Lyophilised composition

Abstract

The present invention provides lyophilised pharmaceutical compositions and processes of making said lyophilised pharmaceutical compositions. More particularly, the present invention provides lyophilised pharmaceutical compositions comprising lipid nanoparticles encapsulating a nucleic acid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads) and processes for making said compositions. The present invention further provides the use of said lyophilised pharmaceutical compositions in medicine.

Description

LYOPHILISED COMPOSITIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority under Article 8(1) to United Kingdom Application No. GB2311382.2, which was filed July 25, 2023 and is hereby incorporated herein by reference in their entireties for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in computer readable form in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on July 25, 2023, is named “70364GP01 P_sequence_listing.xml” and is 12,818 bytes in size.TECHNICAL FIELD

[0003] The present invention relates to the field of lyophilised pharmaceutical compositions and processes for making and using said lyophilised pharmaceutical compositions.BACKGROUND OF THE INVENTION

[0004] A major challenge for nucleic acid containing pharmaceuticals and vaccines is efficient delivery of the payload to the target site while avoiding degradation and clearance from the body. To overcome this barrier, lipid carrier particles (e.g., lipid nanoparticles) that are capable of encapsulating the nucleic acid within a liquid core of the particles, have emerged as one of the most efficient delivery vehicles for said nucleic acids (see EP2591103). The particles protect the nucleic acid from degradation and enable its cellular uptake and intracellular release.

[0005] However, a disadvantage of this approach is the instability of the nucleic acid and lipid carrier particles, (e.g., due to susceptibility to hydrolysis). To avoid such degradation, formulations comprising nucleic acids and lipid carrier particles are typically stored at minus 20 °C to minus 80 °C under RNase free conditions.

[0006] For distribution, pharmaceutical compositions containing the nucleic acid and lipid carrier particles are subject to cold chain storage, in which it is typically maintained at a temperature ranging from -20 °C to -80 °C during its lifecycle. However, maintaining cold chain storage has a number ofdisadvantages, including cost, complexity of distribution, and potential loss of product. In some cases, failure to maintain cold chain storage, e.g., due to refrigeration failure, electrical outages, etc., may necessitate discarding of the pharmaceutical composition. In fact, Globally, about half of the vaccines are wasted due to improper temperature control (WHO, “Monitoring Vaccine Wastage at Country Level: Guidelines for Programme Managers”. World Health Organization; Geneva, Switzerland: 2005). Additionally, once reaching respective endpoints, pharmaceutical compositions that cannot be stored at the designated temperature must be administered within a prescribed timeframe or discarded.

[0007] Accordingly, it is desirable to improve long term stability of pharmaceutical compositions comprising nucleic acids encapsulated within lipid carrier particles, as well as prevent and / or reduce the need for cold chain storage.

[0008] One approach that has been postulated has been the use of lyophilisation (also known as freeze-drying) techniques. Lyophilisation is commonly used in the pharmaceutical industry to increase the stability and shelf life of various products by removing the solvent (most usually water) from drug formulations (Chen et al. J Control Release. 2010 Mar 19;142(3):299-311). However, lyophilisation of nucleic acid containing pharmaceuticals, especially pharmaceutical compositions comprising nucleic acids that are encapsulated within lipid carrier particles (e.g., lipid nanoparticles, also categorized as liposomes) is not straightforward. Lyophilisation processes are time-consuming and can result in high inherent heterogeneity (i.e. , vial-to-vial inconsistencies). Furthermore, lyophilisation is known to negatively impact a number of critical quality attributes (CQAs) most notably loss of biological potency after lyophilisation. Improved methods are required.SUMMARY OF THE INVENTION

[0009] The inventors of the present application have discovered improved processes for lyophilizing pharmaceutical compositions that comprise lipid carrier particles encapsulating a nucleic acid payload. More particularly, the inventors discovered that by lyophilizing said compositions in the form of lyophilised beads (or “LyoBeads”) the result was that a number of CQAs were improved compared to comparator compositions that were lyophilised as cakes i.e., using traditional batch freeze-drying techniques. Most notably, and to the inventor’s surprise, the lyophilised pharmaceutical compositions of the present disclosure (i.e., in the form of LyoBeads) were significantly more potent than the comparator compositions (in the form of cakes).

[0010] A further advantage of the improved processes disclosed herein is that the disclosed processes can be carried out using faster drying protocols than traditional batch freeze-drying resulting in faster manufacturing and distribution of pharmaceuticals and vaccines.

[0011] Accordingly, in a first aspect there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleicacid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads).

[0012] In a further aspect there is provided a method of reconstituting the lyophilised pharmaceutical compositions disclosed herein comprising adding a sterile reconstitution solution to the lyophilised pharmaceutical composition; and reconstituting the lyophilised pharmaceutical composition.

[0013] In a further aspect there is provided a vaccine comprising the lyophilised pharmaceutical compositions disclosed herein. Further provided is a vaccine reconstituted from the lyophilised pharmaceutical compositions disclosed herein.

[0014] In a further aspect there is provided a multicomponent vaccine comprising: a first lyophilised pharmaceutical composition according to the first aspect (“First LyoBead”) and a second lyophilised pharmaceutical composition according to the first aspect (“Second LyoBead”) wherein the first LyoBead comprises lipid nanoparticles encapsulating a first nucleic acid payload and the second LyoBead comprises lipid nanoparticles encapsulating a second nucleic acid payload wherein the first and second nucleic acid payloads encode protein immunogens that are different from one another.

[0015] In a further aspect there is provided a kit comprising a first container and a second container wherein the first container comprises the lyophilised pharmaceutical compositions or vaccines disclosed herein, and the second container comprises a sterile reconstitution solution.

[0016] In a further aspect there is provided a process for preparing the lyophilised pharmaceutical compositions of the first aspect said process comprising: i) introducing an aqueous pre-lyophilisation composition comprising lipid nanoparticles encapsulating a nucleic acid payload into a dispensing tip; ii) positioning the dispensing tip above a cryogenic liquid or a cryogenically cooled surface iii) dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the cryogenically cooled surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv) drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)).

[0017] In a further aspect there is provided a lyophilised pharmaceutical composition obtained from the processes disclosed herein. Further provided is a vaccine comprising the lyophilised pharmaceutical composition disclosed herein.

[0018] In a further aspect there is provided a vessel comprising at least one LyoBead, said at least one LyoBead being prepared by the processes disclosed herein.

[0019] In a further aspect there is provided the use of the lyophilised pharmaceutical compositions, vaccines or kits disclosed herein in the manufacture of a medicament for treating a subject in need thereof. Also provided is the use of the pharmaceutical compositions, vaccines or kitsdisclosed herein in the manufacture of a medicament for prophylaxis in a subject in need thereof.

[0020] In a further aspect there is provided a method for eliciting an immune response in a subject in need thereof comprising administering the pharmaceutical compositions or vaccines disclosed herein to the subject, optionally wherein the subject is a human subject.

[0021] In a further aspect there is provided the pharmaceutical compositions or vaccines disclosed herein for use in medicine.

[0022] In a further aspect there is provided the pharmaceutical compositions or vaccines disclosed herein for use in the treatment or prevention of disease in a subject, optionally wherein the subject is a human subject.BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 : Sequence of the mRNA used in the experiments described herein. Said mRNA encodes for eGFP and nanoLuciferase (#22004 sequence).

[0024] FIG. 2: Photographs depicting the appearance of lyophilised cakes (left) and lyophilised beads (right) in their final container.

[0025] FIG. 3: Lyophilisation cycle - Conservative cycle: primary drying -29°C (DEL5010, DEL5012). Graph plots temperature and pressure which are labelled with datapoints as set forth in columns 4 and 5.

[0026] FIG. 4: Lyophilisation cycle - Aggressive cycle: primary drying 0°C (DEL5086). Graph plots temperature and pressure which are labelled with datapoints as set forth in columns 4 and 5.

[0027] FIG. 5: Lyophilisation cycle - Aggressive cycle: primary drying 15°C (DEL5011 , DEL5013, DEL5085). Graph plots temperature and pressure which are labelled with datapoints as set forth in columns 4 and 5.

[0028] FIG. 6 shows the lyophilisation cycle for the conditions in FIGS. 7 and 8. The graph (top) plots temperature (dotted lines) and pressure (solid lines) which are labelled with datapoints as set forth in columns 4 and 5 in the table (bottom).

[0029] FIG. 7 depicts results of repeating in part Example 2 and Table 12, and it shows that the average mass percentage of mRNA encapsulation in lipid nanoparticles (LNPs) was the same for cake lyophilization and bead lyophilization when comparing a Moderna-like formulation comprising 9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), cholesterol, distearoylphosphatidylcholine (DSPC), and 1 ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG2k) and a formulation comprising 2-(5-((4-((1 ,4-dimethylpiperidine-4- carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1 ,3-diyl dioctanoate (RV94), cholesterol, DSPC, and 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2k). TO conditions are measurements from samples stored at -70°C after lyophilizationuntil testing for encapsulation whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization until reconstitution and testing for encapsulation. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.

[0030] FIG. 8 shows that bead lyophilization unexpectedly increased the percent of baby hamster kidney-21 cells (BHK-21) cells expressing enhanced green fluorescent protein (eGFP) encoded in mRNA delivered with the formulations of FIG. 7 when compared to the same measure but with cake lyophilization of the same formulations. TO conditions are measurements from samples stored at - 70°C after lyophilization until reconstitution and testing for eGFP expression whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization before testing for eGFP expression. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.

[0031] FIG. 9 shows the lyophilisation cycle for the conditions in FIGS. 10-13. The graph (top) plots temperature (dotted lines) and pressure (solid lines) which are labelled with datapoints as set forth in columns 4 and 5 in the table (bottom).

[0032] FIG. 10 depicts the average mass percentage of mRNA encapsulation in LNPs with 4%, 7.5%, 15%, and 20 % weight by volume of sucrose for formulations comprising RV94, cholesterol, DSPC, and DMPE-PEG2k. Bead lyophilization was less affected by varying the amount of sucrose than cake lyophilization was affected by the amount of sucrose. Unexpectedly, the average mRNA encapsulation for bead lyophilization with 4%, 7.5%, 15%, or 20% weight by volume of sucrose was higher than the same sucrose amounts for cake lyophilization. TO conditions are measurements from samples stored at -70°C after lyophilization until testing for encapsulation whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization until reconstitution and testing for encapsulation. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.

[0033] FIG. 11 shows that bead lyophilization unexpectedly increased the percent of BHK-21 cells expressing enhanced green fluorescent protein (eGFP) encoded in mRNA delivered with the formulations of FIG. 10 when compared to the same measure but with cake lyophilization of the same formulations. TO conditions are measurements from samples stored at -70°C after lyophilization until reconstitution and testing for eGFP expression whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization before testing for eGFP expression. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.

[0034] FIG. 12 shows the LNPs’ mean diameter for the formulations in FIG. 10. With 4% weight by volume sucrose, lyophilization in cakes or beads increased the mean diameter. But with 7.5% and 15% weight by volume sucrose, the LNP diameter was less affected across cake and beadlyophilization conditions when compared to non-lyophilized conditions (“bulk liquid”). However, as FIG. 11 demonstrates, the increase in mean LNP diameter does not negatively affect the potency of the formulations. TO conditions are measurements from samples stored at -70°C after lyophilization until reconstitution and testing for diameter whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization before testing for diameter. Error bars are standard deviation.

[0035] FIG. 13 shows the LNPs’ polydispersity index for the formulations in FIG. 10. With 7.5% and 15% weight by volume sucrose, the PDI was largely unchanged for lyobeads or for cake lyophilization, except with the condition stored at room temperature for seven days after lyophilization.DETAILED DESCRIPTION OF THE INVENTION

[0036] Prior to setting forth the invention in detail, it may be helpful to the understanding of one of ordinary skill to define the following terms:

[0037] Unless otherwise explained or defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopaedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0038] The articles "a" and "an" are used herein to refer to one or to more than one ( / .e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0039] “Or” supports, contemplates, and when recited in the claims, claims “one or a combination of” as in “one or a combination of A, B, or C.” To illustrate, “A, B, or C” means A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C, unless otherwise illustrated. That is, “or” supports and contemplates “and” as in “and / or” wherein “and / or” includes any combinations within the list of alternatives without being limited solely to the combination of all alternatives in a list ( / .e., “A, B, or C” includes “A and B” and is not limited to “A, B, and C”).

[0040] Furthermore, the recitation of a list of alternatives, which may be conjoined by “and” and from which at least one alternative is selected, further contemplates, and supports all combinations within the list of alternatives. For example, “X is selected from the group of: A, B, and C” contemplates and supports “X is selected from the group of: A, B, C, and combinations thereof,” “X is selected from at least one of the group of: A, B, and C,” and “X is selected from one or more of thegroup of: A, B, and C.” For further example, “X is selected from the group consisting of A, B, and C” contemplates and supports “X is selected from the group consisting of A, B, C, and combinations thereof,” “X is selected from at least one of the group consisting of A, B, and C,” or “X is selected from one or more of the group consisting of A, B, and C.”

[0041] Each of the following contemplates and supports any of the others: “comprises,” “consists of,” “consists essentially of,” “is / are / being,” “is selected from,” “is at least selected from,” “is selected from the group of,” “is selected from the group consisting of,” “is at least selected from the group consisting of,” “is from at least one of the group consisting of,” and “is from one or more of the group consisting of.” For example and in consideration of the above regarding combinations of listed elements, recitation of “X comprises an A, a B, or a C” in the specification contemplates and supports embodiments wherein “X consists of an A, a B, or a C,” “X consists of an A, a B, a C, or combinations thereof,” “X consists of one or more of an A, a B, or a C,” “X is one or more of an A, a B, or a C,” “X is an A, a B, a C, or combinations thereof,” “X is selected from an A, a B, or a C,” “X is selected from an A, a B, a C, or combinations thereof,” “X is selected from the group consisting of an A, a B, a C, and combinations thereof,” “X is selected from at least one of the group consisting of an A, a B, and a C,” or “X is selected from one or more of the group consisting of an A, a B, and a C.”

[0042] When a specific component of an embodiment is listed — e.g. “X comprises A, B, or C” — then also supported and contemplated are any embodiments which specifically exclude any individual or combinations of components — e.g. “X comprises A, but not B or C” or “X comprises A but does not comprise B or C.”

[0043] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, a quantum of measurement, and the like, is meant to encompass variations of +- 10%, for example +-5%, +-1%, +-0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] “Between” as used herein when referring to a range includes the endpoints of said range, for example between 10 and 100 encompasses both 10 and 100 as well as any and all intermediate values.

[0045] “Sequence,” “region,” or “segment” as used herein, unless otherwise specified, also contemplates, and supports sequences incorporating different forms of nucleic acids, i.e., RNA and DNA, of the same information, or sequences incorporating differing nucleotides found in the different forms of the nucleic acids (j.e. uridines in RNA and thymidines in DNA), as well as sense and antisense (e.g. reverse complementary) information therein.

[0046] Amino acids refers to an amino acid selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N) , aspartic acid (asp,D), cysteine (cys, C) .glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T),tryptophan (trp, W), tyrosine (tyr, Y), valine (val, V).

[0047] A “subject” as used herein is an animal, preferably a mammal, including humans, nonhuman primates, and non-primate mammals such as members of the rodent genus (including but not limited to mice and rats), the Cavia genus (including but not limited to guinea pigs) and members of the order Lagomorpha (including but not limited to rabbits). In an embodiment, the subject is a human.

[0048] "Buffer" refers to a buffered solution that resists changes in pH by the action of its acidbase conjugate components. The pH of the buffer will generally be chosen to stabilize the active material of choice. Generally, this will be in the range of physiological pH, although some proteins, can be stable at a wider range of pHs, for example acidic pH.

[0049] As used herein, “immune response” means the sequence of events occurring at the molecular, cellular or tissue level (i.e . , at any level of biological organisation) in response to an immunogen. In the context of the present disclosure, “immune response” may be the series of cellular (cell mediated) and / or humoral (antibody mediated) events occurring in response to an antigen (e.g., antigens on the surface of bacteria, viruses, fungi etc.) or in response to antigens that are translated from a nucleic acid that encodes said antigen. As used herein, “immunogenicity” or “immunogenic” means the ability of an immunogen to elicit an immune response.

[0050] As used herein, “adjuvant” means a compound or substance (or combination of compounds or substances) that, when administered to a subject in conjunction with an antigen or antigens, for example as part of an immunogenic composition or vaccine, increases or enhances the subject’s immune response to the administered antigen or antigens, compared to the immune response obtained in the absence of adjuvant. With respect to the present disclosure, the adjuvant may additionally mean a compound or substance (or combination of compounds or substances) that, when administered to a subject in conjunction with a pharmaceutical comprising nucleic acid and lipid carrier particles, for example as part of an immunogenic composition or vaccine, increases or enhances the subject’s immune response to the protein (e.g., protein immunogen) encoded by the nucleic acid.

[0051] As used herein the term “immunogenic composition” relates to a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) that is capable of eliciting a specific immune response, e.g., against a pathogen. As such, an immunogenic composition includes one or more antigens (for example, polypeptide antigens) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and / or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject, wholly or partially, against symptoms or conditions induced by a pathogen.

[0052] As used herein, "pharmaceutical composition" refers to preparations which are suitable for administering to a subject that includes a pharmaceutical agent.

[0053] By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, protection or prevention. Administration of an immunologically effective amount elicits an immune response, including a protective immune response .This amount can vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual’s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor’s assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range.

[0054] As used herein “vaccine” refers to a composition that induces an immune response upon inoculation into a subject. In particular the term “vaccine” refers to a composition comprising a nucleic acid that encodes for an immunogen against which an immune response is induced upon inoculation of the composition into a subject. In some embodiments, the induced immune response provides protective immunity.

[0055] The “collapse temperature” or “Tcol” is the temperature at which the composition being dried softens to the point of not being able to support its own structure. The collapse temperature is the maximum temperature that the composition can withstand during primary drying without the composition collapsing. The collapse temperature can be determined using freeze drying microscopy.

[0056] The “glass transition temperature” or Tg’ refers to the temperature at which a composition changes from a glassy, amorphous or vitreous state to a rubbery state. Generally, Tg’ is determined using differential scanning calorimetry and is standardly taken as the temperature at which onset of the change of heat capacity (Cp) of the composition occurs upon scanning through the transition.

[0057] As used herein, the term "sublimation" refers to a process wherein materials change from a solid phase directly to a gaseous phase without passing through a liquid phase. With water, ice turns directly to water vapor without first melting to a liquid form, and then evaporating.

[0058] By “lyophilised” it is meant that a composition has been subjected to a “lyophilisation” or “freeze-drying” procedure which remove water from the composition after the composition is frozen and placed under a vacuum. The lyophilisation or freeze-drying procedure allows ice that forms during freezing of the composition to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, interdependent processes: freezing, primary drying (sublimation), and secondary drying (desorption). In an embodiment, the primary drying step may be referred to as the sublimation step. In an embodiment, the secondary drying step may be referred to as the desorption step.Lyophilised Pharmaceutical Composition

[0059] In a first aspect there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles encapsulating a nucleic acid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads).

[0060] As used herein the term “lyophilised bead” or “LyoBead” refers to a substantially spherical or ovoid structure of freeze-dried material of a given diameter, that is obtained or obtainable when a droplet of an aqueous pre-lyophilisation composition is frozen (i.e. into a frozen droplet) prior to undergoing drying steps (e.g. primary and secondary drying steps) to produce said lyophilised bead. In an embodiment, said given diameter is at least greater than 0.1 mm, such as at least 0.5 mm in diameter.

