evaporator (24) through the feed inlet (32). The fluid is then distributed onto the evaporation surfaces (33), where it forms a thin film that flows horizontally. As the liquid film moves across the surface, heat exchange takes place between the heated gas and the liquid. This heat exchange causes the organic phase from the lipid-based carrier suspension to evaporate, resulting in the concentration of the remaining suspension. The concentrate, now separated from organic solvents, exits the evaporation unit through a concentrate outlet (29) and is transferred to a buffering tank (28) for further processing. The collected concentrate in the buffering tank is then directed to a filtration unit (12) for filtration. This transfer is facilitated by a volumetric pump (30). Furthermore, the system includes additional inlets between the buffering tank and the filtration unit. These additional inlets allow for the introduction of buffers or reagents to the collected concentrate, enabling adjustments such as pH modification or concentration dilution as needed.

The evaporated organic phase is then condensed to exit the system via a permeate outlet (11) to be condensed by a condenser (26) and collected separately.

Figure 8 shows a possible embodiment of a spray evaporation unit. The spray evaporation unit consists of a drying chamber and a cyclone that are interconnected, enabling the transfer of moist air. Both the drying chamber and the cyclone have outlets at the bottom end for the concentrate. The drying chamber is equipped with an inlet for the liquid feed, an atomizer, and an inlet for the air or gas. The cyclone includes an outlet for the permeate. Additionally, the unit is equipped with at least one thermocouple or sensor for temperature measurement, at least one pump, and a vacuum pump.

Figure 9 depicts an embodiment of a formulation production system designed to produce lipid-based carriers utilizing a spray evaporation unit, as described in the current disclosure. The system includes various components and handling apparatuses such as a microfluidic chip (1), a spray evaporation unit (2), vacuum, filters (9 and 12), atomizer (5), pumps (18), inlets (13, 14, 15, 16), air or gas inlet (7), outlets for product, permeate, and waste (11, 17, 10, and 19), thermocouple, air filter (9), and T junctions.

The formulation system enables the formulation of biological compounds, including nucleotides, within a lipid-based carrier. To achieve this, the system comprises a microfluidic mixing unit (1) that is connected to a spray evaporation unit (2).

The formulation of biological compounds, such as nanoparticles, is achieved within the microfluidic mixing unit (1) as described in figure 7. Once the formulation is achieved, the fluid containing the particles is transferred to the spray evaporation unit (2) via tubing. The spray evaporation unit (2) consists of a drying chamber (3) and a cyclone (4) that are interconnected, enabling the transfer of moist air. The fluid containing the particles is introduced into the drying chamber through an atomizer (5), which facilitates the formation of atomized droplets comprising a mixture of aqueous and organic phases. Within the drying chamber (3), these atomized droplets come into contact with hot air or heated gas that is supplied through an inlet (7). The contact with the hot air or gas promotes rapid evaporation, resulting in a concentrate composed of concentrated particles in liquid form. Once the desired evaporation is achieved, the concentrate and the air are separated in the cyclone. The concentrate, from which the organic solvent has been removed, is collected through a cyclone outlet (11). Additionally, some of the concentrate settles down in the drying chamber due to gravity and is collected through a product outlet (20). The concentrate collected through the cyclone outlet (11) is then directed to a filtering unit (12) for further processing. Furthermore, the system may include additional inlets between the cyclone outlet and the filtering unit to allow for the addition of buffers or reagents to the collected concentrate. This enables adjustments such as pH modification or dilution of the concentrate.

The air, now containing moisture from the evaporation process, is typically discharged from the spray dryer through an outlet (19), which can be connected to further filtering units (9) with a waste outlet (10). Vacuuming, purge gas and/or heating separately or in combination is optionally applied before and/or during the evaporation process to improve the efficiency of solvent separation.

Figure 10 shows a schematic overview of a pervaporation unit where a vacuum is applied to the gas phase (A), a purge gas or dry air is flown (B), and the liquid or the gas is warmed (C). To accelerate the evaporation rate and create a sink condition, all presented means vacuum, purge gas and heating or any combination thereof can be used during the spraying process.

Figure 11. shows the effect of the pH, the buffer concentration, the temperature and the storage time on the LNP hydrodynamic diameter (size) and PDI in the acidic buffer (citrate pH 3 to 6) or in the reconstituted buffer (TRIS pH 7.4). The dashed red line shows the responses for the selected factors (i.e., pH 3.8, 1 mM citrate, 20°C and 10 days storage).

Figure 12. (A) shows the hydrodynamic diameter of SM-102-LNP and CX5-LNP stored at 37°C in TRIS sucrose pH 7.4 buffer. Figure 12 (B) shows the hydrodynamic diameter of SM-102-LNP and CX5-LNP produced in citrate 1 or 5 mM at pH 3.8, stored at 37°C in citrate 2 or 10 mM at pH 3.8 and reconstituted in TRIS sucrose pH 7.4.