[0061] In an embodiment, a plurality of lyophilised beads is greater than one LyoBead (i.e. two or more LyoBeads). In an embodiment said pharmaceutical composition is presented as a single unit dose of said pharmaceutical composition. As such in an embodiment said plurality of LyoBeads is a number of LyoBeads sufficient for one unit dose of said pharmaceutical composition. In an embodiment, the lyophilised pharmaceutical composition is in the form of a plurality of LyoBeads, wherein said plurality of LyoBeads corresponds to between 2 and 250 LyoBeads, 2 and 150 LyoBeads, 2 and 100 LyoBeads, 2 and 75 LyoBeads or 2 and 50 LyoBeads. It will be understood by the person skilled in the art that the number of LyoBeads sufficient for one unit dose of said pharmaceutical composition is dependent upon a number of factors including but not limited to the concentration of the nucleic acid, the total dose intended for administration, the dose volume and the size / volume of the LyoBeads.

[0062] A plurality of lyophilised beads are obtained or obtainable when a given volume of an aqueous pre-lyophilisation composition is taken and single droplet aliquot(s) of said aqueous pre- lyophilisation composition are frozen to form a plurality of frozen droplet(s) prior to undergoing drying steps (e.g. primary and secondary drying steps) to produce said plurality of lyophilised beads. As used herein a lyophilised bead is differentiated from a lyophilised cake in that, with the same given volume, a lyophilised cake is obtained when the entirety of said volume is frozen (i.e., into a single frozen mass) prior to undergoing drying steps (e.g. primary and secondary drying steps).

[0063] In an embodiment the plurality of lyophilised beads (LyoBeads) are contained within a vessel. In an embodiment, the vessel has a limited internal volume which is typically between 2 and 50 mL which is sufficient for containing a single unit dose (i.e., a ready-to-use quantity) of the lyophilised pharmaceutical composition disclosed herein to a subject (e.g., a human). In an embodiment the internal volume of the vessel is 3mL ± 0.5 mL. In an embodiment, the vessel is at least partially made of a material which is translucent for electromagnetic radiation, in particular infrared, ultraviolet, and / or visible light. In an embodiment the vessel is plastic or glass. In anembodiment the vessel is glass. In an embodiment the vessel is a container that is configured to contain said plurality of LyoBeads. In an embodiment the vessel is capable of containing a single unit dose of the pharmaceutical compositions disclosed herein. Said, single unit dose comprises said plurality of lyophilised beads.

[0064] In an embodiment the vessel is a vial, optionally wherein said vial has a diameter of between about 10 and 40mm. In an embodiment, the vial is substantially cylindrically shaped. In an embodiment the vial is a siliconized glass vial. In an embodiment, the vial is a non-siliconized glass vial. Alternative vessels may be used and include containers, plates, syringes, tubes etc.

[0065] In an embodiment said LyoBeads (or an average taken therefrom) are spherical or nearly spherical shaped bodies wherein said bodies are structures of freeze-dried material. In an embodiment, said nearly spherical shaped bodies includes bodies that are ovoid.

[0066] In an embodiment the LyoBeads are spherical shaped bodies wherein the sphericity (s) of said bodies is measured by a parameter that is defined as a ratio of ratios. In particular, it is the ratio of the surface area to volume of the body divided by the ratio of the surface area to volume of an idealized spherical body having the same displacement volume as the body. Thus, considering the ratio of surface area to volume of a perfect spherical body of volume (V), to be s0, and the ratio of the surface area to volume of a body of the present disclosure also having volume V to be sp, the sphericity of the body is given by the formula: s = Sp / s0. In an embodiment the spherical shaped bodies of the present disclosure have a sphericity in the range 0.7 and 1 .0, 0.8 and 1 .0 or 0.9 and 1 .0 (where 1.0 indicates a perfectly spherical body).

[0067] In an embodiment the LyoBeads have an average diameter of between 0.1 and 50 mm, such as between 0.1 and 25 mm, between 0.5 and 20 mm, between 1 and 15 mm or between 2 and 10 mm. In an embodiment, the LyoBeads have an average diameter of 1 ± 0.5 mm, 2.5 ± 0.5 mm, 5 ± 0.5 mm or 10 ± 0.5 mm. In an embodiment the average diameter of the LyoBeads is greater than the average diameter of any particulate matter present within a lyophilised cake (for more information see section below entitled “comparator lyophilizate”).

[0068] Although it is an object of the present disclosure that said LyoBeads are uniform in diameter, it will be appreciated by the skilled person that some degree of non-uniformity may be present. However, in an embodiment the average diameter of the LyoBeads disclosed herein fall within a close range, for example within ± 0.25 mm, ± 0.5 mm, ± 0.75mm or ± 1 mm of one another. Thus, for example, a plurality of LyoBeads is composed of LyoBeads, all of whose average diameters lie in the range 4 ± 0.5 mm.

[0069] In an embodiment, the LyoBeads have a percentage residual moisture of less than 2, less than 1 .5, less than 1 , less than 0.75, less than 0.5, less than 0.4 or less than 0.3 as calculated by Karl Fisher titration. In an embodiment the residual moisture content is determined using the gravimetric, Karl Fischer or thermogravimetric methods, for example those methods disclosed in May et al 1982,Journal of Biological Standardization, vol 10, issue 3. In an embodiment the residual moisture content is determined by the Karl Fischer method, for example as described in Example 1 herein. In an embodiment, the LyoBeads have lower percentage residual moisture than a comparator lyophilizate (described further below) wherein the comparator lyophilizate is in the form of a cake (i.e. , not in the form of a plurality of lyophilised beads) as calculated by Karl Fisher titration. In an embodiment, the LyoBeads are dryer than said comparator lyophilizate.

[0070] In an embodiment, there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles encapsulating a nucleic acid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads) and wherein the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or hybrids thereof. In an embodiment the nucleic acid is DNA, optionally for example a DNA antisense oligonucleotide. However, in a preferred embodiment, the nucleic acid is RNA. It will be understood by a person of skill in the art that a nucleic acid in accordance with the present disclosure may be combined or used in conjunction with one or more other nucleic acids.

[0071] In an embodiment the RNA is present at a concentration of between 2.5 and 300 pg / ml, 5 and 250 pg / ml, 10 and 100 pg / ml, 25 and 100 pg / ml, 25 and 75 pg / ml, or 50 and 75 pg / ml. In an embodiment the RNA is present at a concentration of 40 ± 5 pg / ml, 50 ± 5 pg / ml, 60 ± 5 pg / ml or 70 ± 5 pg / ml. In an embodiment the RNA is present at a concentration of 60 ± 5 pg / ml (e.g., 60 pg / ml).

[0072] In an embodiment the RNA is a therapeutic oligonucleotide, optionally wherein the therapeutic oligonucleotide is a small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0073] In an embodiment the RNA is a siRNA. In an embodiment said siRNA comprises or consists of a first stand and a second strand strands, wherein the first strand is an antisense strand capable of binding to a target mRNA sequence and wherein the second strand is a sense strand. In an embodiment the sense strand and / or the antisense strand comprises or consists of 15-27 nucleotides. In an embodiment, at least one nucleotide in the siRNA is modified, optionally wherein the modification comprises locked nucleic acid (LNA), unlocked nucleic acid (UNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, phosphate backbone, DNA, fluorescent probe, ligand modification or combination thereof. In an embodiment the modification comprises a thio-modified phosphate backbone. In an embodiment, said siRNA is synthesized in vitro.

[0074] In a separate embodiment the RNA is a shRNA. shRNA, also referred to as small hairpin RNA or Hairpin Vector, is an artificial RNA molecule with a tight hairpin or stem-loop turn that can be used to silence target gene expression.

[0075] In a preferred embodiment the nucleic acid is messenger RNA (mRNA). In an embodiment the mRNA is an artificial (or recombinant) ribonucleic acid encoding at least one protein, optionally wherein the protein is a protein immunogen, which may be translated in a cell (j.e., mRNA). In an embodiment, the mRNA is neither, nor comprised within, a viral vector or virus-based vaccine (suchas a live-attenuated virus vaccine).

[0076] In an embodiment, the mRNA will comprise, in the 5’ to 3’ direction: 5’ Cap, 5’ UTR, open reading frame encoding at least one protein (optionally a protein immunogen), 3’UTR, and 3’ poly-A tail (in particular, the 5’ Caps; 5’ UTRs, 3’UTRs and 3’ poly-A tails as detailed elsewhere herein). In an embodiment, at least one nucleotide in the mRNA is modified as described elsewhere herein.

[0077] In an embodiment, there is therefore provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles encapsulating an mRNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads). The mRNA molecules can have various lengths but are typically 500-25,000 ribonucleotides long e.g., 1000-25,000, 1000-20,000, 1000-15,000, 1000-10,000, 1000-5000, 1000- 3000, 1000-2500, 1000-2500 or 1000-2000 ribonucleotides long.

[0078] In an embodiment the mRNA is either self-amplifying mRNA (SAM) or non-self-amplifying mRNA.

[0079] In an embodiment the mRNA is self-amplifying and may be referred to as self-amplifying mRNA (SAM). As used herein, the terms self-amplifying mRNA and self-replicating mRNA may be used interchangeably. Where the mRNA is self-amplifying, it is generally larger and typically contains the basic elements described above (a cap, 5' UTR, 3' UTR, and poly(A) tail of variable length) but will have a large open reading frame (ORF) at the 5' end that may encode (i) an RNA-dependent RNA polymerase which can transcribe RNA from the self-amplifying mRNA and (ii) at least one heterologous protein, optionally a protein immunogen.

[0080] A self-amplifying mRNA molecule is typically a positive-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of the encoded protein or may be transcribed to provide further transcripts with the same sense as the delivered RNA, which are translated to provide in situ expression of the encoded protein. The overall result of this sequence of transcriptions is substantial amplification in the number of the introduced RNAs, and so the encoded protein (potentially in addition to further proteins as detailed above) becomes a major polypeptide product of the cells.

[0081] Self-amplifying mRNA can be produced using replication elements derived from, e.g., alphaviruses, and substituting sequences encoding the structural viral proteins with that encoding at least one heterologous protein. The polymerase can be an alphavirus replicase e.g., comprising one or more of alphavirus proteins nsP1 , nsP2, nsP3 and nsP4. Or, such alphavirus-based self-amplifying mRNA can use a replicase from, for example, a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus (EEEV), or a Venezuelan equine encephalitis virus (VEEV). Mutant or wild-type virus sequences can be used e.g., the attenuated TC83 mutant of VEEV has been used for selfamplifying RNA (see WO 2005 / 113782). Thus, a typical self-amplifying mRNA encodes at least one protein and may have two open reading frames. The first (5') open reading frame encodes a replicase, in particular an alphavirus replicase (e.g., as detailed above); the second (3') open reading frame encodes the at least one protein (optionally a protein immunogen). Further open reading frames may also be present, encoding (i) one or more further proteins and / or (ii) accessory polypeptides.

[0082] Self-amplifying mRNA may alternatively comprise separate RNA molecules: (i) a RNA construct for expressing an RNA-dependent RNA polymerase and (ii) a RNA replicon that can be replicated by the replicase in trans, wherein the RNA construct for expressing alphavirus replicase comprises a 5'-cap for driving translation of the replicase. Generally, the RNA will comprise, in the 5’ to 3’ direction: 5’ Cap, 5’ UTR, open reading frame encoding at least one protein (optionally a protein immunogen), 3’UTR, and 3’ poly-A tail (in particular, the 5’ Caps; 5’ UTRs, 3’UTRs and 3’ poly-A tails as detailed above throughout this subsection). SAM molecules can have various lengths, but are typically 5000 to 25000 ribonucleotides long, such as 8000 to 15000 ribonucleotides long, for example 9000 to 12000 ribonucleotides long.

[0083] In an embodiment, the nucleic acid of the present disclosure is provided in purified or substantially purified form. By purified or substantially purified it is meant that said nucleic acid is substantially free from other nucleic acids (e.g., free or substantially free from naturally occurring nucleic acids, such as further nucleic acids expressed by a host cell) or from other proteins or cellular debris. In an embodiment said nucleic acids is at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90%, or 95% pure (by weight).

[0084] In an embodiment, the nucleic acid is suitable for the expression of at least one protein in vitro from a host cell ( / .e., the nucleic acid is, or is part of, an expression vector). Suitable nucleic acid expression vectors (in particular, DNA expression vectors) can comprise, for example, (1) an origin of replication; (2) a selectable marker gene; (3) one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, or a terminator), and / or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell.

[0085] However, in a preferred embodiment, the nucleic acid is for the expression of at least one protein in vivo in a subject ( / .e., the nucleic acid is, or is part of, a nucleic acid-based vaccine). In such preferred embodiments, in addition to a sequence encoding the at least one protein, the nucleic acid may comprise one or more heterologous sequences, such as a sequence encoding a further protein (e.g., as detailed below) and / or a control sequence, in particular a promoter or an internal ribosome entry site.

[0086] In an embodiment the nucleic acid is RNA (e.g. mRNA) and said RNA encodes for at least one protein, optionally a protein immunogen. In an embodiment the pharmaceutical composition of thepresent disclosure is an RNA (e.g. mRNA) vaccine composition.

[0087] In an embodiment, the nucleic acid (e.g., mRNA) encodes a single protein. Said protein may be an immunogen i.e. , a protein that elicits an immune response against a bacterium, a virus, a fungus, or a parasite (or, in some embodiments, against an allergen; and in other embodiments, against a tumor antigen). Following administration of the nucleic acid to a subject the immunogen is translated in vivo and can elicit an immune response in said subject. The immune response may comprise an antibody response (usually including IgG) and / or a cell-mediated immune response. The protein immunogen will typically elicit an immune response which recognises the corresponding bacterial, viral, fungal or parasite (or allergen or tumour) polypeptide, but in some embodiments the polypeptide may act as a mimotope to elicit an immune response which recognises a bacterial, viral, fungal or parasite saccharide. The immunogen will typically be a surface polypeptide e.g., an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc. It will be understood that the present disclosure is not limited by the protein that is encoded by the nucleic acid. However, without limiting the disclosure in any way, in some embodiments the protein elicits an immune response against one of these bacteria:- Neisseria meningitidis: useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein. A combination of three useful polypeptides is disclosed in reference (Giuliani et al. (2006) Proc Natl Acad Sci U S A 103(29): 10834-9.)- Streptococcus pneumoniae’, useful polypeptide immunogens are disclosed in W02009 / 016515. These include, but are not limited to, the RrgB pilus subunit, the beta-N-acetyl- hexosaminidase precursor (spr0057), spr0096, General stress protein GSP-781 (spr2021 , SP2216), serine / threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA.- Streptococcus pyogenes’, useful immunogens include, but are not limited to, the polypeptides disclosed in WO02 / 34771 and WG2005 / 032582.- Moraxella catarrhalis.- Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.- Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in WO2010 / 119343, such as a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and / or the sta011 lipoprotein.- Clostridium tetani’. the typical immunogen is tetanus toxoid.- Cornynebacterium diphtheriae: the typical immunogen is diphtheria toxoid.- Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in WG2006 / 110413 and WG2005 / 111066.Pseudomonas aeruginosa- Streptococcus agalactiae-. useful immunogens include, but are not limited to, the polypeptides disclosed in W02009 / 016515.- Chlamydia trachomatis: Useful immunogens include, but are not limited to, MOMP, PepA, LcrE (e.g., as disclosed in W02006 / 138004), ArtJ, DnaK, CT398, OmpH-like, L7 / L12, OmcA, AtoS, CT547, Eno, HtrA (e.g., as disclosed in W02009 / 109860) and MurG (e.g. as disclosed in W02005 / 002619).- Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in W002 / 02606.- Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and / or urease (WO03 / 018054).- Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and / or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E.coli (UPEC) and meningitis / sepsis-associated E.coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in W02006 / 091517 and W02008 / 020330. Useful MNEC immunogens are disclosed in W02006 / 089264. A useful immunogen for several E.coli types is AcfD (W02009 / 104092).- Bacillus anthracis- Yersinia pestis Useful immunogens include, but are not limited to, those disclosed in WC2009 / 031043 and WC2007 / 049155.- Staphylococcus epidermis- Clostridium perfringens or Clostridium botulinums- Legionella pneumophila- Coxiella burnetii- Brucella, such as B. abortus, B.canis, B.melitensis, B.neotomae, B.ovis, B.suis, B.pinnipediae.- Francisella, such as F.novicida, F.philomiragia, F.tularensis.- Neisseria gonorrhoea.- Treponema pallidum- Haemophilus ducreyi- Enterococcus faecalis or Enterococcus faecium- Staphylococcus saprophyticus- Yersinia enterocolitica- Mycobacterium tuberculosis- Rickettsia- Listeria monocytogenes- Vibrio cholerae- Salmonella typhi- Borrelia burgdorferi- Porphyromonas gingivalis- Klebsiella

[0088] It will be understood that the present disclosure is not limited by the protein that is encoded by the nucleic acid. However, without limiting the disclosure in any way, in some embodiments the protein elicits an immune response against one of these viruses:- Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus, such as the hemagglutinin, neuraminidase or matrix M2 proteins. Where the immunogen is an influenza A virus hemagglutinin it may be from any subtype e.g., H1 , H2, H3, H4, H5, H6, H7, H8, H9, H10, H11 , H12, H13, H14, H15 or H16.- Paramyxoviridae viruses: Viral immunogens include, but are not limited to, those derived from Pneumoviruses (e.g., respiratory syncytial virus, RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g. measles).- Poxviridae: Viral immunogens include, but are not limited to, those derived from Orthopoxvirus such as Variola vera, including but not limited to, Variola major and Variola minor.- Picornavirus'. Viral immunogens include, but are not limited to, those derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one embodiment, the enterovirus is a poliovirus e.g., a type 1 , type 2 and / or type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie A or B virus.- Bunyavirus: Viral immunogens include, but are not limited to, those derived from an Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus.- Heparnavirus’. Viral immunogens include, but are not limited to, those derived from a Heparnavirus, such as hepatitis A virus (HAV).- Filovirus'. Viral immunogens include, but are not limited to, those derived from a filovirus, such as an Ebola virus (including a Zaire, Ivory Coast, Reston, or Sudan ebolavirus) or a Marburg virus.- Togavirus'. Viral immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. This includes rubella virus.- Flavivirus'. Viral immunogens include, but are not limited to, those derived from a Flavivirus, such as Tick-borne encephalitis (TBE) virus, Dengue (types 1 , 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus.- Pestivirus'. Viral immunogens include, but are not limited to, those derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV).- Hepadnavirus’. Viral immunogens include, but are not limited to, those derived from a Hepadnavirus, such as Hepatitis B virus. A composition can include hepatitis B virus surface antigen (HBsAg).- Other hepatitis viruses: A composition can include an immunogen from a hepatitis C virus, delta hepatitis virus, hepatitis E virus, or hepatitis G virus.- Rhabdovirus’. Viral immunogens include, but are not limited to, those derived from a Rhabdovirus, such as a Lyssavirus (e.g., a Rabies virus) and Vesiculovirus (VSV).- Caliciviridae: Viral immunogens include, but are not limited to, those derived from Calciviridae, such as Norwalk virus (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.- Coronavirus: Viral immunogens include, but are not limited to, those derived from a SARS coronavirus (e.g., SARS-CoV-2), avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV). The coronavirus immunogen may be a spike polypeptide. Useful Coronavirus antigens (e.g., SARS-CoV-2 antigens) include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein. In an embodiment, the Coronavirus antigen is a SARS-CoV-2 spike protein. Said SARS-CoV-2 spike protein may be from any variant, e.g., Omicron (such as Omicron BA.1 , BA.2, BA3, BA.4 or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, lota, Zeta, Mu, Lambda, Beta, Gamma, or Delta. In an embodiment, said SARS-CoV-2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g., two) mutations to proline resides. Said one or more mutations may be introduced to stabilize said SARS-CoV-2 spike protein in its pre-fusion conformation.- Retrovirus’. Viral immunogens include, but are not limited to, those derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus.- Reovirus’. Viral immunogens include, but are not limited to, those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus.- Parvovirus: Viral immunogens include, but are not limited to, those derived from Parvovirus B19.- Herpesvirus’. Viral immunogens include, but are not limited to, those derived from a human herpesvirus, such as, by way of example only, Herpes Simplex Viruses (HSV) (e.g., HSV types 1 and 2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).- Papovaviruses’. Viral immunogens include, but are not limited to, those derived from Papillomaviruses and Polyomaviruses. The (human) papillomavirus may be of serotype 1 , 2, 4, 5, 6, 8, 11 , 13, 16, 18, 31 , 33, 35, 39, 41 , 42, 47, 51 , 57, 58, 63 or 65 e.g. from one or more of serotypes 6, 11 , 16 and / or 18.- Adenovirus’. Viral immunogens include those derived from adenovirus serotype 36 (Ad-36).

[0089] As described above, the nucleic acid (e.g., mRNA) may encode a single protein or alternatively, the nucleic acid may encode multiple proteins. Multiple proteins (e.g., multiple immunogens) can be presented as a single polypeptide (fusion polypeptide) or as separate polypeptides. If proteins are expressed as separate polypeptides, then one or more of these may be provided with an upstream internal ribosome entry site (IRES) or an additional viral promoter element. Alternatively, multiple proteins may be expressed from a polyprotein that encodes individual proteins fused to a short autocatalytic protease or as inteins.

[0090] In an embodiment, the nucleic acid of the present disclosure encodes a primary immunogen and at least one further protein. The at least one further protein may be a nanoparticle, e.g. a ferritin nanoparticle (e.g. which is encoded, along with the primary immunogen, by a single open reading frame, resulting in expression of a single polypeptide). In embodiments, the at least one further protein is an antigen; and as such may comprise, or may be, a viral, bacterial, fungal, parasitic, tumour, or allergenic (i.e., from, or derived from, an allergen) antigen; typically encoded by a separate open reading frame to the primary immunogen. The at least one further protein will typically be a pathogen antigen. The at least one further protein will typically be an antigen that is a surface polypeptide e.g., a spike glycoprotein, a haemagglutinin, an adhesin, a fusion protein, or an envelope glycoprotein. In a particular embodiment, the at least one further protein is an antigen from, or derived from, a virus, in particular a virus causing respiratory disease, in particular a Coronavirus. In a further particular embodiment, the at least one further protein is an antigen from, or derived from RSV, in particular the F protein.

[0091] In an embodiment the nucleic acid of the present disclosure is a plurality of mRNA polynucleotides said mRNA polynucleotides encoding different proteins. In an embodiment said plurality of mRNA polynucleotides are mixed prior to encapsulation within the lipid carrier particles (e.g., within lipid nanoparticles). In an embodiment, the plurality of mRNA polynucleotides are separately encapsulated within the lipid carrier particles and then subsequently compounded and / or mixed to produce the final bulk.

[0092] In an embodiment, the RNA (e.g. mRNA) comprises a 5’ cap. In an embodiment said 5’ cap is a 7-methylguanosine which may be added via enzymatic means or a non-enzymatic reaction. In an embodiment, the RNA may have the following exemplary 5’ caps:- a 7-methylguanosine linked 5’-to-5’ to a 5’ first ribonucleotide by a triphosphate bridge (also referred to as “Cap 0”);- a 7-methylguanosine linked 5’-to-5’ to a 5’ first ribonucleotide by a triphosphate bridge, and wherein the first 5’ ribonucleotide comprises a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 1”);- a 7-methylguanosine linked 5’-to-5’ to a 5’ first ribonucleotide by a triphosphate bridge, which5’ first ribonucleotide is linked to a second 5’ ribonucleotide, and wherein the first and second 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 2”);- or a 7-methylguanosine linked 5’-to-5’ to a 5’ first ribonucleotide by a triphosphate bridge, which 5’ first ribonucleotide is linked to a second 5’ ribonucleotide and wherein the first, second and third 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O-Me).

[0093] In an embodiment, the 5’ cap is a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’- methylated ribose (2’-O-Me), e.g., the 5’ end of the RNA has the structure m7G(5')ppp(5')(2'OMeA)pG. In an embodiment, this cap is added non-enzymatically through the use of the following reagent:

[0094] Said reagent is known in the art as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES).

[0095] In other embodiments, the cap may be added resulting in the 5’ end of the RNA (e.g. mRNA) having the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. In an embodiment this cap may be added non-enzymatically through the use of the following reagent:

[0096] Said reagent is known in the art as CLEANCAP Reagent AG (3’OMe) (TRILINK BIOTECHNOLOGIES).

[0097] In an embodiment, the RNA comprises a 3’ poly-adenosine (“poly-A”) tail. In an embodiment said 3’ poly-A tail comprises between 10 and 700 A ribonucleotides. The poly-A tail may comprise at least two non-contiguous stretches of A ribonucleotides (also referred to as a “split poly-A tail”), or a (in particular, only one) contiguous stretch of A ribonucleotides. The total number of A ribonucleotides (“As”) in at least two non-contiguous stretches may be, for example, 10-700, such as 10-600, 10-500, 20-500, 50-500, 70-500, 100-500, 20-400, 30-300, 40-200, 50-150, 70-120, 100-120, or, in particular, 100-120. The total number of As in a (in particular, only one) contiguous stretch may be, for example, 10-700; such as 10-600, 20-600 or in particular 40-600 (such as 50-600, 80-600, 80-550, 100-500; or 40-70, 50-65 or 55-65). Wherein at least two non-contiguous stretches of As are used, these may be of differing length. For example, a first stretch may be 10-150 As in length, such as 10-100, 10-50, 15-50, 20-50, 20-40, 25-40, or, in particular 25-35 As in length. For example, a second stretch may be 10-150 As in length, such as 10-150, 20-120, 30-100, 40-90, 50-90, 60-90, 65-90, 70-90, or, in particular, 80-90 As in length. The first stretch may be located 5’ or 3’ relative to the second stretch. However, in a particular embodiment, the first stretch is located 5’ relative to the second stretch. In a further particular embodiment, the polyA tail comprises, in the 5’ to 3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 80-90 As in length respectively. In a further particular embodiment, the polyA tail comprises, in the 5’-3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 65-90 As in length respectively. In some embodiments, the at least two non-contiguous stretches of As is from, or is part of, the 3’ untranslated region (UTR), e.g., as detailed below.

[0098] In an embodiment the RNA is nucleoside modified. Therefore, RNA analogues, such as those containing modified backbones (e.g., peptide nucleic acids (PNAs) or phosphorothioates) or modified bases, are within the scope of the present disclosure. Thus, in an embodiment, the nucleic acid is RNA, optionally nucleoside modified RNA. The nucleic acid may be linear, circular and / or branched, but will generally be linear. Typically, the nucleic acid will be in recombinant form, i.e., a form which does not occur in nature.