Claims

1. A method for manufacturing a lipid-based carrier, comprising: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming a lipid-based carrier suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said lipid-based carrier in said suspension; and c. collecting said concentrated lipid-based carrier suspension.

2. The method according to claim 1, wherein said concentration of said lipid- based carrier in said concentrated lipid-based carrier suspension is at most 30 times higher than a concentration of said lipid-based carrier in said lipid- based carrier suspension.

3. The method according to claim 1 or 2, wherein the aqueous phase solution comprises one or more polynucleotides.

4. The method according to any of the previous claims, wherein a second aqueous phase solution is added to said collected concentrated lipid-based carrier suspension during or after the evaporation process, wherein said second aqueous phase solution optionally comprises one or more polynucleotides.

5. The method according to any of the previous claims, wherein said lipid-based carrier comprises said one or more polynucleotides.

6. The method according to any of the previous claims, wherein a total surface to volume ratio of said organic phase solution and said gas is maximized.

7. The method according to any of the any of the previous claims, wherein the evaporation process is spray evaporation or film evaporation, such as thin film evaporation or falling film evaporation.

8. The method according to claim 7, wherein the spray evaporation comprises spraying said lipid-carrier based suspension through a nozzle wherein said nozzle preferably has a diameter of 0.5 mm to 2.5 mm.

9. The method according to claim 8, wherein the temperature of said nozzle is between 20 and 50°C, resulting in an outlet aerosol temperature of between 4 and 40°C.

10. The method according to any of the claims 8 or 9, wherein said spraying of said lipid-carrier based suspension comprises a flow rate ranging from 3 mL/min to 5 L/min.

11. The method according to any of the claims 7 to 10, wherein the spray evaporation comprises applying a gas pressure of between 2 and 6 bar.

12. The method according to claim 7, wherein said film evaporation is carried out at a temperature ranging between 15 to 40°C, and preferably at a flow rate of said lipid-carrier based suspension ranging from 1 to 150 mL/min.

13. The method according to claims 7 or 12, wherein the film evaporation comprises applying a vacuum pressure of between 0.0001 and 1 bar.

14. The method according to any of the previous claims, wherein said mixing in step a occurs by means of impingement jet mixing and/or microfluidic mixing, wherein said microfluidic mixing is laminar microfluidic mixing or chaotic microfluidic mixing.

15. The method according to any of the previous claims, wherein the formed lipid- based carrier suspension is diluted before step b.

16. The method according to any of the previous claims, wherein the lipid-based carrier manufacturing does not involve tangential filtration through a membrane, such as a TFF membrane.

17. The method according to any of the previous claims, wherein the collected concentrated lipid-based carrier suspension is mixed with a solution containing a buffer, a cryoprotectant, or a mixture thereof, to protect the lipid-based particles from aggregation and degradation during collection or to make said collected suspension an injectable solution at physiological pH of between 7.35 and 7.45.

18. The method according to any of the previous claims, wherein said organic phase solution further comprises one or more calixarenes.

19. The method according to any of the previous claims, wherein the aqueous phase solution in step a comprises a buffer having a pH below 7 and/or a low salt concentration.

20. The method according to any of the previous claims, wherein said collected concentrated lipid-based carrier suspension is stored in a storage buffer.

21. The method according to claim 20, wherein said storage buffer comprises the same pH and/or salt concentration as obtained after the mixing in step a.

22. The method according to any of the previous claims 20-21, wherein said storage buffer has a pH between 3-6.

23. The method according to any of the previous claims 20-22, wherein said storage buffer has a pH between 3-4.

24. The method according to any of the previous claims 20-23, wherein said storage buffer has an ionic strength less than about 10 mM.

25. The method according to any of the previous claims 20-24, wherein said storage buffer comprises a monovalent salt.

26. The method according to any of the previous claims 20-25, wherein said storage buffer is a 1 mM citrate buffer having a pH of 3.8.

27. The method according to any of the previous claims 20-25, wherein said stored concentrated lipid-based carrier suspension is buffer-exchanged or diluted with a buffer to make said stored suspension an injectable solution at physiological pH of between 7.35 and 7.45.

28. The method according to claim 27, wherein said stored concentrated lipid- based carrier suspension is diluted in a 20 mM TRIS buffer having a pH of

7.4.

29. The method according to claim 27, wherein said stored concentrated lipid- based carrier suspension is diluted in a 40 mM TRIS buffer having a pH of

8.4.

30. The method according to any of the previous claims 27-29, wherein said stored concentrated lipid-based carrier suspension is diluted between 2 and 200 times.

31. The method according to any of the previous claims, wherein said evaporation process generates a continuous process of manufacturing said formulation.

32. A method for manufacturing a lipid nanoparticle (LNP), comprising: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming an LNP suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said LNP in said suspension; and c. collecting said concentrated LNP suspension.