[0099] In an embodiment, the RNA comprises (in addition to any 5' cap structure) one or more modified ribonucleotides, i.e., ribonucleotides that are modified in structure relative to standard A, C, G or U ribonucleotides. In other embodiments, the RNA does not comprise modified ribonucleotides, i.e., the RNA contains standard A, C, G or U ribonucleotides only (except for any 5’ cap structure, if present, e.g., as detailed above).[000100] In an embodiment, wherein one or more modified ribonucleotides are used, said one or more modified ribonucleotides may be, or may comprise, N1-methylpseudouridine (“I m^P”); pseudouridine (“M^”); N1-ethylpseudouridine; 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine (m6A); N6- threonylcarbamoyladenosine; 1 ,2'-0-dimethyladenosine; 1 -methyladenosine; 2'-0-methyladenosine; 2'-0-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2- methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-0-methyladenosine; 2'-0-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O- dimethyladenosine; N6,2'-0-dimethyladenosine; N6,N6,2'-0-trimethyladenosine; N6,N6- dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6- threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza- adenosine; N1-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; .alpha. -thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2- (halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'- Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8- (alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8- (thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6- (isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1 -Deazaadenosine TP; 2'Fluoro-N6- Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP; 2'0-methyl-N6-Bz-deoxyadenosine TP; 2'-a- Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-a- Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; 2'- Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2'-b- aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy- 2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'- Deoxy-2'-b-mercaptoadenosine TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-lodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2- Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3- Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4'- Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2- aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2- aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3- methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O- methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-0-methylcytidine; Lysidine;N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'-OMe- Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; .alpha. -thio- cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3- (deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5- (alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine: 5-bromo-cytidine;5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5- methyl-cytidine: 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1 -methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-th io- 1 -methyl-1 -deaza-pseudoisocytidine; 4-th io- 1 -methyl- pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo- pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl- cytidine TP; 2'0-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy- 2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'- Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'- Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP; 2'- Deoxy-2'-b-thiomethoxycytidine TP; 2'-0-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'- Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5- (2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5- Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'-Homo-cytidine TP; 5- Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-0-dimethylguanosine; N2-methylguanosine; Wyosine; 1 ,2'-0-dimethylguanosine; 1 -methylguanosine; 2'-0-methylguanosine; 2'-0-ribosylguanosine (phosphate); 2'-0-methylguanosine; 2'-0-ribosylguanosine (phosphate); 7-aminomethyl-7- deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-0-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O- trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1-methyl-guanosine; .alpha. -thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2'-Azido-2'- deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine;6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8- (alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio- guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio- 7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio- guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'0-methyl- N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b- Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'- Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b- aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy- 2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'- Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1 ,2'-O- dimethylinosine; 2'-0-methylinosine; 7-methylinosine; 2'-0-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5- carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5- taurinomethyluridine; Dihydrouridine; (3-(3-amino-3-carboxypropyl)uridine; 1 -methyl-3-(3-amino-5- carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'- O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-0-methyluridine; 3-(3-amino-3- carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5- (carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-0-methyluridine; 5- carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-0-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-caboxymethylaminomethyluridine; 5- carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycaeoonylmethyl-2'-0- methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5- methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl- 2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5- Oxyacetic acid-methyl ester-Uridine TP; N1-methyl-pseudo-uridine; N1-ethyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5- (iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-0-methyluridine TP; 5- (iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; .alpha. -thio-uridine; 1 (aminoalkylamino- carbonylethylenyl)-2(thio)-pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouridine; 1(aminoalkylaminocarbonylethylenyl)-pseudouridine; 1 (aminocazbonylethylenyl)-2(thio)-pseudouridine; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouridine; 1 (aminocarbonylethylenyl)-4 (thio)pseudouridine; 1 (aminocarbonylethylenyl)-pseudouridine; 1 substituted 2(thio)-pseudouridine; 1 substituted 2, 4-(dithio)pseudouridine; 1 substituted 4 (thio)pseudouridine; 1 substituted pseudouridine;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouridine; 1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio)pseudouridine; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP; 2'-Azido- deoxyuridine TP; 2'-0-methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a- aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5 (1 ,3- diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouridine; 5- (alkyl)-2,4 (dithio)pseudouridine; 5-(alkyl)-4 (thio)pseudouridine; 5-(alkyl)pseudouridine; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5- (dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1 ,3-diazole-1-alkyl)uracil; 5- (methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5- (methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2- (thio)pseudouridine; 5-(methyl)-2,4 (dithio)pseudouridine; 5-(methyl)-4 (thio)pseudouridine; 5- (methyl)pseudouridine; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-1-2-ethanoic acid; Pseudouridine; 4-Thio- pseudo-UTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1- taurinomethyl-1 -methyl-uridine; 1 -taurinomethyl-4-thio-uridine; 1 -taurinomethyl-pseudouridine; 2- methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine;2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4- methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio- pseudouridine; 5-aza-uridine; Dihydropseudouridine; (,+-.)1-(2-Hydroxypropyl)pseudouridine TP; (2R)- 1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo- vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2- Bromo-vinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1 -(2, 2, 3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-Diethoxyethyl)pseudouridine TP; 1-(2,4,6- Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP; 1-(2- Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP; 1-(3,4-Bis- trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3- carboxypropyl)pseudo-UTP; 1 -(3-Amino-propyl)pseudo-UTP; 1 -(3-Cyclopropyl-prop-2- ynyl)pseudouridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1- (4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1- (4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4- Fluorobenzyl)pseudouridine TP; 1-(4-lodobenzyl)pseudouridine TP; 1-(4- Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy- benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4- Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1-(4- Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP; 1-(4- Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino- pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP; 1 ,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2- Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouri- dine TP; 1 -{3-[2-(2-Aminoethoxy)-ethoxy]- propionyl} pseudouridine TP; 1 -Acetylpseudouridine TP; l-Alkyl-6-(1-propynyl)-pseudo-UTP; 1 -Alkyl-6- (2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6- homoallyl-pseudo-UTP; 1-Alkyl-6-vinyl-pseudo-UTP; 1 -Allylpseudouridine TP; 1-Aminomethyl- pseudo-UTP; 1 -Benzoylpseudouridine TP; 1 -Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo- UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1- Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1- Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1- Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1- Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1- Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1 -Homoallylpseudouridine TP; 1 -Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio- pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1- Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4- morpholino)-pseudo-UTP; 1 -Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1 -Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6- bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6- cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1-Methyl-6-ethoxy-pseudo-UTP; 1- Methyl-6-ethylcarboxylate-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo- UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP; 1-Methyl-6-hydroxy- pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy- pseudo-UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6-trifluoromethoxy-pseudo-UTP; 1- Methyl-6-trifluoromethyl-pseudo-UTP; 1 -Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP;1-Phenyl-pseudo-UTP; 1 -Pivaloylpseudouridine TP; 1 -Propargylpseudouridine TP; 1 -Propyl-pseudo- UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1- Thiomethoxymethylpseudouridine TP; 1 -Thiomorpholinomethylpseudouridine TP; 1- Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'- anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'- OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b- Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-a-mercaptouridine TP; 2'- Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'- Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'- Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine TP;2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-0-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo- UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP; 5- iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5- Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4- Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6- Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro- pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6- Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6- Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-lodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo- UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4- methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3- (2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2- ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1- methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid; Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7- heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4- demethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1 ,3-(diaza)-2-(oxo)- phenthiazin-1-yl; 1 ,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1 ,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'- deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'- deoxyribose; 2'-fluoro-modified bases; 2'-0-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo- pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3- (methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4- (methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5- (methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6- chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenthiazin-1 -yl; 7-(aminoalkylhydroxy)-1 -(aza)-2-(thio)-3-(aza)-phenoxazin-1 -yl; 7-(aminoalkylhydroxy)-1 ,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1 ,3-(diaza)-2-(oxo)- phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1 ,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7- (guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7-(guanidiniumalkylhydroxy)-1- (aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1 ,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7- (guanidiniumalkyl-hydroxy)-l ,3-(diaza)-2-(oxo)-phenthiazin-1 -yl; 7-(guanidiniumalkylhydroxy)-1 ,3- (diaza)-2-(oxo)-phenoxazin-1 -yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1 ,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on- 3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1 ,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2- amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara- guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-Amino-pentaoxanonadecyl)adenosine TP.[000101] In an embodiment, the one or more modified ribonucleotides detailed above is, or comprise, I m^P and / or MT In a preferred embodiment, wherein one or more modified ribonucleotidesare used, said one or more modified ribonucleotides comprises or consists of I mMT In such embodiments, the RNA may comprise I m^P and / orand neither standard U ribonucleotides nor other modified U ribonucleotides ( / .e. there are no standard U nucleotides, nor modified U ribonucleotides other than 'I m'-P and / orin the RNA; i.e. 100% U substitution). In particular, the RNA may comprise 1 m and / orand neither standard U ribonucleotides nor other modified ribonucleotides (i.e. there are no standard U nucleotides, nor modified ribonucleotides of any type - A, C, G or U substitutable - other than I m^Pin the RNA; i.e. 100% U substitution with no other modified nucleotides being allowed). The RNA may compriseand neither standard U ribonucleotides nor other modified U ribonucleotides (i.e., 100% U substitution with ^P). In particular, the RNA may compriseand neither standard U ribonucleotides nor other modified ribonucleotides (i.e., 100% U substitution withwith no other modified nucleotides being allowed). In an embodiment, the RNA comprises I m'P, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e., 100% U substitution with I m^P). In an embodiment, the RNA comprises I m^P, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e., 100% U substitution with I m^P with no other modified nucleotides being allowed).[000102] In some embodiments, the percentage of standard As substituted with A-substitutable modified nucleotide (e.g. those above) is at least: 0.1 %, 0.5%, 0.8%, 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard As substituted with m6A may be 0.1-5%, in particular 0.5-2%, in particular 0.8-1.2%, such as about 1 % (or 1 %); in these embodiments the RNA may be circular RNA. Low substitution levels with m6A (e.g., 1%) have been shown to inhibit innate immune activation (Chen et al. Mol Cell. 2019 76, 1: 96-109). In some embodiments, the percentage of standard Cs substituted with cytosine-substitutable modified nucleotide (e.g. those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard Gs substituted with G- substitutable modified nucleotide (e.g., those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In an embodiment, the percentage of standard Us substituted with U-substitutable modified nucleotide (e.g., those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% e.g. with I m'P and / or MT[000103] Nucleic acids of the present disclosure may be codon optimized e.g.; codon optimized RNA. In some embodiments, nucleic acids of the present disclosure may be codon optimised for expression in human cells. Codon optimisation refers to the use of specific codons, which, while not altering the sequence of the expressed protein (given genetic code redundancy), may increase translation efficacy and / or half- life of the nucleic acid.[000104] Therefore, in an embodiment, the RNA is codon optimised. Codon optimisation mayprovide an elevated GC content, relative to non-codon optimised RNA encoding the same protein(s). The GC content (the percentage of all ribonucleotides (or, defined alternatively, all “nitrogenous bases”) in the RNA which are G or C) of the RNA may be at least 10%, such as at least 20%, 30%, 35% or at least 40%, 45%, 46%, 47%, 48%, 49%, or at least 50%. The GC content of the RNA may be 10-70%, such as 20-65%, 30-65%, 35-65%, 40-60%, 45-55%, 46-53%, 47-51%, or 48-50%. Codon optimisation may provide an elevated C content relative to non-codon optimised RNA encoding the same protein(s). The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be least 30%, such as at least 40%, 50%, 55% or at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72% or at least 72%. The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be 30-80%, such as 40-90%, 45-90%, 50-80%, 55-80%, 60-80%, 65-75%, 66-75%, 67-75%, 68-75%, 69-75%, 70-74%, 71-74% or 72-74%[000105] Where the translation of the mRNA is intended to be driven by mammalian UTRs, they may be selected from the 5’and 3’ UTRs of RNA transcripts of the following genes (i.e. the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein (“FAP”), H4 clustered histone 15 (“HIST2H4A"), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene,, interleukin-2 gene (“IL-2”), and transferrin. In some embodiments, the RNA comprises a 5’ and a 3’ UTR selected from:- SEQ ID NO: 1 and 2, respectively,- SEQ ID NO: 3 and 4, respectively,- SEQ ID NO: 5 and 6, respectively,- SEQ ID NO: 7 and 8, respectively,- SEQ ID NO: 9 and 10, respectively, and- RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 1 , 3, 5, 7 or 9 (for the 5’ UTR) and RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 2, 4, 6, 8 or 10 (for the 3’ UTR) (in particular, the pairing of 5’ and 3’ UTRs having such identity to SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, and SEQ ID NO: 9 and 10, respectively);[000106] RNA sequences according to SEQ ID NO: 1 and 2, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 (and RNA sequences having such identity thereto e.g., at least 95% or greater) are more preferred; and RNA sequences according to SEQ ID NO: 1 and 2 (and RNA sequences having such identity thereto e.g., at least 95% or greater) are even more preferred.[000107] Both the 3’ and 5’ UTR may influence expression of the encoded protein through a variety of mechanisms. Without wishing to be bound by this theory, the 5’ UTR may affect the expression of the encoded protein e.g., via pre-initiation complex regulation, closed-loop regulation,upstream open reading frame regulations ( / .e. reinitiation), provision of internal ribosome entry sites, and provision of microRNA binding sites. Without wishing to be bound by this theory, the 3’ UTR may affect the expression of the encoded protein e.g., via providing regulation regions that post- transcriptionally influence expression; e.g. influencing translation efficiency, localisation of the RNA, stability of the RNA, polyadenylation, and circularization of the RNA.[000108] In one specific embodiment, the RNA is circular RNA.[000109] The RNA can be prepared by any means known to the person skilled in the art. In an embodiment the RNA is produced by in vitro transcription (IVT). IVT can use a (DNA) template created and propagated in plasmid form in bacteria or created synthetically (for example by gene synthesis and / or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA- dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the replicating RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the poly-A tail is usually encoded within the DNA template).[000110] Nucleic acid (especially RNA) by themselves and unprotected, may be degraded by the subject’s nucleases and may require a carrier to facilitate target cell entry. Accordingly, the present disclosure provides the nucleic acid (e.g., RNA) and a carrier.[000111] The carrier may be lipid-based (e.g., a lipid nanoparticle) In particular embodiments, the carrier is non-virion, i.e., free or substantially free of viral capsid.[000112] The pharmaceutical composition disclosed herein comprises lipid carrier particles encapsulating a nucleic acid (e.g., RNA) payload. The lipid carrier particles provide a means to protect the nucleic acid (e.g., RNA) payload, and deliver it to target cells for protein expression. With a CNE, the nucleic acid (e.g., RNA) which encodes the protein (optionally the protein immunogen) is complexed with a CNE particle, in particular comprising an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the emulsion particles. The negatively charged phosphate of the nucleic acid complexes with the positively charged molecule of the cationic lipid on the surface of the CNE particle; CNEs do not encapsulate the nucleic acid.[000113] In an embodiment, a lipid-based carrier is a lipid inorganic nanoparticle ( LION”).[000114] In a preferred embodiment the lipid carrier particles are lipid nanoparticles (LNPs). Thus, in an embodiment there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid nanoparticles encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads). In an embodiment the lipid nanoparticles encapsulating an RNA payload is lipid nanoparticle encapsulated RNA. In an embodiment the lipid nanoparticles encapsulating an RNA payload is RNA containing lipid nanoparticles.[000115] LNPs are non-virion liposome particles within which nucleic acid (e.g., RNA) can be encapsulated. LNP delivery systems and methods for their preparation involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. In an embodiment, RNA is encapsulated within the liposomes, and the liposome forms an outer layer around an aqueous RNA-containing core. This encapsulation has been found to protect RNA from RNase digestion.[000116] A plurality of such LNPs (said LNPs encapsulating an RNA payload) will be part of the pharmaceutical composition disclosed herein comprising free and / or encapsulated nucleic acid (e.g., RNA), and in some embodiments the LNPs encapsulate at least: 75%, 76%, 77%, 78%, 89%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 100% of the total number of nucleic acid (e.g., RNA) molecules in the composition. Measuring the percentage of the total nucleic acid encapsulated within the LNPs can be done according to the RiboGreen assay, for example as described in Muramatsu H, et al. Mol Ther. 2022 30(5):1941 -1951 or as described in Example 1 herein.[000117] In an embodiment at least 80% of the LNPs in the pharmaceutical composition are between 20 and 200 nm, 40 and 190 nm, 60 and 180 nm or, in particular, 80 and 160 nm in diameter. In a particular embodiment, substantially all, or all, LNPs in the composition are between 20 and 200 nm, 40 and 190 nm, 60 and 180 nm or, in particular, 80 and 160 nm in diameter.[000118] The LNP can comprise multilamellar vesicles (MLV), small uniflagellar vesicles (SUV), or large unilamellar vesicles (LUV).[000119] The amount of nucleic acid (e.g., RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particle being used. For RNA molecules, in general, an LNP may include 1-500 RNA molecules, e.g. <200, <100, <50, <20, <10, <5, or 1-4. Generally, an LNP includes fewer than 10 different species of RNA e.g. fewer than 5, 4, 3, or 2 different species. In an embodiment, the LNP includes a single RNA species ( / .e. all RNA molecules in the particle have the same sequence).[000120] LNPs according to the present disclosure may be formed from a single lipid (e.g. a cationic lipid) or, in particular, from a mixture of lipids. In particular, the mixture comprises various classes of lipids, such as: a) a mixture of cationic lipids and sterols, b) a mixture of cationic lipids and neutral lipids, c) a mixture of cationic lipids and polymer-conjugated lipids, d) a mixture of cationic lipids, sterols and polymer-conjugated lipids, or e) a mixture of cationic lipids, neutral lipids and polymer-conjugated lipids;f) a mixture of cationic lipids, sterols and neutral lipids; or g) a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids Further classes of lipids, such as anionic lipids, may also be present in a mixture of lipids.[000121] The LNP can comprise multilamellar vesicles (MLV), small uniflagellar vesicles (SUV), or large unilamellar vesicles (LUV).[000122] In an embodiment the LNPs comprise a cationic lipid, a neutral lipid and a PEGylated lipid.[000123] In an embodiment, the cationic lipid has a pKa of 5.0-10.0, 5.0-9.0, 5.0-8.5, 5.0-8.0, 5.0-7.9, 5.0-7.8, 5.0-7.7, or 5.0-7.6. The pKa of the cationic lipid is distinct to the pKa of the LNP as a whole (sometimes called “apparent pKa"). pKa may be determined via any well-known method, such as via a toluene nitrosulphonic acid (TNS) fluorescence assay or acid base titration.[000124] In an embodiment, the cationic lipid is an ionizable amino lipid. In an embodiment the cationic lipid is unsaturated.[000125] In an embodiment, the cationic lipid comprises a tertiary or quaternary amine group. In an embodiment, the cationic lipid comprises a tertiary amine group. Exemplary cationic lipids comprising tertiary amine groups include: 1 ,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin- DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy- 3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-dilinoleyloxy- 3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1 ,2-propanediol (DLinAP), 3-(N,N- dioleylamino)-1 ,2-propanediol (DOAP), 1 ,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl[1 ,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)[1 ,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA); or MC3 (see, e.g. US20100324120).[000126] In some embodiments, the cationic lipid has the structure of lipid RV28, RV31 , RV33,RV37, RV39, RV42, RV44, RV73, RV75, RV81 , RV84, RV85, RV86, RV88, RV91 , RV92, RV93, RV94, RV95, RV96, RV97, RV99, or RV101 , as disclosed in WO 2021 / 038508. In a further embodiment, the cationic lipid has the structure:[000127] In an embodiment, the LNP comprises a cationic lipid and the cationic lipid is RV39. In an embodiment, RV39 is 2,5-bis((9Z,12Z)- octadeca-9,12-dien-1-yloxy)benzyl 4- (dimethylamino)butanoate. In an embodiment, RV39 has the structure:[000128] In an embodiment the LNP comprises a cationic lipid and the cationic lipid is RV94. In an embodiment, RV94 is 2-(5-((4-((1 ,4-dimethylpiperidine-4- carbonyl)oxy)hexadecyl)oxy)-5- oxopentyl)propane-1 ,3-diyl dioctanoate. In an embodiment, RV94 has the structure:[000129] In another embodiment, the cationic lipid has the structure:[000130] In another embodiment, the cationic lipid has the structure:[000131] In an embodiment the lipids in the LNP comprises (in mole %) 20-80, 25-75, 30-70, 35- 65%, 30-60, 35-55 or 35-50% cationic lipid; such as about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid. In an embodiment the lipids in the LNP may comprise (in mole %) at least 20, 25 or at least 35%, or at least 40% cationic lipid. In an embodiment the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60% or no more than 50% cationic lipid.[000132] In an embodiment, the N:P ratio is in the range of (including the endpoints) 1 :1-20:1 , 2:1-10:1 , 3: 1-9:1 , or 4: 1-8:1 , for example within the range 4.5:1 -7.5:1 , 4.5:1 -6.5:1 or 5.0:1 -6.5:1 . In an embodiment, the N:P ratio is between 7:1 and 9:1 e.g. about 8:1. The N:P ratio is the molar ratio of protonatable nitrogen atoms in the LNP’s cationic lipids to phosphates in the nucleic acid (a.k.a “N:P” ratio).[000133] In an embodiment, the polymer-conjugated lipid is a polyethylene glycol-conjugated (PEGylated) lipid. In an LNP, the polyethylene glycols (PEGs) of such PEGylated lipids may have average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5, 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3, 1.8-2.2 or 1.9-2.1 kDa. In an embodiment the PEGs of such PEGylated lipids may have an average molecular weight of about 2.0 (or 2.0 kDa). The average molecular weight of such PEGs may be expressed as the median molecular weight. Alternatively, in an LNP, at least 80% of the PEGs of such PEGylated lipids may have molecular weight of 0.5-11 .0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1 .0- 4.0, 1 .0-3.5, 1 .0-3.0, 1 .2-2.8, 1 .4-2.6, 1 .5-2.5, 1 .6-2.4, or 1 .7-2.3, 1 .8-2.2 or 1 .9-2.1 kDa.[000134] The PEGylated lipid may have the structure:[000135] In an embodiment, the N:P ratio is in the range of (including the endpoints) 1 :1-20:1 , 2:1-10:1 , 3: 1-9:1 , or 4: 1-8:1 , for example within the range 4.5:1 -7.5:1 , 4.5:1 -6.5:1 or 5.0:1 -6.5:1 . In anembodiment, the N:P ratio is between 7:1 and 9:1 e.g. about 8:1. The N:P ratio is the molar ratio of protonatable nitrogen atoms in the LNP’s cationic lipids to phosphates in the nucleic acid (a. .a “N:P” ratio).[000136] Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide and 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, 1 ,2- dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)] and 1 ,2-dimyristoyl- rac-glycerol-3-methoxypolyethylene glycol. In a preferred embodiment, the PEGylated lipid is 1 ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) or 1 ,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2k). In an embodiment, the PEGylated lipid prevents particle aggregation and increases circulation lifetime. In an embodiment the PEGylated lipid has the structure:[000137] The lipids in the LNP may comprise (in mole %) 0.1-8.0, 0.4-7.0, 0.6-6.0, 0.8-4.0 or 0.8-3.5% or 1.0-3.0% polymer-conjugated lipid (e.g., PEGylated lipid); such as about 1.0 (or 1.0%), about 1.5% (or 1.5%), about 2.0% (or 2.0%) or about 2.5% (or 2.5%) polymer-conjugated lipid (e.g., PEGylated lipid). In an embodiment the lipids present in the LNPs comprise (in mole %) between 1 % and 3%, such as between 1 .2% and 2.8%, between 1 .4% and 2.6% or between 1 .6% and 2.4%, optionally 2%. The lipids in the LNP may comprise (in mole %) at least 0.1 , 0.5 or at least 0.8%, or at least 1 % polymer-conjugated lipid (e.g., PEGylated lipid). The lipids in the LNP may comprise (in mole %) no more than 8.0, 6.0 or 4.0% or no more than 3.0% polymer-conjugated lipid (e.g., PEGylated lipid).[000138] In an embodiment, the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to the skilled person may also be used. In an embodiment the neutral lipid is DSPC. In an embodiment the neutral lipid has the structure:[000139] In an embodiment the lipids in the LNP may comprise (in mole %) 0-15.0, 0.1-15.0, 2.0-14.0, 5.0-13.0, 6.0-12.0 or 7.0-11.0%, 8.0-11.0% or 9.0-11.0% neutral lipid, such as about 9.4% (or 9.4%), about 9.6% (or 9.6%), about 9.8% (or 9.8%) or about 10.0% (or 10%) neutral lipid. In an embodiment the lipids in the LNP may comprise (in mole %) at least 0.1 , 5.0 or at least 7.0%, or at least 8.0% or at least 9.0% neutral lipid. In an embodiment the lipids in the LNP may comprise (in mole %) no more than 15.0, 13.0 or no more than 12.0%, or no more than 11.0% neutral lipid. Neutral lipid may also herein be referred to as zwitterionic lipids.[000140] In an embodiment the LNPs further comprise a sterol. In an embodiment the LNPs further comprise a cholesterol-based lipid. In an embodiment the LNPs further comprise cholesterol. In an embodiment the cholesterol stabilizes the nanoparticles by adding mechanical strength and elasticity and increases lipid packing density compared to an LNP lacking cholesterol.[000141] Further exemplary sterols include cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, symosterol, lathosteriol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3p-ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)- cholest-8(9)-en-3p-ol (dihydro T-MAS-d6), zymostenol, sitostanol, campestanol, camperstanol, 7- dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholest-8(9)-en-3p-ol (dihyrdro T-MAS), A5- avensterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, oxysterols, deuterated sterols, fluorinated sterols, sulfonated sterols, phosphorylated sterols, A-ring substituted sterols, cholest-5- ene-3B,4B-diol, 5a-cholestan-3B-ol, 4-cholesten-3-one, cholesta-8(9),24-dien-3-one, cholesta- 8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5a-cholestan-3B-ol, cholesteryl phosphocholine, cholesteryl-d7 pentadecanoate, cholesteryl-d7 palmitate, B-ring substituted sterols, cholestanol, 5B,6B-epoxy-d7, 3B-hydroxy-5-cholestene-7-one, 6a-hydroxy-5a-cholestane, cholestanol, 5a, 6a- epoxy, cholest-5-en-3B,7a-diol, cholest-5-en-3B,7B-diol, cholestanol, 5a,6a-epoxy-d7, A5,7- cholesterol, cholesta-5,8(9)-dien-3B-ol, cholesta-5,8(14)-dien-3B-ol, 7a-hydroxy-4-cholesten-3-one, zymostenol-d7, zymostenol, 7-dehydrodesmosterol, 3b,5a-dihydroxy-cholestan-6-one, D-ring substituted sterols, 3B-hydroxy-5a-cholest-8(14)-en-15-one, 3B-hydroxy-5a-cholestane-15-one, 5a- cholest-8(14)-ene-3B,15a-diol, 5a-cholest-8(14)-ene-3B,15B,-diol, lanosterol-95, 5a-7,24- cholestadiene, 14-dehydro zymostenol, ergosta-5,7,9(11),22-tetraen-3B-ol, cholest-5-ene-3B,25-diol, cholest-(25R)-5-ene-3B,27-diol, 24(R / S),25-epoxycholesterol, 24(S),25-epoxycholesterol, 24(R / S),25- epoxycholesterol-d6, cholest-5-ene-3B,22(S)-diol, cholest-5-ene-3B,22(R)-diol, cholest-5-ene- 3B,24(S)-diol, cholest-5-ene-3B,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N- dimethyl-3B-hydroxycholenamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3B-hydroxycholenamide, 25,27-dihydroxycholesterol, 5-cholestene-3p,20a-diol, 24S,25-epoxy-5a-cholest-8(9)-en-3p-ol, 24(S / R),25-epoxylanost-8(9)-en-3p-ol, 7-keto-27-hydroxycholesterol, 7a,27-dihydroxy-4-cholesten-3-one, 7a,27-dihydroxycholesterol, 7B,27-dihydroxycholesterol, 5a,6B-dihydroxycholestanol, 7a, 25- dihydroxycholesterol, 7p,25-dihydroxycholesterol, 7a,24(S)-dihydroxycholesterol, 7a,24(S)-dihydroxy- 4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7a,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol, or 24S,27- dihydroxycholesterol.[000142] In an embodiment the cholesterol has the structure:[000143] In an embodiment, the lipids in the LNP comprise, or have, from 35 mole% to 65 mole%, optionally from 40 mole% to 60 mole%, optionally 45 mole% to 55 mole%, optionally 45 mole% to 50 mole% cholesterol. In an embodiment, the lipids in the LNP may comprise (in mole %) 20-80, 25-80, 30-70, 30-60, 35-60, 40-60%, 40-50% or 41-49% sterol; such as about 42% (or 42%), about 43% (or 43%), about 44% (or 44%), about 46% (or 46%), or about 48% (or 48%) sterol. In an embodiment the lipids in the LNP may comprise (in mole %) at least 20, 30 or at least 35%, or at least 40% or at least 41 % sterol. In an embodiment the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60%, or no more than 50% sterol.[000144] In an embodiment the lipids in the LNP may have the following mole % in combination: 30-60% cationic lipid (such as 35-55%, or 35-45%), 35-70% sterol (such as 40-55%, or 41-49%), 0.8- 4.0% polymer-conjugated lipid (such as 0.8-3.5%, or 1.0-3.0%), and 0-15% neutral lipid (such as 6.0- 12.0% or 8.0-11.0%).[000145] In a particular embodiment the LNP comprises the cationic lipid RV94, the neutral lipid DSPC, cholesterol and a PEGylated lipid. In an embodiment the LNP comprises the cationic lipid RV94, the neutral lipid DSPC, cholesterol and a PEGylated lipid wherein the cationic lipid is present at a molar percentage of 40% ± 5%, the neutral lipid is present at a molar percentage of 10% ± 5%, cholesterol is present at 48% (w / v) ± 5% and the PEGylated lipid is present at 2% (w / v) ± 0.5%.[000146] Such LNPs encapsulating a nucleic acid (e.g., RNA) payload may be formed by admixing a first solution comprising the nucleic acids with a second solution comprising lipids which form the LNP. The admixing may be performed by any suitable means available to the skilled person, e.g., a T-mixer, microfluidics, or an impinging jet mixer. Admixing may be followed by filtration. Thefiltration may be performed by any suitable means available to the skilled person, e.g., tangential-flow filtration or cross-flow filtration.[000147] Accordingly, in a further aspect, the present disclosure provides a method of preparing an LNP encapsulating a nucleic acid (e.g., RNA) payload encoding at least one protein (optionally a protein immunogen), comprising admixing a first solution comprising the nucleic acid and a second solution comprising lipids which form the LNP (e.g. using the means as set out in the foregoing paragraph); and optionally filtering the obtained admixture (e.g. using the means as set out in the foregoing paragraph).[000148] The pharmaceutical composition of the present disclosure is lyophilised. However, the lyophilised pharmaceutical compositions disclosed herein are lyophilised from an aqueous prelyophilisation composition comprising lipid carrier particles (e.g. LNPs) encapsulating a nucleic acid (e.g. RNA) payload. Said aqueous pre-lyophilisation composition is formulated for stable lyophilisation of the pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating a nucleic acid (e.g. RNA) payload. As used herein therefore the term “aqueous pre-lyophilisation composition” refers to the pharmaceutical formulation in its aqueous state immediately prior to lyophilisation (i.e., immediately prior to undergoing any freezing or drying steps), otherwise referred to as the Final Liquid Bulk.[000149] In an embodiment the aqueous pre-lyophilisation composition comprises an amorphous sugar. The amorphous sugar may be a cryoprotectant and / or a lyoprotectant. The term “cryoprotectant” refers to a class of excipients which prevents freeze damage of what is being frozen. In the present disclosure the term cryoprotectant means a class of excipients that prevent freeze damage to the nucleic acid and lipid carrier particles. The term “lyoprotectant” refers to a class of excipients that prevent drying damage of what is being freeze-dried. In the present disclosure the term lyoprotectant means a class of excipients that prevent drying damage to the pharmaceutical composition comprising lipid carrier particles encapsulating a nucleic acid payload.[000150] In an embodiment the amorphous sugar is selected from sucrose, trehalose, mannose, mannitol, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, isomaltose, maltilol, palatinit, stachyose, melezitose, dextran, or a combination thereof. In one embodiment, the amorphous sugar is selected from sucrose, trehalose, lactose, raffinose, dextran and combinations thereof. In one embodiment, the amorphous sugar is selected from sucrose or trehalose. In a preferred embodiment the amorphous sugar is sucrose.[000151] In an embodiment the aqueous pre-lyophilisation composition comprises an amorphous sugar, wherein said amorphous sugar is sucrose. In an embodiment, the aqueous pre- lyophilisation composition comprises sucrose in a concentration greater than or equal to 5% (w / v). In an embodiment the aqueous pre-lyophilisation composition comprises sucrose in a concentration of between 0.5% and 25% (w / v), 1 % and 20% (w / v) or 2.5% and 10% (w / v). In an embodiment sucroseis present in the aqueous pre-lyophilisation composition in a total amount of less than 30% (w / v), less than 28% (w / v), less than 26% (w / v), less than 24% (w / v), less than 22% (w / v), less than 20% (w / v), less than 18% (w / v), less than 16% (w / v) or less than 14% (w / v), less than 12% (w / v), less than 10% (w / v), or less than 8% (w / v), In an embodiment, the aqueous pre-lyophilisation composition comprises sucrose in a concentration of 7.5% (w / v).[000152] In a further embodiment the aqueous pre-lyophilisation composition further comprises salt. As used herein “salt” refers to ionic compounds that result from the neutralization reaction of an acid and a base, compose of a related number of cations and anions such that the product is without net charge, for example sodium chloride. The component ions can either be inorganic or organic and can be monoatomic or polyatomic. In an embodiment the salt is sodium chloride.[000153] In an embodiment the aqueous pre-lyophilisation composition further comprises sodium chloride. In an embodiment the aqueous pre-lyophilisation composition comprises sodium chloride in an amount less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 15mM, less than 10mM or less than 7.5mM. In an embodiment the aqueous pre-lyophilisation composition comprises sodium chloride in a concentration of between 0.1 mM and 50mM, 0.5mM and 40mM, 1 mM and 30mM, 1 mM and 25mM, 1 mM and 20mM, 1 mM and 10mM, 1 mM and 8mM, 1 mM and 7.5mM, 2.5mM and 7.5mM or 4mM and 6mM. In an embodiment the aqueous pre-lyophilisation composition comprises sodium chloride at a concentration of 5mM ± 0.5mM (or 5mM). In an embodiment, the composition is not completely devoid of salt (e.g., not completely devoid of sodium chloride).[000154] In an embodiment, the aqueous pre-lyophilisation composition further comprises a surfactant. The surfactant may act to decrease the surface tension and may also increase the solubility of other formulation components. In an embodiment the aqueous pre-lyophilisation composition further comprises a surfactant wherein the surfactant is selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g., polysorbate 80 and / or polysorbate 20), octoxinal surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxyl castor oil surfactants, N-octyl-glucoside surfactants, macrogol 15 hydroxy stearate, and combinations thereof. In an embodiment, the surfactant is selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g., polysorbate 80 and / or polysorbate 20), in particular polysorbate surfactants such as polysorbate 80. In an embodiment, the aqueous pre-lyophilisation composition comprises a surfactant wherein the surfactant is present in an amount of at least 0.001 % (w / v), at least 0.005% (w / v), at least 0.01 % (w / v), and / or up to 0.5% (w / v). In an embodiment the surfactant is present in an amount less than 0.25% (w / v) or less than 0.1% (w / v). In another embodiment, the surfactant is present in an amount of about 0.02% (w / v). According to specific embodiments, the surfactant is polysorbate 80 or poloxamer 188 present in the aqueous mixture in an amount between 0.005% and 0.5% (w / v), such as about 0.02% (w / v).[000155] In a further embodiment, the aqueous pre-lyophilisation composition further comprises a buffer, optionally wherein the buffer is a Tris buffer, a histidine buffer, a citrate buffer, a HEPES buffer or a phosphate buffer. The pH is typically adjusted in view of the therapeutic components of the composition. Suitably, the pH of the aqueous pre-lyophilisation composition is at least 6, at least 6.5, at least 7 or at least 7.5. Alternatively stated, the pH of the aqueous pre-lyophilisation composition may be less than 10, less than 9.5, less than 9 or less than 8.5. In other embodiments, pH of the aqueous pre-lyophilisation composition is between 6 and 10, between 7 and 9.5, between 7.5 and 9.5, or, about 8, for example 8 ± 1 or 8 ± 0.5. An appropriate buffer may be selected from Tris, succinate, borate, Tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate, phosphate, or combinations thereof. In one embodiment, the aqueous pre-lyophilisation composition further comprises a buffer and the buffer is Tris, succinate, or borate. In a further embodiment, the buffer is Tris. In a particular embodiment, the aqueous pre-lyophilisation composition further comprises a Tris buffer and wherein the pH of the aqueous composition is 8 ± 0.5.[000156] In an embodiment, the buffer is present in the aqueous pre-lyophilisation composition in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM or at least 5 mM. In an embodiment, the buffer is present in the aqueous pre-lyophilisation composition in an amount of less than 50 mM, less than 40 mM, or less than 30 mM. For example, the buffer may be present in the aqueous pre- lyophilisation composition in an amount of 0.5 mm to 50 mM, 1 mM to 50 mM or 2 mM to 30 mM. In one embodiment, the buffer is present in the aqueous pre-lyophilisation composition in an amount of 20 ± 2 mM. According to specific embodiments, the buffer is T ris, present in the aqueous pre- lyophilisation composition in an amount between 15 and 25 mM, optionally 20 mM.[000157] In an embodiment, the aqueous pre-lyophilisation composition also comprises histidine in an amount of up to or about 20 mM, such as at a concentration of about 10 mM. According to further embodiments, the aqueous pre-lyophilisation composition also comprises bivalent metal ions, such as Mg2+, Ca2+, or Mg2+or Ca2+in the form of a salt, such as MgCI2, CaCI2or MgSO4. In one embodiment the bivalent metal ion is Mg2+. Typical amounts wherein the bivalent metal ions are present in the aqueous pre-lyophilisation composition are between 0.5 and 10 mM, such as 1 or 2 mM, or 1 mM in particular.[000158] Lyophilised pharmaceutical compositions of the present disclosure are typically reconstituted. Thus, in an embodiment, said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition. As used herein the term “aqueous post-reconstitution composition” refers to the solution obtained following reconstitution of the lyophilised pharmaceutical compositions disclosed herein. It will be appreciated by the skilled person that the aqueous post-reconstitution composition is the solution obtained following reconstitution wherein no substantial further changes take place. In an embodiment the aqueous post-reconstitution composition is the composition that is administered to a subject. In anembodiment, the lyophilised pharmaceutical compositions disclosed herein are reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition immediately prior to administration to a subject, optionally a human being.[000159] In an embodiment the lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution. The sterile reconstitution solution may be any medium wherein said medium is safe for administration to a subject, taking into consideration the route of administration. The precise composition of the sterile reconstitution solution will depend on both the contents of the lyophilised pharmaceutical composition being reconstituted and the subsequent use of the reconstituted contents. It is however envisaged that sterile reconstitution solution used for reconstitution is at an acceptable and physiologically relevant pH, temperature and osmolarity such that the sterile reconstitution solution itself does not impact any of the critical quality attributes of the pharmaceutical composition (such as the percentage of nucleic acid that remains encapsulated within the lipid carrier particles or the biological potency of the nucleic acid). Thus, in an embodiment, the lyophilised pharmaceutical composition is reconstituted with a sterile reconstitution solution, wherein said sterile reconstitution solution has no impact on the critical quality attributes of the pharmaceutical composition. In an embodiment, the sterile reconstitution solution is water or saline. In an embodiment the lyophilised pharmaceutical composition is reconstituted with sterile water for injection. In an embodiment, the lyophilised pharmaceutical composition is reconstituted with saline, optionally wherein said saline is at a concentration of 0.4% ± 0.1 % sodium chloride or 0.9% ± 0.1 % sodium chloride.[000160] The lyophilisation (i.e., freeze-drying) process is known to introduce mechanical forces which has the potential to deform structures present in the freeze-dried composition. In the case of RNA-LNP vaccines this is a problem since breakdown or degradation of the LNP may leave the RNA exposed and susceptible to hydrolysis. To the extent that the pharmaceutical compositions disclosed herein are mRNA vaccines, the potency of the mRNA as a vaccine is largely dependent upon maintenance of the complete intact mRNA molecule. Even a minor degradation reaction, anywhere along an RNA strand, can severely slow or stop proper translation performance of that strand and thus result in the incomplete expression of the target immunogen. It will be appreciated by the person skilled in the art that reduced expression of the protein encoded for by the RNA has a negative impact on immunogenicity. It is therefore an object of the present disclosure that the lyophilised pharmaceutical composition comprising lipid carrier particles encapsulating a nucleic acid (e.g., RNA) payload does not suffer with a loss of potency having undergone a freeze-drying procedure.[000161] In an embodiment, the lyophilised pharmaceutical compositions disclosed herein do not suffer with a significant loss of potency when compared to the aqueous pre-lyophilisation composition (i.e., the final liquid bulk). Thus, in an embodiment the potency of the nucleic acid (e.g. RNA) in the aqueous post-reconstitution composition is at least 90%, at least 91 %, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% such as at least 99% compared to the potency of the nucleic acid (e.g. RNA) in the aqueous pre-lyophilisation composition (otherwise known at the Final Liquid Bulk). Thus, in an embodiment, the potency of the nucleic acid in the aqueous post-reconstitution composition is compared against the potency of the aqueous pre- lyophilisation composition and is found to have not suffered any substantial loss in potency or biological functionality. This is advantageous considering the lyophilised beads have substantially improved shelf-life, stability etc. In an embodiment, the aqueous pre-lyophilisation composition (final liquid bulk) corresponds to a sample of the same lot of aqueous pre-lyophilisation composition used to make the lyophilised pharmaceutical composition. [000162] In an embodiment, the potency of the nucleic acid (e.g., RNA) is increased in the aqueous post-reconstitution composition compared to the potency of the nucleic acid in a comparator composition reconstituted from a comparator lyophilizate. The comparator composition reconstituted from a comparator lyophilizate is described further below.[000163] There is thus provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads), wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition and wherein the potency of the RNA is increased in the aqueous post-reconstitution composition compared to a comparator composition reconstituted from a comparator lyophilizate.[000164] In an embodiment the potency of the nucleic acid is increased in the post-reconstitution composition by more than 5%, more than 7.5%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% compared to a comparator composition reconstituted from a comparator lyophilizate. In an embodiment the potency of the nucleic acid is increased in the post- reconstitution composition by more than 10% compared to a comparator composition reconstituted from a comparator lyophilizate. Percentage increase in potency is calculated as follows: a) subtracting the potency of the nucleic acid in the post-reconstitution composition from the potency of the nucleic acid in a comparator composition reconstituted from a comparator lyophilizate. b) dividing the value obtained in a) by the potency of the nucleic acid in a comparator composition reconstituted from a comparator lyophilizate; and c) multiplying the value obtained in b) by 100.[000165] As used herein the term “potency” (in reference to the potency of nucleic acids) refers to the ability of the nucleic acid to provide a meaningful therapeutic benefit when administered to a subject. The ability of the nucleic acid to provide said therapeutic benefit when administered to a subject may however be measured, determined, identified, quantified, confirmed and / or validated by a surrogate measure for example by assessing either a) expression of the protein encoded for by thenucleic acid or b) the ability of the protein encoded for by the nucleic acid to induce an immune response in a subject.[000166] Thus, in an embodiment, said potency is determined by measuring the expression of a protein encoded for by the nucleic acid. In an embodiment, potency is determined by measuring the expression of a protein (optionally a protein immunogen) encoded for by the nucleic acid following transfection of the nucleic acid or the aqueous post-reconstitution composition into a eukaryotic cell (i.e., in vitro). In this respect an increase in potency corresponds to increased translation of the protein encoded for by the nucleic acid following transfection of the nucleic acid into a eukaryotic cell.