33. The method according to claim 32, wherein said organic phase solution further comprises one or more calixarenes.

34. The method according to claim 32, wherein the one or more lipids in the organic phase solution consist of a sterol, a phospholipid, a PEGylated lipid and an ionizable cationic lipid.

35. The method according to claim 34, wherein said ionizable cationic lipid has a pKa between 5 and 7.

36. The method according to any of the previous claims 32-35, wherein said aqueous phase solution comprises one or more polynucleotides.

37. The method according to any of the previous claims 32-36, wherein said aqueous phase solution has a pH below 7.

38. The method according to any of the previous claims 32-37, wherein said aqueous phase solution has a pH less than the pKa of the ionizable lipid.

39. The method according to any of the previous claims 32-38, wherein said mixing in step a occurs by means of impingement jet mixing and/or microfluidic mixing, wherein said microfluidic mixing is laminar microfluidic mixing or chaotic microfluidic mixing.

40. The method according to claim 39, wherein chaotic microfluidic mixing occurs by means of one or more herringbone structures.

41. The method according to any of the previous claims 39-40, wherein the total flow rate (TFR) is between 1 ml/min and 500 ml/min.

42. The method according to any of the previous claims 39-42, wherein the flow rate ratio (FFR) is between 5/1 and i.

43. The method according to any of the previous claims 39-42, wherein the flow rate ratio (FFR) is greater than 1 : 1.

44. The method according to any of the previous claims 32-43, wherein said LNPs have a mean particle diameter below 500 nm.

45. A method for storing a lipid-based carrier, said method comprising storing said lipid-based carrier suspension in a storage buffer having a pH below 7 and/or a low salt concentration.

46. The method according to claim 46, wherein the lipid-based carrier suspension is manufactured by: a. mixing an aqueous phase solution with an organic phase solution comprising one or more lipids in one or more organic solvents and thereby forming a lipid-based carrier suspension; b. removing at least part of said organic phase from the suspension by an evaporation process, wherein said one or more organic solvents is transported from said organic phase solution to a gas and wherein said organic phase solution is in direct contact with said gas and thereby increasing a concentration of said lipid-based carrier in said suspension; and c. collecting said concentrated lipid-based carrier suspension.

47. The method according to claim 46, wherein said storage buffer comprises the same pH and/or salt concentration as obtained after the mixing in step a.

48. The method according to any of the previous claims 45-47, wherein said storage buffer has a pH between 3-6.

49. The method according to any of the previous claims 45-481, wherein said storage buffer has a pH between 3-4.

50. The method according to any of the previous claims 45-49, wherein said lipid- based carrier is a lipid nanoparticle (LNP), said LNP comprising an ionizable lipid.

51. The method according to claim 50, wherein said storage buffer has a pH less than the pKa of the ionizable lipid.

52. The method according to any of the previous claims 45-51, wherein said storage buffer has an ionic strength less than about 10 mM.

53. The method according to any of the previous claims 45-52, wherein said storage buffer comprises a monovalent salt.

54. The method according to any of the previous claims 45-53, wherein said storage buffer is a 1 mM citrate buffer having a pH of 3.8.

55. The method according to any of the previous claims 45-54, wherein said stored concentrated lipid-based carrier suspension is buffer-exchanged or diluted with a buffer to make said stored suspension an injectable solution at physiological pH of between 7 and 9, preferably between 7 and 8.

56. The method according to claim 55, wherein said stored concentrated lipid- based carrier suspension is diluted in a 20 mM TRIS buffer having a pH of

7.4.

57. The method according to claim 55, wherein said stored concentrated lipid- based carrier suspension is diluted in a 40 mM TRIS buffer having a pH of

8.4.

58. The method according to any of the previous claims 55-57, wherein said stored concentrated lipid-based carrier suspension is diluted between 2 and 200 times.

59. The method according to any of the previous claims 45-58, wherein said lipid- based carrier suspension is stored at a temperature at least above 4°C for at least 48 hours.

60. The method according to any of the previous claims 45-59, wherein said lipid- based carrier suspension is stored at a temperature above 37°C for at least 2 weeks.

61. A system for manufacturing a formulation comprising one or more lipid-based carriers comprising polynucleotides, wherein, said system comprises one or more mixing units, wherein said mixing units are impingement jet mixing units and/or microfluidic mixing units and wherein said mixing units are suited to mix at least one aqueous solution comprising polynucleotides with an organic phase solution comprising one or more lipids and/or one or more polymers thereby forming a formulation comprising one or more carriers, wherein said one or more microfluidic mixing units are fluidly connected to a spray-drying apparatus or a film evaporation apparatus for removal of at least a part of the organic solvents from said formulation, and wherein said system optionally further comprises one or more filtering unit, one or more condenser, and/or one or more buffering tank fluidly connected to spraydrying or evaporation apparatus.