[000167] Therefore in an embodiment, there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads), wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition and wherein following transfection of the aqueous post-reconstitution solution to a eukaryotic cell increased expression of a protein encoded for by the RNA is observed in comparison to a comparator composition reconstituted from a comparator lyophilizate. In an embodiment said increased expression of the protein encoded for by the RNA is an increase in expression of more than 5%, more than 7.5%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% compared to a comparator composition reconstituted from a comparator lyophilizate.[000168] In a further embodiment, potency is calculated by measuring the expression of a protein encoded for by the nucleic acid following administration to a subject. Therefore in an embodiment, there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads), wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition and wherein following administration of the aqueous post-reconstitution solution to subject increased expression of a protein encoded for by the RNA is observed in comparison to a comparator composition reconstituted from a comparator lyophilizate.[000169] In both of these embodiments measuring the expression of the protein encoded for the by the nucleic acid (e.g., RNA) may be conducted by flow cytometry, immunoblotting or ELISA using a mAb that is capable of binding to said protein. In a particular embodiment, the mAb that is capable of binding to said protein (i.e., protein encoded for by the nucleic acid) is functional and / or binds to a functional epitope. Thus, the expression of the protein encoded for by the nucleic acid provides an indication that functionally important epitopes are conformationally preserved.[000170] In an embodiment potency is calculated by measuring the immunogenicity of the pharmaceutical composition following administration to a subject. In an embodiment, immunogenicity of the pharmaceutical composition is measured following administration to a subject, by analyzing the titers of antibodies raised against the protein encoded for by the nucleic acid. In an embodiment, immunogenicity is measured by assessing the functionality of the antibody response induced against the protein encoded for the by nucleic acid e.g., using a neutralization assay. Therefore in an embodiment, there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads), wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition and wherein following administration of the aqueous post-reconstruction composition to a subject, an improved immune response against the protein encoded for by the nucleic acid is observed compared to the immune response observed following administration of a comparator composition reconstituted from a comparator lyophilizate.[000171] In an alternative embodiment, potency is calculated by measuring percentage knockdown in gene expression. Potency is calculated in this way if the nucleic acid present in the pharmaceutical composition is present for the purpose of silencing a target gene (e.g., siRNA, shRNA etc.) as opposed to being present for the purpose of encoding a protein, such as a protein immunogen. Methods used to measure percentage knockdown in gene expression are known in the art. One such method of measuring percentage knockdown in gene expression is to transfect cells (e.g., in vitro) with the nucleic acid (e.g., siRNA) and compare the expression of the target gene (i.e. , the gene that is targeted for silencing by the siRNA) with the expression of the same target gene in a negative control sample. Expression of the target gene may be determined using polymerase chain reaction (PCR) based techniques such as RT-PCR or Q-PCR. Percentage knockdown may be calculated using the comparative CTmethod (also known as the AACTmethod). In this method, data are normalized using a control transcript (e.g., 18S rRNA or GAPDH), and the normalized expression value (ACT) for the target gene of interest is compared to the equivalent ACTfor a negative control sample.Comparator Lyophilizate[000172] As described above, the lyophilised pharmaceutical compositions of the present disclosure comprising lipid carrier particles (e.g., LNPs) encapsulating an RNA payload are reconstituted in a sterile reconstitution solution to form an aqueous post-reconstitution composition and the potency of the RNA is increased in the aqueous post-reconstitution composition compared to a comparator composition reconstituted from a comparator lyophilizate.[000173] The comparator lyophilizate is thus reconstituted to form a comparator composition. The comparator lyophilizate is reconstituted using the same sterile reconstitution solution as is used to reconstitute the lyophilised beads.[000174] The comparator lyophilizate is a lyophilizate that has been produced from an identical aqueous pre-lyophilisation composition. However, the comparator lyophilizate is not in the form of a plurality of LyoBeads. The comparator lyophilizate is in the form of a lyophilised cake. In an embodiment the comparator lyophilizate is identical with the exception that the comparator lyophilize is in the form of a lyophilised cake. Thus in an embodiment there is provided a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g. LNPs) encapsulating an RNA payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads) wherein said lyophilised pharmaceutical composition is reconstituted to form an aqueous post-reconstitution composition and wherein the potency of the nucleic acid is increased in the aqueous post-reconstitution composition compared to a comparator composition reconstituted from a comparator lyophilizate wherein the comparator lyophilizate is identical with the exception that the comparator lyophilize is in the form of a lyophilised cake.[000175] As used herein lyophilised beads (“LyoBeads”) and “lyophilised cakes” differ from one another.[000176] As used herein the term “lyophilised cake” refers to a structure of freeze-dried material that is obtained when the entirety of a given volume of aqueous pre-lyophilisation composition is frozen into a single frozen mass prior to undergoing drying steps (e.g., primary and secondary drying steps) and wherein said given volume is greater than 400 pL e.g., greater than 500pL. Should the lyophilised cake be considered to comprise particles or fragments of freeze-dried material (i.e., particulate material) said particles or fragments present within the lyophilised cake will not be interpreted to be lyophilized beads by virtue of their diameter being significantly less than the given diameter of the lyophilised beads. Thus, particles present within the lyophilised cake are distinguishable from lyophilised beads by virtue of their diameter. In an embodiment, the lyophilised cake comprises particles wherein said particles are less than 2 mm in diameter, such as less than 1 .8 mm in diameter, less than 1 .6 mm in diameter, less than 1 .4 mm in diameter, less than 1 .2 mm in diameter, less than 1 mm in diameter, less than 0.8 mm in diameter, less than 0.6 mm in diameter, less than 0.4 mm in diameter, less than 0.2mm in diameter, less than 0.1 mm in diameter. In an embodiment the lyophilised cake comprises particles wherein said particles are less than 500 pmeters. In an embodiment the lyophilised cake comprises particles wherein said particles are less than 250 pmeters. In an embodiment the lyophilised cake comprises particles wherein said particles are less than 100 pmeters.[000177] If the same given volume of aqueous pre-lyophilisation composition is used to produce lyophilised cakes and lyophilised beads, the lyophilised beads will be in the form of a plurality of substantially spherical or ovoid structures of freeze-dried material of a given diameter, and the lyophilised cake will be present as a singular mass of freeze-dried material.[000178] In an embodiment, the comparator lyophilizate is produced by the steps of a) taking a volume of the aqueous pre-lyophilisation composition disclosed herein and subjecting the entirety of said volume to freezing conditions such that the entire volume of said aqueous pre- lyophilisation composition freezes into a single frozen mass; and b) drying the single frozen mass to form a lyophilised cake using an identical drying protocol to the drying protocol used to produce the lyophilised beads (LyoBeads).[000179] In an embodiment during step a) the volume of aqueous pre-lyophilisation composition that is taken and subjected to freezing conditions is between 0.1 and 3 milliliters, between 0.25 and 2 milliliters or between 0.4 and 1.2 milliliters. Preferably during step a) the volume of aqueous pre- lyophilisation composition that is taken and subjected to freezing conditions is greater than 0.4 milliliters. Said freezing conditions result in the entirety of said volume freezing into a single frozen mass. As already described above, this is different to the approach taken during the manufacture of the lyophilised beads in that, if the same volume of aqueous pre-lyophilisation composition is taken and subjected to freezing conditions, the result is a plurality (e.g., between 2 and 250, between 2 and 200, between 2 and 150, between 2 and 100 etc.) of frozen droplets as opposed to a single frozen mass.[000180] In an embodiment during step a) the volume of the aqueous pre-lyophilisation composition is transferred into a vessel(s) prior to subjecting the entirety of said volume to said freezing conditions. Thus, the aqueous pre-lyophilisation composition is transferred into vessels prior to freezing, whereas during the manufacture of the lyophilised beads the frozen droplets of aqueous pre-lyophilisation composition are transferred into the vessels (i.e. post-freezing).[000181] In an embodiment, the single frozen mass and the lyophilised cake are present substantially at the base of the vessel(s). Both the single frozen mass and lyophilised cake cover the base of the vessel(s) such that substantially no air gaps exist. This is different to the plurality of lyophilised pellets where air gaps exist between each adjacent bead.[000182] The comparator lyophilizate is identical to the LyoBeads with the exception that the comparator lyophilizate is in the form of a lyophilized cake. Thus, the comparator lyophilizate has been subjected to identical drying protocols. In an embodiment, during step b) said identical drying protocol comprises identical primary and identical secondary drying steps. In an embodiment, step b) comprises drying the single frozen mass under the same drying conditions used to produce the lyophilised beads.[000183] In an embodiment, the LyoBeads have an increased surface area to volume ratio in comparison to the surface area to volume of the comparator lyophilizate. In an embodiment, the surface area to volume ratio is increased by greater than 1.5 times, greater than 2 times, greater than 3 times or greater than 4 times compared to the surface to area volume ratio prior to step i). In an embodiment the surface area to volume ratio is increased by between 1x and 10x, between 2x and 8x, between 3x and 7x or between 3x and 5x compared to the surface area to volume ratio of the comparator lyophilizate.[000184] In a further aspect of the present disclosure there is provided a method of reconstituting the lyophilised pharmaceutical composition of the first aspect, comprising adding a sterile reconstitution solution to the lyophilised pharmaceutical composition and reconstituting the lyophilised pharmaceutical composition. The result is the production of the aqueous postreconstitution composition.[000185] In one embodiment, the sterile reconstitution solution is introduced into the vessel containing said lyophilised pharmaceutical composition in an amount sufficient to provide the desired concentration of the various components of the pharmaceutical composition or vaccine in the aqueous post-reconstitution solution, such that it is ready for administration (e.g., via injection) to a subject. In an embodiment the vessel will have enough of the lyophilised pharmaceutical composition (e.g., a sufficient number of LyoBeads) for a single unit dose. In another embodiment, the vessel will have enough of the lyophilised pharmaceutical composition for multiple doses, such as 2-10 doses or 3-8 doses or 4-8 doses or 5-6 doses. In an embodiment, the sterile reconstitution solution is introduced into the vessel and the vessel is shaken. Following this the aqueous post-reconstitution composition is inspected for expected and desired visual properties, such as degree of transparency, color, etc. The solution is then ready for being withdrawn from the vial by techniques known in the art, such as by inserting a syringe needle into the vial and withdrawing an appropriate amount of injectable solution into the syringe. In a separate embodiment, the lyophilised pharmaceutical composition and the liquid for reconstitution are present in separate chambers of a dual chamber syringe and are then combined prior to administration. Although sterile water for injection may be a suitable sterile reconstitution solution, other aqueous solutions such as buffers containing other additives, excipients and / or adjuvants may be included in the solution for reconstitution if these other additives, excipients and / or adjuvants are not present, or are not present in sufficient quantities, in the lyophilised pharmaceutical composition. Reconstitution can take place at any suitable temperature but will typically take place at room temperature.[000186] In a further aspect there is provided an immunogenic composition comprising the lyophilised pharmaceutical composition of the first aspect disclosed herein. Further provided is an immunogenic composition reconstituted from the lyophilised pharmaceutical compositions disclosed herein. In a further aspect there is provided a vaccine comprising the lyophilised pharmaceuticalcomposition of the first aspect disclosed herein. There is further provided a vaccine reconstituted from the lyophilised pharmaceutical composition of the first aspect disclosed herein.[000187] One advantage of the lyophilised pharmaceutical compositions disclosed in the first aspect (i.e. wherein said lyophilised pharmaceutical composition is in the form of a plurality of LyoBeads) is that it makes it easier to produce a multi-component or multi-valent vaccine RNA vaccine. By multi-component or multi-valent vaccine, it is meant that a vaccine composition can more easily be produced that comprises different mRNA molecules, for example mRNA molecules encoding for different protein immunogens.[000188] Thus in a further aspect there is provided a multicomponent vaccine comprising: a first lyophilised pharmaceutical composition according to the first aspect (“First LyoBead”) and a second lyophilised pharmaceutical composition according to the first aspect (“Second LyoBead”) wherein the first LyoBead comprises lipid carrier particles encapsulating a first nucleic acid payload and the second LyoBead comprises lipid carrier particles encapsulating a second nucleic acid payload wherein the first and second nucleic acid payloads encode protein immunogens that are different from one another. In an embodiment there is provided a multicomponent vaccine comprising: a first lyophilised pharmaceutical composition according to the first aspect (“First LyoBead”) and a second lyophilised pharmaceutical composition according to the first aspect (“Second LyoBead”) wherein the first LyoBead comprises LNPs encapsulating a first RNA payload and the second LyoBead comprises LNPs encapsulating a second nucleic acid payload wherein the first and second nucleic acid payloads encode protein immunogens that are different from one another. Said multicomponent vaccine is contained within a container such that there is provided a container comprising said first and second LyoBeads. The relative amounts of the first and second LyoBeads is dependent upon the immune response that it is desired to induce. However, in an embodiment the multicomponent vaccine comprises equal amounts of first and second LyoBeads.[000189] Further contemplated is a kit comprising a first container and a second container wherein the first container comprises the lyophilised pharmaceutical composition of the first aspect or the vaccine the present disclosure and the second container comprises a sterile reconstitution solution. In an embodiment said kit further comprises a sterile needle i.e. for injecting.[000190] After reconstitution, the resulting formulation (referred to herein as the aqueous postreconstitution composition) will have acceptable properties for injection, for example acceptable and physiologically relevant temperature, pH, osmolarity etc.[000191] Instructions for reconstitution at the clinic site and for administration of the reconstituted vaccine composition to the subject may be included in, on or associated with the various components of the kit. In an embodiment the kit will comprise one unit dose i.e., one first container, one second container and one sterile needle thus enabling administration of the pharmaceutical composition or vaccine to one subject. In an alternative embodiment the kit comprises a plurality (e.g., greater than 5,greater than 10, greater than 50, greater than 100 or greater than 500) of unit doses i.e., greater than 5, 10, 50, 100 or 500 first containers, greater than 5, 10, 50, 100 or 500 second containers and greater than 5, 10, 50, 100 or 500 sterile needles. In an embodiment, the kit will always comprise the same number of first containers, second containers and sterile needles.Processes[000192] In a further aspect there is provided a process for preparing the lyophilised pharmaceutical composition of the first aspect said process comprising: i. introducing an aqueous pre-lyophilisation composition comprising lipid carrier particles encapsulating a nucleic acid payload into a dispensing tip; ii. positioning the dispensing tip above a cryogenic liquid or a cryogenically cooled surface, iii. dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the cryogenically cooled surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv.drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)). [000193] The process of this aspect of the disclosure is a process for preparing the lyophilised pharmaceutical composition of the first aspect. To avoid undue repetition the features of the lyophilised pharmaceutical composition of the first aspect shall not be repeated with respect to the process of the present aspect. However, in brief, there is provided a process for preparing a lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles (e.g., LNPs) encapsulating a nucleic acid (e.g., RNA) payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads) said process comprising: i. introducing an aqueous pre-lyophilisation composition comprising lipid carrier particles (e.g., LNPs) encapsulating a nucleic acid (e.g., RNA) payload into a dispensing tip; ii. positioning the dispensing tip above a cryogenic liquid or a cryogenically cooled surface iii. dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the cryogenically cooled surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv.drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)). [000194] As described in detail within the section above (entitled lyophilised pharmaceutical composition) the aqueous pre-lyophilisation composition comprises the lipid carrier particles encapsulating a nucleic acid (e.g. RNA) payload. To avoid undue repetition features of the aqueous pre-lyophilisation composition will not be repeated however, in brief, the aqueous pre-lyophilisation composition further comprises additional constituents such as an amorphous sugar, a buffer, a salt etc.[000195] In an embodiment step i) comprises introducing an aqueous pre-lyophilisation composition comprising LNPs encapsulating a nucleic acid payload into a nucleic acid payload wherein the aqueous pre-lyophilisation composition comprises an amorphous sugar. In an embodiment the amorphous sugar is selected from sucrose, trehalose, mannose, mannitol, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, isomaltose, maltilol, palatinit, stachyose, melezitose, dextran, or a combination thereof. In one embodiment, the amorphous sugar is selected from sucrose or trehalose. In a preferred embodiment the amorphous sugar is sucrose. In an embodiment the aqueous pre-lyophilisation composition comprises sucrose in a concentration of between 0.5% and 25% (w / v), 1% and 20% (w / v) or 2.5% and 10% (w / v). In a further embodiment the aqueous pre-lyophilisation composition further comprises a salt. In an embodiment the salt is sodium chloride. In an embodiment the aqueous pre-lyophilisation composition comprises sodium chloride at a concentration of between 0.1 mM and 50mM, 0.5mM and 40mM, 1 mM and 30mM, 1 mM and 25mM, 1 mM and 20mM, 1 mM and 10mM, 1 mM and 8mM, 1 mM and 7.5mM, 2.5mM and 7.5mM or 4mM and 6mM. In an embodiment, the aqueous pre-lyophilisation composition may further comprise a surfactant. In an embodiment the aqueous pre-lyophilisation composition further comprises a surfactant wherein the surfactant is selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g., polysorbate 80 and / or polysorbate 20), octoxinal surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxyl castor oil surfactants, N- octyl-glucoside surfactants, macrogol 15 hydroxy stearate, and combinations thereof. In a further embodiment, the aqueous pre-lyophilisation composition further comprises a buffer, optionally wherein the buffer is a Tris buffer, a histidine buffer, a citrate buffer, a HEPES buffer or a phosphate buffer. In an embodiment, the buffer is Tris. In a particular embodiment, the aqueous pre-lyophilisation composition further comprises a T ris buffer and wherein the pH of the aqueous composition is 8 ± 0.5. In an embodiment, the aqueous pre-lyophilisation composition may further comprise histidine in an amount of up to or about 20 mM, such as at a concentration of about 10 mM.[000196] In an embodiment, during step iii) said single droplet aliquot(s) are exposed to freezing conditions such that they freeze to form a frozen droplet(s). Said freezing conditions expose the single droplet aliquot(s) to a temperature below the freezing point of the aqueous pre-lyophilization composition.[000197] In an embodiment, the cryogenic liquid (referred to in step ii and iii) has a boiling point of -75°C or lower (such as -80°C or lower, -90°C or lower or -100°C or lower). In an embodiment the cryogenic liquid is liquid nitrogen, liquid helium, liquid oxygen, or liquid carbon dioxide. In an embodiment the cryogenic liquid is liquid nitrogen. A cryogenic liquid is defined as a liquefied gas having a normal boiling or sublimation point below about -75°C and in some cases, this point is below about -150°C.[000198] In an embodiment, the cryogenically cooled surface (referred to in step ii and iii) is a solid flat surface i.e., comprises no cavities or wells. In an embodiment, the cryogenically cooled surface is at a temperature of -90°C or lower. In an embodiment, the cryogenically cooled surface is at a temperature within a range of about -90 °C to about -130 °C, about -110 °C to about -150 °C, about -150 °C to about -195 °C or -180 °C to about -196 °C. In an embodiment, the cryogenically cooled surface is a metal surface, optionally wherein the metal surface comprises a conductive, inert metal such as gold, silver, stainless steel, aluminum or copper. In an embodiment, the metal surface is comprised of aluminum.[000199] In an alternative embodiment the cryogenically cooled surface can be replaced by a hydrophobic surface. Thus, in an alternative embodiment, there is provided a process for preparing the lyophilised pharmaceutical composition of the first aspect said process comprising: i. introducing an aqueous pre-lyophilisation composition comprising lipid carrier particles encapsulating a nucleic acid payload into a dispensing tip; ii. positioning the dispensing tip above a cryogenic liquid or a hydrophobic surface iii. dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the hydrophobic surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv.drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)). [000200] The hydrophobic surface may comprise a chemically inert plastic such as polytetrafluoroethylene (PTFE), polypropylene and the like. The hydrophobic surface may be bonded to a different material or may comprise the top surface of a thin film made using the hydrophobic material (e.g., PTFE, polypropylene). The hydrophobic surface is preferably made by a deposition method such as chemical vapor deposition (CVD) onto a metallic slide surface. Another method of making a surface is laser deposition of carbon / silicon dioxide coating material.[000201] In an embodiment the hydrophobic surface is a diamond film, a silicon-oxide film or a PTFE surface. In an embodiment the hydrophobic surface is a diamond-Si02 slide deposited by chemical vapor deposition or a Teflon-coated glass slide. To freeze the liquid droplet, the film containing the dispensed droplet is chilled to a temperature that is below the freezing point of the liquid composition comprising the biological material, and preferably to a temperature of about 5 °C to 25°C below the freezing point.[000202] In a further alternative embodiment freezing of the single droplet aliquot(s) (i.e. to form a frozen droplet(s)) takes place by immersion of the single droplet aliquot(s) into a cold fluid, more particularly, by immersion of the single drop aliquot(s) into a stream of cold gas or vapor. In one embodiment, the cold gas or vapor is for example air, carbon dioxide, nitrogen, helium, oxygen, or argon but is preferably gaseous nitrogen. The cold fluid can have a temperature ranging from about - 40°C to about -250°C.Thus in an alternative embodiment step ii) involves positioning the dispensingtip above a chamber comprising a cold gas or vapor and step iii) involves dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the chamber comprising said cold gas or vapor in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s).[000203] In an embodiment step i) comprises introducing an aqueous pre-lyophilisation composition comprising lipid carrier particles (e.g., LNPs) encapsulating a nucleic acid (e.g. RNA) payload into a dispensing tip wherein said dispensing tip has an open end configured to dispense single droplet aliquot(s) of the aqueous pre-lyophilisation composition (during step iii). In an embodiment the dispending tip has an open end configured to dispense single droplet aliquot(s) of the aqueous pre-lyophilisation composition (during step iii) and a second end, wherein the second end is connected to a container comprising the aqueous pre-lyophilisation composition.[000204] In an embodiment, during step ii) the dispensing tip (or open end thereof) is positioned far enough above the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface) such that the aqueous pre-lyophilisation composition does not freeze within said dispensing tip. In an embodiment, during step ii) the dispensing tip (or open end thereof) is positioned not far enough above the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface) such that the single droplet aliquots disperse and do not form frozen droplets when they drop into the cryogenic liquid or onto the cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface).[000205] In an embodiment, during step ii) the dispensing tip (or open end thereof) is positioned at least 0.1 cm, at least 0.5 cm, at least 1 cm, at least 5 cm or at least 10 cm above the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface). In an embodiment, during step ii) the dispensing tip (or open end thereof) is positioned not greater than 50 cm (e.g., not greater than 45cm, not greater than 40cm, not greater than 35 cm or not greater than 30cm) above the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface). In an embodiment during step ii) the dispensing tip (or open end thereof) is positioned between 0.1 cm and 10 cm, between 0.5 and 8cm, between 0.75 and 7cm, between 1 and 6cm, between 1 .5 and 5cm or between 2 and 5cm above the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface).[000206] In an embodiment, the during step ii) the dispensing tip (or open end thereof) is intermittently moved to a position greater than 10cm away from the cryogenic liquid or cryogenically cooled surface (or in an alternative embodiment above the hydrophobic surface) to ensure that the aqueous pre-lyophilisation composition does not freeze within said dispensing tip (or open end thereof).[000207] In an embodiment, during step iii) single droplet aliquot(s) of the aqueous pre- lyophilisation composition are dispensed into the cryogenic liquid or onto the cryogenically cooled surface (or in an alternative embodiment onto the hydrophobic surface) in a manner such that thesingle droplet aliquot(s) freezes to form a frozen droplet(s). In an embodiment said single droplet aliquot(s) are of a volume of between 0.5 and 500 microliters, such as between 1 and 250 microliters, 1.5 and 200 microliters, 2 and 150 microliters or between 2.5 and 100 microliters. In an embodiment said single droplet aliquot((s) are of a volume between 0.5 and 50 microliters, such as in the range of between 1 to 30 microliters, in the range of between 2 and 25 microliters, or in the range of between 5 and 20 microliters. In an embodiment said single droplet aliquot((s) are of a volume of 10 ± 5 microliters, 15 ± 5 microliters, 20 ± 5 microliters, 25 ± 5 microliters, 30± 5 microliters, 35 ± 5 microliters, 40± 5 microliters, 45 ± 5 microliters, 50 ± 5 microliters.[000208] The speed at which the single droplet aliquot(s) of the aqueous pre-lyophilisation composition are dispensed and the gap distance (i.e., the distance between the dispensing tip, or open end thereof, and the cryogenic liquid or cryogenically cooled surface or an upper surface thereof) will depend upon the volume of the liquid droplet, and the shape of the open end of the dispensing tip and may be readily determined experimentally. In an embodiment, the single droplet aliquots are dispensed at a speed sufficient to allow droplet formation and avoid that the aqueous pre- lyophilisation composition is dispensed in a continuous flow. In an embodiment the single droplet aliquot(s) are dispensed at a speed of about 0.5ml / min to about 5ml / min, for example between 0.6ml / min and 2.5ml / min or between 0.7ml / min and 1 .5 ml / min. In an embodiment the single droplet aliquot(s) are dispensed at a speed of about 3 ml / min to about 75 ml / min, about 5 ml / min to about 75 ml / min, about 3ml / min to about 60 ml / min, about 20 ml / min to about 75 ml / min, about 20 ml / min to about 60 ml / min, respectively.[000209] The person skilled in the art has a choice in terms of how to ensure that each vessel of lyophilised pharmaceutical composition comprises a single unit dose of the pharmaceutical composition. In an embodiment, between step iii) and iv) the frozen droplet(s) are transferred onto tray(s) and dried in batches according to the primary and secondary drying cycles described below. Once primary and secondary drying cycles have been completed, a plurality of LyoBeads sufficient for one unit dose of the pharmaceutical composition may then be transferred into vessels, optionally with the aid of an automated dispenser instrument.[000210] However, in a separate embodiment between step iii) and step iv) the frozen droplet(s) are transferred into a vessel(s) wherein each vessel comprises a plurality of frozen droplets. In an embodiment each vessel comprises a sufficient number of frozen droplets such that each vessel comprises one unit dose of pharmaceutical composition. In an embodiment, between 2 and 250 frozen droplets, 2 and 150 frozen droplets, 2 and 100 frozen droplets, 2 and 50 frozen droplets or 2 and 25 frozen droplets are transferred into each vessel.[000211] In an embodiment between step iii) and step iv) the frozen droplet(s) are transferred into a vessel(s) followed by said vessel(s) (i.e. comprising said frozen droplet(s)) being transferred to a lyophilisation chamber that has been pre-cooled to a temperature sufficient to maintain the frozendroplet(s) in their frozen state. In an embodiment, the frozen droplet(s) are transferred into vessel(s) [000212] In an embodiment, said vessel(s) (i.e. comprising said frozen droplet(s)) are transferred to a lyophilisation chamber that has been pre-cooled to a temperature sufficient to maintain the frozen droplet(s) in their frozen state, wherein said pre-cooled temperature is less than -20°C (e.g. such as less than -25°C, less than -30°C or less than -35°C). After the vessel(s) have been transferred into the pre-cooled lyophilisation chamber, the primary drying step may commence. The primary drying step does not however need to start immediately, since the lyophilisation chamber can be maintained at temperatures below -20°C for extended periods of time (e.g., up to 24 hours). In an embodiment, prior to initiation of the primary during step the temperature is lowered to between -35 °C and -75°C for a period of between 30 and 180 minutes.[000213] In an embodiment, during step iv) the frozen droplet(s) is subjected to a primary drying step, and after the primary drying step, is subjected to secondary drying step, both primary and secondary drying steps taking place within a lyophilisation chamber. In the primary drying step, the ice formed during freezing of the single droplet aliquot is removed by sublimation at sub-ambient temperatures (although greater than the freezing temperature) under vacuum. After primary drying, any residual amounts of liquid which could not be removed by sublimation is removed by secondary drying, i.e., desorption. The temperature during secondary drying is near or greater than ambient temperature.[000214] In an embodiment, the frozen droplets can be subjected to an annealing step. Said annealing step involves raising the temperature of lyophilisation chamber to an annealing temperature. In an embodiment the annealing temperature is a temperature between the temperature at which the ice crystals formed within the frozen droplets melts and the glass transition temperature of the composition (Tg’). In an embodiment, the annealing step may take place following the transfer of the vessel(s) to a lyophilisation chamber that has been pre-cooled to a temperature sufficient to maintain the droplets in their frozen state, but prior to commencement of primary drying. In an embodiment, the annealing temperature is applied for a period of 15 minutes to 12 hours, such as between 30 minutes and 10 hours. In an embodiment, the annealing temperature is applied for 60 minutes ± 30 minutes.[000215] The primary drying step commences by ramping the temperature within the lyophilisation chamber to a primary drying temperature and applying a vacuum (i.e., decreasing the pressure inside the lyophilisation chamber). In an embodiment, the primary drying step comprises adjusting the temperature of the lyophilisation chamber to a primary drying temperature of between - 40 °C and 40 °C (such as between -40°C and 20°C, between -35°C and 5°C between, -30°C and -5°C or between -30 °C and -10°C with a ramp rate from between 0.1 °C / min and 2.0 °C / min (such as between 0.1 °C / min and 1 °C / min, between 0.2 °C / min and 0.8 °C / min or between 0.4°C / min and 0.6 °C / min). In an embodiment, the primary drying step comprises adjusting the temperature of thelyophilisation chamber to a primary drying temperature between -35°C and -20°C with a ramp rate from between 0.1 °C / min and 1.0 °C / min. In an embodiment, the primary drying temperature is -28 °C, -29 °C or -30 °C. In an embodiment, the ramp rate is 0.5 °C / min.[000216] During the primary drying step, the frozen droplet(s) should be maintained in the solid state below the collapse temperature (“Tc”) of the aqueous pre-lyophilisation composition (i.e., the product temperature should be below the Tc). The Tcis the temperature above which the LyoBead loses macroscopic structure and collapses. Thus, in an embodiment the primary drying temperature is a temperature which maintains the product temperature (i.e. the actual temperature of the frozen droplets undergoing sublimation) below the Tc of the aqueous pre-lyophilisation composition (e.g. below between -35 °C and -32 °C). Determining the collapse temperature is within the remit of the skilled person, for example using freeze drying microscopy.[000217] In an embodiment, during the primary drying step the lyophilization chamber is held at the primary drying temperature for a period of time sufficient to sublimate a major portion of the ice within the frozen droplet(s). In an embodiment during the primary drying step the lyophilization chamber is held at the primary drying temperature for a period of time sufficient to remove a major proportion of vapor, as detected by Pirani gauge measurement.[000218] In an embodiment, during the primary drying step the lyophilization chamber is held at the primary drying temperature for between 4 and 25 hours (e.g., between 6 and 24 hours) at a pressure of between 10 and 150 microbars (pbar), for example at a pressure of between 10 pbar and 100 pbar. In an embodiment, the lyophilisation chamber is held at the primary drying temperature for between 11 and 22 hours at a pressure of between 65 pbar and 85 pbar, for example at a pressure of 75 pbar. In an embodiment, the lyophilisation chamber is held at the primary drying temperature for 21 hours ± 2 hours (e.g., 21 hours).[000219] As mentioned above, during step iv) the frozen droplet(s) is subjected to a primary drying step, and after the primary drying step, is subjected to secondary drying step, both primary and secondary drying steps taking place within a lyophilisation chamber. In an embodiment, the primary drying step is immediately followed by the secondary drying step. At the end of the primary drying step, the composition will typically have 5 to 15% moisture content. This remaining water fraction is removed by a secondary drying step, also referred to as desorption step. Thus, in an embodiment, the secondary drying step comprises adjusting the temperature of the lyophilisation chamber to a secondary drying temperature under vacuum. Since substantially all of the free ice has been removed during primary drying, the composition temperature can now be increased considerably without fear of melting or collapse. In an embodiment, the secondary drying temperature is above 0°. [000220] In an embodiment, the secondary drying step comprises adjusting the temperature of the lyophilization chamber to a secondary drying temperature ranging from 0 °C to 40 °C (such as between 5 °C and 35 °C or between 10 °C and 30 °C) with a ramp rate ranging from 0.05 °C / min up to1.0 °C / min. In an embodiment, the secondary drying step comprises adjusting the temperature of the lyophilization chamber to a secondary drying temperature ranging from 5 °C to 20 °C with a ramp rate ranging from 0.05 °C / min up to 1.0 °C / min. In an embodiment, the secondary drying temperature is 14 °C, 15 °C or 16 °C. In an embodiment, the ramp rate is 0.1 °C / min.[000221] Secondary drying is continued until the lyophilised pharmaceutical composition has acceptable moisture content for long term storage. Accordingly, in an embodiment the secondary drying step is effected for a period of time sufficient to result in the lyophilised pharmaceutical composition having a moisture content of between 0.01% and 3% such as between 0.1 % and 3% e.g., between 0.2 and 3%, between 0.25 and 2.5% (w / w), between 0.5 and 2% (w / w), between 0.5 and 1 .25% (w / w), or between 0.5 and 1 % (w / w). In most cases, the more dry the composition, the longer its shelf life will be. However, certain complex biological compositions may actually become too dry for optimum storage results and the secondary drying process (the desorption step) should be controlled accordingly. Residual moisture content is determined by techniques known to the skilled person such as the Karl Fisher method.[000222] In an embodiment, during the secondary drying step the lyophilization chamber is held at the secondary drying temperature for between 5 and 25 hours at a pressure of between 10 pbar and 150 pbar, for example at a pressure of between 10 pbar and 100 pbar. In an embodiment, during the secondary drying step the lyophilization chamber is held at the secondary drying temperature for between 10 and 14 hours at a pressure of between 65 pbar and 85 pbar, for example at a pressure of 75 pbar.[000223] As an alternative to the primary and secondary drying cycles disclosed above, it is foreseen that both primary and secondary drying may take place significantly faster (i.e. fast-drying cycle). This is possible because the frozen droplet(s) that are formed during step iii) of the process, comprise an increased surface area to volume ratio. Said increased surface area to volume ratio is in comparison to the surface area to volume ratio of an identical volume of aqueous pre-lyophilisation composition that has not been frozen as droplets (e.g. frozen as a single solid mass). Without wishing to be bound by theory, said increased surface area to volume ratio enables primary and secondary drying to occur much more rapidly due to having a greater surface area exists for the water molecules to leave the frozen droplets compared to situations where the surface area to volume ratio is not increased. In an embodiment, the primary drying step lasts for a period of time sufficient to sublime a major portion or substantially all of the ice crystals within the frozen droplets. In an embodiment, the secondary drying step lasts for a period of time sufficient to desorb a major portion or substantially all of the remaining water molecules within the frozen droplets. In an embodiment primary and / or secondary drying step takes between 25 mins and 300 minutes e.g., between 20 minutes and 240 minutes, between 30 minutes and 200 minutes or between 45 minutes and 180 minutes. In an embodiment, the primary and / or secondary drying step is 90 minutes ± 30 minutes, 90 minutes ± 20minutes or 90 minutes ± 10 minutes. In an embodiment the primary and / or secondary drying step is approximately 90 minutes. In an embodiment, the primary and / or secondary drying step is less than 30 hours, less than 25 hours, less than 20 hours, less than 15 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2.5 hours or less than 2 hours.[000224] In an embodiment at least one step in process disclosed herein is controlled via automated instruments. For example, in an embodiment, the dispensing tip is connected to an automated droplet dispending device or unit that is capable of controlling the droplet dispensing speed, the droplet dispensing volume and / or the gap between the open end of the dispensing tip and the cryogenic liquid or a cryogenically cooled surface. Said automated instruments facilitate greater control of process parameters leading to enhanced reproducibility and reduced batch-to-batch inconsistency.[000225] In an embodiment during step iv) the temperature of the lyophilised pharmaceutical composition and / or the vessel(s) comprising the composition is measured using a sensor. In an embodiment the sensor is an infrared camera, contact probe, thermocouple or quartz probe.[000226] It will be acknowledged that the process may comprise further steps following step iv). In an embodiment the method further comprises (i.e. , following step iv) plugging the vessel, optionally wherein the vessel is plugged with a stopper. In an embodiment the stopper is siliconized and is configured to be positioned at least partially in the vessel. In an embodiment, following plugging the vessel, the vessel is capped to secure the stopper in respect of the vessel. In an embodiment, the method further comprises labelling of the vessel. In an embodiment, the vessel comprising the pharmaceutical composition is stored under conditions suitable for long-term stability.[000227] In an embodiment, there is further provided a process for preparing a packaged lyophilised pharmaceutical composition said process comprising: collecting an amount of the lyophilized pharmaceutical compositions disclosed herein in the form of a plurality of LyoBeads; and packaging that amount into a container. As described earlier, said amount of the lyophilized pharmaceutical composition in the form of a plurality of LyoBeads relates to the amount of LyoBeads corresponding to one unit dose of the pharmaceutical composition or vaccine.[000228] In an embodiment there is further provided a lyophilised pharmaceutical composition obtained from the process of the disclosure. In an embodiment there is provided a vaccine comprising the lyophilised pharmaceutical composition obtained from the process of the disclosure. Further provided is a vessel comprising at least one LyoBead, said at least one LyoBead being prepared by the process of the disclosure.Medical Use[000229] The lyophilised pharmaceutical compositions or vaccines of the present disclosure are for use in medicine.[000230] As such, there is provided the use of the lyophilised pharmaceutical compositions, vaccines or kits disclosed herein in the manufacture of a medicament for treating a subject in need thereof. In an embodiment there is provided the use of the lyophilised pharmaceutical compositions, vaccines or kits disclosed herein in the manufacture of a medicament for prophylaxis in a subject in need thereof.[000231] In a further aspect there is provided a method for eliciting an immune response in a subject in need thereof comprising administering the lyophilised pharmaceutical composition (i.e., following reconstitution) or vaccines of the present disclosure to the subject, optionally wherein the subject is a human subject. It will be understood that said administration of the lyophilised pharmaceutical compositions or vaccines of the disclosure is following reconstitution in a sterile reconstitution solution (i.e., to form the aqueous post-reconstitution composition). In an embodiment, the pharmaceutical composition or vaccine is for eliciting an immune response in vivo against an immunogen of interest. In an embodiment, the immune response is protective and involves antibodies and / or cell-mediated immunity. By raising an immune response, the subject can be protected against various diseases and / or infections e.g., against bacterial and / or viral diseases as discussed above. RNA-containing compositions are immunogenic and are more preferably vaccine compositions. Vaccines according to the invention may either be prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but will typically be prophylactic.[000232] In a further aspect there is provided the lyophilised pharmaceutical compositions or vaccines of the present disclosure for use in medicine. Further provided is the lyophilised pharmaceutical compositions or vaccines of the disclosure for use in the treatment or prevention of disease in a subject, optionally wherein the subject is a human subject. In an embodiment, the subject is a mammal, such as a human or a large veterinary mammal. In an embodiment, the subject is a human. The pharmaceutical composition and vaccines prepared according to the disclosure may be used to treat both children and adults. Thus, a human patient may be less than 1 year old, less than 5 years old, 1 -5 years old, 5-15 years old, 15-55 years old, or older than 55 years old.[000233] Pharmaceutical compositions and vaccines of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by any method of administration known to the skilled person such as via parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly or to the interstitial space of a tissue) or mucosally, such as by rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, pulmonary or other mucosal administration. Injection may be via a needle (e.g., a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml.The pharmaceutical compositions and vaccines disclosed herein may be used to elicit systemic and / or mucosal immunity.[000234] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In an alternative embodiment, two primary doses are administered about two months apart, e.g., about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g., about 6, 8, 10 or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, e.g., about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third primary dose, e.g., about 6, 8, 10, or 12 months after the third primary dose.[000235] In some embodiments, the subject is human. In some embodiments, the subject is a mammal, such as a human or a large veterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where the formulation or recombinant RNA is for eliciting a protective immune response, the subject is preferably a human, such as a child (e.g. a toddler or infant), a teenager, and the recombinant RNA or the formulation comprising the recombinant RNA is formulated as a vaccine. Where the composition or recombinant RNA is used as a treatment or for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults, with the provisio that the amount of recombinant RNA or formulation comprising the recombinant RNA may be scaled up to provide an unit dose consistent with the state of the immune system of the subject (i.e. the elderly having more difficulty eliciting certain immune responses) and the average body weight of a subject of that age or the actual body weight of the subject.[000236] In some embodiments, the recombinant RNA or formulation comprising the recombinant RNA is administered to the subject intramuscularly, intradermally, subcutaneously, transcutaneously, topically, intraperitoneally, intrathecally, pulmonarily (i.e. inhaled), intracerebroventricularly, intravenously, intra-arterially, onto a mucosa (i.e. vaginally), buccally, sublingually, intranasally, optically, to the cornea, or into the eyeball.[000237] In some embodiments, a method for treating cancer in a subject is provided, the subject having a tumor of the cancer, the method comprising administering to the subject the formulation comprising the recombinant RNA or the recombinant RNA or an unit dose thereof. In some embodiments, the recombinant RNA comprises a sequence that encodes a heterologous polypeptide comprising a polyepitopic peptide comprising two or more immunogenic neo-epitopes anda linker, the linker linking the two or more immunogenic neo-epitopes, the immunogenic neo-epitopes being from a first sample comprising cells from the tumor from the subject, each neo-epitope being: (a) encoded in mRNA in the first sample, (b) occurring in a protein-coding region therein, (c) being predicted to bind to a major histocompatibility complex, and (d) being capable of introducing a difference in the amino acid sequence of the neo-epitope when compared to a reference amino acid sequence or genetic sequence predicted to encode the reference amino acid sequence obtained from a second sample from a non-cancerous cell from the subject. In some embodiments, the recombinant RNA is produced from a method comprising: obtaining a first nucleic acid sequence from the first sample, obtaining a second nucleic acid sequence from the second sample, comparing the first nucleic acid sequence to the second nucleic acid sequence thereby obtaining at least two somatic mutations present in the tumor cells, identifying from the at least two somatic mutations (a)-(d), and producing the recombinant RNA.[000238] In one aspect, a method for treating cancer in a subject is provided, the method comprising administering to the subject the recombinant RNA, a formulation comprising the recombinant RNA, or an unit dose thereof, wherein the recombinant RNA comprises a sequence encoding a heterologous polypeptide comprising IL-12sc, IL-15sushi, IFNa, or GM-CSF. In some embodiments, the method further comprises administering an anti-PD-1 / PD-L1 checkpoint inhibitor. In some embodiments, the cancer is melanoma, head and neck squamous cell cancer (HNSCC), cutaneous squamous cell carcinoma (CSCC), or advanced anti-PD-1 / PD-L1 naive cancers thereof.[000239] In some embodiments, a method for treating cancer in a subject is provided, the method comprising administering to the subject the recombinant RNA, a formulation comprising the recombinant RNA, or an unit dose thereof. In some embodiments, the recombinant RNA comprises a sequence encoding a heterologous polypeptide comprising autogene, cevumeran, or atezolizumag. In some embodiments, the cancer is melanoma, head and neck squamous cell cancer (HNSCC), cutaneous squamous cell carcinoma (CSCC), non-small cell lung cancer (NSCLC), or advanced anti- PD-1 / PD-L1 naive cancers thereof.[000240] In some embodiments, the unit dose (e.g. administered to the human) comprises at least, or is at least: 5 pg, 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg,70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 105 pg, 110 pg, 115 pg, 120 pg, 125 pg, 130 pg,135 pg, 140 pg, 145 pg, 150 pg, 155 pg, 160 pg, 165 pg, 170 pg, 175 pg, 180 pg, 185 pg, 190 pg,195 pg, 200 pg, 205 pg, 210 pg, 215 pg, 220 pg, 225 pg, 230 pg, 235 pg, 240 pg, 245 pg, 250 pg,255 pg, 260 pg of nucleic acid payload (e.g. mRNA molecules). In some embodiments, the unit dose comprises no more than, or is no more than: 260 pg, 255 pg, 250 pg, 245 pg, 240 pg, 235 pg, 230 pg, 225 pg, 220 pg, 215 pg, 210 pg, 205 pg, 200 pg, 195 pg, 190 pg, 185 pg, 180 pg, 175 pg, 170 pg, 165 pg, 160 pg, 155 pg, 150 pg, 145 pg, 140 pg, 135 pg, 130 pg, 125 pg, 120 pg, 115 pg, 110 pg,105 pg, 100 pg, 95 pg, 90 pg, 85 pg, 80 pg, 75 pg, 70 pg, 65 pg, 60 pg, 55 pg, 50 pg, 45 pg, 40 pg, 35 pg, 30 pg, 25 pg, 20 pg, or 15 pg of nucleic acid payload (e.g. mRNA molecules).[000241] In some embodiments, the unit dose (e.g. administered to the human) comprises, or is, from 5 pg to 259 pg; from 25 pg to 255 pg; from 50 pg to 250 pg; from 80 pg to 245 pg; from 85 pg to 240 pg; from 90 pg to 230 pg; from 95 pg to 220 pg; from 100 pg to 210 pg; from 105 pg to 200 pg; or from 110 pg to 195 pg of nucleic acid payload (e.g. mRNA molecules).[000242] In some embodiments, the unit dose comprises, or is, from 5 pg to 50pg; from 7.5 pg to 45pg; from 10 pg to 40 pg; from 12.5 pg to 35 pg; from 15 pg to 30 pg; from 17.5 pg to 25 pg; or from 20 pg to 25 pg of each of the nucleic acid payloads (e.g. the first nucleic acid payload and the second nucleic acid payload; e.g. each of the mRNA molecules).[000243] In some embodiments, the unit dose comprises, or is, from 0.1 pg of each of the nucleic acid payloads e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human) to 1 .5 pg of each of the nucleic acid payloads (e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human). In some embodiments, the unit dose comprises, or is, from 0.5 pg of each of the nucleic acid payloads e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human) to 1 .2 pg of each of the nucleic acid payloads e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human). In some embodiments, the unit dose comprises or is from 0.75 pg of each of the nucleic acid payloads e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human) to 1 .0 pg of each of the nucleic acid payloads e.g. each of the mRNA molecules) per kg body weight of the subject (e.g. human).[000244] Embodiments are further described in the following numbered clauses:1. A lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid carrier particles encapsulating a nucleic acid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads).2. The lyophilised pharmaceutical composition of clause 1 wherein the LyoBeads are spherical shaped bodies.3. The lyophilised pharmaceutical composition of clause 1 or clause 2 wherein the LyoBeads are between 0.1 and 25 mm in diameter, such as between 0.5 and 20 mm, between 1 and 15 mm or between 2 and 10 mm.4. The lyophilised pharmaceutical composition of clauses 1-3 wherein the LyoBeads have a percentage residual moisture of less than 2, less than 1 .5, less than 1 , less than 0.75, less than 0.5, less than 0.4 or less than 0.3 as calculated by Karl Fisher titration.5. The lyophilised pharmaceutical composition of any preceding clause wherein the nucleic acidis ribonucleic acid (RNA). The lyophilised pharmaceutical composition of clause 5 wherein the RNA is a therapeutic oligonucleotide optionally wherein the therapeutic oligonucleotide is a small interfering RNA (siRNA) or short hairpin RNA (shRNA). The lyophilised pharmaceutical composition of clause 5 wherein the RNA is messenger RNA (mRNA). The lyophilised pharmaceutical composition of clause 7 wherein the mRNA is either selfamplifying mRNA (SAM) or non-self-amplifying mRNA. The pharmaceutical composition of clause 7 or clause 8 wherein the mRNA encodes for at least one protein, optionally a protein immunogen. The lyophilised pharmaceutical composition of any preceding clause wherein said pharmaceutical composition is an RNA vaccine. The lyophilised pharmaceutical composition of clauses 5-10 the RNA is present at a concentration of between 5 and 250pg / ml, 10 and 100 pg / ml, 25 and 100 pg / ml, 25 and 75 pg / ml, or 50 and 75 pg / ml. The pharmaceutical composition of clause 11 wherein the RNA is present at a concentration of 60 pg / ml. The pharmaceutical composition of clauses 5-12 wherein the RNA comprises one or more modified ribonucleotides, optionally wherein said modified ribonucleotides comprise N1- methylpseudouridine (I m'-P). The pharmaceutical composition of any preceding clause wherein the lipid carrier particles are lipid nanoparticles (LNPs). The pharmaceutical composition of clause 14 wherein the LNPs comprise a cationic lipid, a neutral lipid, and a PEGylated lipid. The pharmaceutical composition of clause 15 wherein the cationic lipid is RV94. The pharmaceutical composition of clause 15 wherein the neutral lipid is DSPC. The lyophilised pharmaceutical composition of clause 15 wherein the PEGylated lipid is 1 ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) or 1 ,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2k) . The pharmaceutical composition of clause 15 wherein the lipids present in the LNPs comprise (in mole %) 0.1-8.0, 0.4-7.0, 0.6-6.0, 0.8-4.0, 0.8-3.5% or 1.0-3.0% PEGylated lipid. The pharmaceutical composition of clause 19 wherein the lipids present in the LNPs comprise (in mole %) between 1 % and 3%, such as between 1.2% and 2.8%, between 1.4% and 2.6% or between 1.6% and 2.4%, optionally 2% PEGylated lipid. The pharmaceutical composition of clause 15-20 wherein the LNPs further comprise cholesterol.The pharmaceutical composition of clause 21 wherein the lipids present in the LNPs comprise from 35 mole% to 65 mole% of the cholesterol, optionally from 40 mole% to 60 mole% of the cholesterol, optionally 45 mole% to 55 mole% of the cholesterol, optionally 45 mole% to 50 mole% of the cholesterol. The pharmaceutical composition of any preceding claim wherein the LyoBeads are contained within a vessel(s). The lyophilised pharmaceutical composition of any preceding clause wherein said lyophilised pharmaceutical composition is lyophilised from an aqueous pre-lyophilisation composition comprising lipid carrier particles encapsulating a nucleic acid payload. The pharmaceutical composition of clause 24, wherein the aqueous pre-lyophilisation composition comprises sucrose in a concentration of between 0.5% and 25% (w / v), 1 % and 20% (w / v) or 2.5% and 10% (w / v). The pharmaceutical composition of clause 25 wherein the aqueous pre-lyophilisation composition comprises sucrose in a concentration of 7.5% (w / v). The pharmaceutical composition of clauses 24-26 wherein the aqueous pre-lyophilisation composition further comprises a salt, optionally sodium chloride. The pharmaceutical composition of clause 24-27 wherein the aqueous pre-lyophilisation composition comprises sodium chloride in a concentration of between 0.1 and 50 mM, 0.5 and 40 mM, 1 and 30 mM, 1 and 25 mM, 1 and 10 mM, 1 and 7.5 mM or 2.5 and 7.5 mM. The pharmaceutical composition of clause 28 wherein the aqueous pre-lyophilisation composition comprises sodium chloride in a concentration of between 1 and 20 mM, 1 and 10 mM, 2 and 8 mM or 4 and 6 mM. The pharmaceutical composition of clause 29 wherein the aqueous pre-lyophilisation composition comprises sodium chloride in a concentration of 5 mM. The pharmaceutical composition of any of clauses 24-30, wherein the aqueous pre- lyophilisation composition further comprises a buffer, optionally wherein the buffer is a Tris buffer, a histidine buffer, a citrate buffer, a HEPES buffer or a phosphate buffer. The pharmaceutical composition of clause 31 wherein the buffer is Tris. The pharmaceutical composition of clause 32 wherein the concentration of Tris is between 15 and 25 mM, optionally 20 mM. The pharmaceutical composition of clause 24-33 wherein the pH of the aqueous pre- lyophilisation composition ranges from pH 6 to pH 10, optionally wherein the pH of the aqueous pre-lyophilisation composition is pH 8. The lyophilised pharmaceutical composition of any preceding clause wherein said lyophilised pharmaceutical composition is reconstituted to form an aqueous post-reconstitution composition.The lyophilised pharmaceutical composition of clauses 35 wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution. The lyophilised pharmaceutical composition of clause 36 wherein the sterile reconstitution solution is water or saline. The lyophilised pharmaceutical composition of clauses 35-37 wherein the potency of the nucleic acid in the aqueous post-reconstitution composition is at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, such as at least 99% compared to the potency of the nucleic acid in the aqueous pre-lyophilisation composition (i.e., final liquid bulk). The lyophilised pharmaceutical composition of clauses 35-37 wherein the potency of the nucleic acid is increased in the aqueous post-reconstitution composition compared to a comparator composition reconstituted from a comparator lyophilizate. The lyophilised pharmaceutical composition of clause 39 wherein the potency of the nucleic acid is increased in the aqueous post-reconstitution composition by more than 5%, more than 7.5%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% compared to a comparator composition reconstituted from a comparator lyophilizate. The lyophilised pharmaceutical composition of clauses 39 and 40 wherein the potency of the nucleic acid payload is increased in the aqueous post-reconstitution composition by more than 10% compared to a comparator composition reconstituted from a comparator lyophilizate. The lyophilised pharmaceutical composition of clauses 39-41 wherein potency is calculated by measuring the expression of a protein encoded for by the nucleic acid following transfection into a eukaryotic cell. The lyophilised pharmaceutical composition of clauses 39-41 wherein potency is calculated by measuring the expression of a protein encoded for by the nucleic acid following administration to a subject. The lyophilised pharmaceutical composition of clauses 42 and 43 wherein measuring the expression of the protein encoded for the by the nucleic acid is conducted by flow cytometry, immunoblotting or ELISA using a mAb that is capable of binding to said protein. The lyophilised pharmaceutical composition of clauses 39-41 wherein potency is calculated by measuring the immunogenicity of the pharmaceutical composition following administration to a subject. The lyophilised pharmaceutical composition of clauses 39-41 wherein potency is calculated by measuring percentage knockdown in gene expression. The lyophilised pharmaceutical composition of clauses 39-46 wherein the comparator lyophilizate is not in the form of LyoBeads. The lyophilised pharmaceutical composition of clauses 39-47 wherein the comparatorlyophilizate is identical with the exception that the comparator lyophilize is in the form of a lyophilised cake. The lyophilised pharmaceutical composition of clauses 39-48 wherein the comparator lyophilizate is produced by the steps of a) taking a volume of the aqueous pre-lyophilisation composition disclosed in claims 24- 34 and subjecting the entirety of said volume to freezing conditions such that the entire volume of said aqueous pre-lyophilisation composition freezes into a single frozen mass; and b) drying the single frozen mass to form a lyophilised cake using an identical drying protocol to the drying protocol used to produce the LyoBeads. The lyophilised pharmaceutical composition of clause 49 wherein during step a) the volume of aqueous pre-lyophilisation composition is between 0.1 and 3 milliliters, between 0.25 and 2 milliliters or between 0.4 and 1.2 milliliters. The lyophilised pharmaceutical composition of clause 49 or 50 wherein during step a) the volume of the aqueous pre-lyophilisation composition is transferred into a vessel(s) prior to subjecting the entirety of said volume to said freezing conditions. The lyophilised pharmaceutical composition of clauses 49-51 wherein the single frozen mass and the lyophilised cake are present substantially at the base of the vessel(s). The lyophilised pharmaceutical composition of clauses 49-52 wherein during step b) said identical drying protocol comprises identical primary and identical secondary drying steps. A method of reconstituting the lyophilised pharmaceutical composition of any of clauses 1-53 comprising adding a sterile reconstitution solution to the lyophilised pharmaceutical composition; and reconstituting the lyophilised pharmaceutical composition. A vaccine comprising the lyophilised pharmaceutical composition of any of clauses 1-53. A vaccine reconstituted from the lyophilised pharmaceutical composition of any of clauses 1- 53. A multicomponent vaccine comprising: a first lyophilised pharmaceutical composition according to clauses 1-53 (“First LyoBead”) and a second lyophilised pharmaceutical composition according to clauses 1-53 (“Second LyoBead”), wherein the first LyoBead comprises lipid carrier particles encapsulating a first nucleic acid payload and the second LyoBead comprises lipid carrier particles encapsulating a second nucleic acid payload, wherein the first and second nucleic acid payloads encode protein immunogens that are different from one another. A kit comprising a first container and a second container wherein the first container comprisesthe lyophilised pharmaceutical composition of clauses 1-53 or the vaccine of clauses 54 or 56 and the second container comprises a sterile reconstitution solution. The kit of clause 58 further comprising a sterile needle. A process for preparing the lyophilised pharmaceutical composition of clauses 1-53 said process comprising: i) introducing an aqueous pre-lyophilisation composition comprising lipid carrier particles encapsulating a nucleic acid payload into a dispensing tip; ii) positioning the dispensing tip above a cryogenic liquid or a cryogenically cooled surface iii) dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the cryogenically cooled surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv) drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)). The process of clause 60 wherein the cryogenic liquid has a boiling point of -90°C or lower. The process of clauses 60 and 61 wherein the cryogenic liquid is liquid nitrogen, liquid helium, liquid oxygen, or liquid carbon dioxide. The process of clauses 60-62 wherein the dispensing tip has an open end configured to dispense said single droplet aliquot(s). The process of clause 60-63 wherein during step ii) the dispensing tip is positioned at least 0.1 cm, at least 0.5 cm, at least 1 cm, at least 5 cm or at least 10 cm above the cryogenic liquid or cryogenically cooled surface. The process of clauses 60-64 wherein during step ii) the dispensing tip is positioned not greater than 50 cm above the cryogenic liquid or cryogenically cooled surface. The process of clause 60-65 wherein the single droplet aliquot(s) are of a volume of between 0.5 and 500 microliters, such as between 1 and 250 microliters, 1.5 and 200 microliters, 2 and 150 microliters or between 2.5 and 100 microliters. The process of clauses 60-66 wherein between step iii) and step iv) the frozen droplet(s) are transferred into a vessel(s) followed by said vessel(s) being transferred to a lyophilisation chamber that has been pre-cooled to a temperature sufficient to maintain the frozen droplet(s) in their frozen state. The process of clauses 60-67 wherein during step iv) the frozen droplet(s) is subjected to a primary drying step, and after the primary drying step, is subjected to secondary drying step, both primary and secondary drying steps taking place within a lyophilisation chamber The process of clause 68 wherein prior to initiation of the primary drying step the lyophilisation chamber is at a temperature of less than -20°C.The process of any clause 68 or clause 69 wherein the primary drying step comprises adjusting the temperature of the lyophilisation chamber to a primary drying temperature of between -40 °C and 40 °C with a ramp rate from between 0.1 °C / min and 1 .0 °C / min The process of clause 70 wherein the primary drying step comprises adjusting the temperature of the lyophilisation chamber to a primary drying temperature between -35°C and -20°C with a ramp rate from between 0.1 °C / min and 1 .0 °C / min The process of clause 70 or clause 71 wherein the primary drying temperature is -28 °C, -29 °C, -30 °C, -31 °C, or -32 °C. The process of clause 69 or clause 70, wherein the ramp rate is 0.5 °C / min. The process of clauses 68-73, wherein during the primary drying step the lyophilization chamber is held at the primary drying temperature for between 4 hours and 25 hours at a pressure of between 10 and 150 pbar, for example at a pressure of between 10 and 100 pbar. The process of clause 74 wherein the lyophilization chamber is held at the primary drying temperature for between 6 hours and 22 hours, such as between 9 hours and 22 hours, optionally between 11 hours and 22 hours, at a pressure of between 65 and 85 pbar, for example at a pressure of 75 pbar. The process of clause 74, further comprising holding the lyophilisation chamber at the primary drying temperature for 21 hours ± 2 hours, optionally 21 hours. The process of clause 68 wherein the secondary drying step comprises adjusting the temperature of the lyophilization chamber to a secondary drying temperature ranging from 0 °C to 40 °C with a ramp rate ranging from 0.05 °C / min up to 1 .0 °C / min, optionally from 0.1 °C / min up to 1 .0 °C / min. The process of clause 77 wherein the secondary drying step comprises adjusting the temperature of the lyophilization chamber to a secondary drying temperature ranging from 5 °C to 20 °C with a ramp rate ranging from 0.05 °C / min up to 1 .0 °C / min. The process of clause 77 or clause 78 wherein the secondary drying temperature is 14 °C, 15 °C or 16 °C. The process of clause 77 or clause 78 wherein the ramp rate is 0.1 °C / min. The process of clauses 76-80, wherein during the secondary drying step the lyophilization chamber is held at the secondary drying temperature for between 5 and 25 hours at a pressure of between 10 and 150 pbar, for example at a pressure of between 10 and 100 pbar. The process of clause 77-81 wherein during the secondary drying step the lyophilization chamber is held at the secondary drying temperature for between 7 hours and 14 hours, optionally between 10 and 14 hours, at a pressure of between 65 and 85 pbar, for example at a pressure of 75 pbar. The process of clause 60-82 further comprising step v) of plugging the vessel(s).84. A process for preparing a packaged lyophilised pharmaceutical composition said process comprising collecting an amount of the lyophilized pharmaceutical composition according to claims 1-53 in the form of a plurality of LyoBeads; and packaging that amount into a container.85. A lyophilised pharmaceutical composition obtained from the process of clauses 60-83.86. A vaccine comprising the lyophilised pharmaceutical composition of clause 85.87. A vaccine reconstituted from the lyophilised pharmaceutical composition of clause 85.88. A vessel comprising a plurality of LyoBeads, said LyoBeads being prepared by the process of clauses 60-8389. The use of the lyophilised pharmaceutical composition of clauses 1-53 or clause 85, the vaccine of clauses 55-57 or clauses 86-87 or the kit of clauses 58-59 in the manufacture of a medicament for treating a subject in need thereof.90. The use of the lyophilised pharmaceutical composition of clauses 1-53 or clause 85, the vaccine of clauses 55-57 or clauses 86-87 or the kit of clauses 58-59 in the manufacture of a medicament for prophylaxis in a subject in need thereof.91 . A method for eliciting an immune response in a subject in need thereof comprising administering the pharmaceutical composition of clauses 1-53 or clause 85 or the vaccine of clauses 55-57 or clauses 86-87 to the subject, optionally wherein the subject is a human subject.92. The pharmaceutical composition of clauses 1-53 or clause 85 or the vaccine of clauses 55-57 or clauses 86-87 for use in medicine.93. The pharmaceutical composition of clauses 1-53 or clause 85 or the vaccine of clauses 55-57 or clauses 86-87 for use in the treatment or prevention of disease in a subject, optionally wherein the subject is a human subject.[000245] The invention is further illustrated by the following exemplary and non-limiting examples.EXAMPLESExample 1 : Materials and MethodsThe mRNA utilised encodes for eGFP and nanoLuciferase (Nano-Luc) with UTR4 (see FIG. 1 and SEQ ID NO: 11). The mRNA stock solution (1000pg / mL) was obtained in citrate buffer.Lipids 1 ,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC; CAT#850365C), Cholesterol (CAT#700100P or [#W004591 , Sigma-Aldrich])), 1 ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMGPEG2k; CAT#880151 P) and 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (DMPE-PEG2k; CAT#880150P) were purchased from Merck-Avanti Polar Lipids. The cationic lipid utilised was RV94 (WuXi AppTec CAT#LXB512)The organic phase was prepared by solubilizing the lipids DSPC, Cholesterol, DMG-PEG2k or DMPE- PEG2k and RV94, individually in ethanol. The lipids were then mixed to obtain a molar ratio of 10:48:2:40 respectively.The aqueous phase of mRNA was prepared by diluting the mRNA with Citrate buffer 75mM pH 6. Lipids and mRNA stock solutions were prepared on the day of the experiment, and mRNA stock was kept on ice before use.The two phases were prepared at an ethanol: aqueous volume ratio of 1 :2, and mRNA and lipids combined at an N / P ratio of 8.Each phase was loaded into a syringe (Becton Dickinson) and locked onto the NxGen microfluidic cartridge for mixing using a NanoAssemblr Ignite instrument (Cytiva-Precision Nanosystems, Vancouver, Canada). The Ignite was set to operate with the following settings: volume ratio- 2:1 ; flow rate- 12 mL / min; start waste volume- 0.35 mL and end waste volume- 0.05 mL.The resulting LNPs were maturated on ice for at least 30 minutes. They were then dialyzed against custom buffer 20mM Tris 5mM NaCI 7.5%w / w Sucrose pH 8 using a PD-10 desalting column (Cytiva, CAT#17085101) at room temperature. The LNP solution in its final buffer was allowed to rest on ice for at least one hour. Following resting, the LNP solution was diluted to its final target concentration of 60pg / mL with the same buffer and filtrated in sterile conditions over 0.22pm PES syringe filter. The final bulk was then stored at -70°C before use.Using the materials and protocol described above, two mRNA-LNP 60pg / mL final bulks were produced and used in this study:22NPT28: DSPC / Cholesterol / DMG-PEG / LXB512 LNPs, mRNA eGFP, Nano-Luc22NPT35: DSPC / Cholesterol / DMPE-PEG / LXB512 LNPs, mRNA eGFP / Nano-LucTwo pilot scale freeze-dryers were used: a LyoVac GT6 (Finn-Aqua Santasalo-Sohlberg SPRL, Brussels, Belgium),A REVO model (Millrock Technology, Kingston, NY, United States)Glass tubing vials (3 mL) provided by Muller + Muller (Holzminden, Germany) that were siliconized and depyrogenized in-house were used throughout the experiments described herein. Stoppers FM460 Igloo 13mm (Datwyler Pharma Packing) sterilized by autoclave in-house were used.For the freeze-drying of the cakes, vials were arranged on a 140-vial bottomless freeze-dryer tray and were filled with 700pL volume of mRNA-LNP final liquid bulk or buffer control (20mM Tris, 5mM NaCI, 7.5%w / w Sucrose pH 8), using a calibrated dispenser pipette. A stopper was inserted loosely into the vial neck. The border rows were kept empty, but no specific position was used for filled vials.Product temperature was measured using product temperature probes as follows:Integrated thermocouple for the REVO Millrock freeze-dryer andQuartz wireless sensors for the GT6 freeze-dryer (Tempris GmbH, Holzkirchen, Germany)The vials were loaded on the freeze dryer shelf at room temperature.Freezing Ramp: For lyophilisation of the cakes, the temperature in the lyo chamber was decreased at a ramp of 1 °C / min. The temperature was decreased to a first plateau at 5°C for 30 minutes and then to a second plateau at -5°C for 30 minutes, before the temperature reached the target temperature of the final freezing plateau.Freezing: A final freezing plateau of 1 hour at -40 or -65°C was performed. It could be extended to match experimental time constraint.Primary and Secondary Drying: The pressure was decreased to 75 pbar and the shelf temperature was increased by 0.5°C / min. Primary drying was carried out with a shelf temperature set-up of -29, 0 and 15°C. When primary drying was finished according to Pirani pressure probe value, shelf temperature was increased at 0.1 °C / min to 15°C, keeping chamber pressure at 75pbar. After 12 hours of secondary drying, the vials were automatically stoppered in the freeze-dryer under 825mbar of dry nitrogen and stored at 4°C pending unloading.The lyophilisation cycles used in this study are shown graphically in FIG 3, FIG. 4 and FIG. 5.The dry product vials were visually inspected, then capped and labelled, and stored at - 70°C. (see FIG. 2).During the freezing ramp of the vials (cakes) the beads were prepared.The mRNA-LNP final liquid bulk solution, or buffer control (20mM Tris 5mM NaCI 7.5%w / w Sucrose pH 8), was manually dripped using a multichannel pipette with 200pL tips in a liquid nitrogen bath under laminar flow. The droplets froze in the bath, moving at the surface of the liquid to finally drop at the bottom of the bath once deep-frozen. The pipette tips were placed between 3 and 10 cm above the liquid nitrogen bath surface when dropping the formulation.Doses of 700pL were made one at the time in the bath. Frozen beads of one dose were manually picked up with inox pincers and transferred into one 3mL siliconized glass vial filled with and stored in a liquid nitrogen bath. Once all beads of one single dose were conditioned in one vial, a new 700pL dose was made in the bath. Once all deep-frozen bead doses were transferred, a stopper was inserted loosely into each vial neck.The deep-frozen bead vials were placed in the middle of a pre-cooled 140-vial tray and quickly loaded onto the cold shelf of the freeze-dryer, alongside the vials (cakes) starting the freezing step i.e. at the point of the freezing plateau at -40 or -65°C. Thus vials filled with the pipette, to produce cakes, undergo the full freeze-drying cycle described below, while the vials filled with deep-frozen beads are loaded in the freeze-dryer during the freezing step (i.e. at the start of the freezing plateau).Primary and Secondary Drying Steps were conducted according to the same protocol as described above for the cakes.Determination of the residual moisture by Karl FisherThe residual moisture content of the freeze-dried cakes was determined using a Karl-Fisher titrator (831 KF Coulometer, Metrohm Belgium) connected to 774 Oven Sample Processor (Metrohm AG, Herisau, Switzerland). 7mL capped vials (Metrohm Belgium), containing 20-25 mg crushed freeze- dried cake were placed in the oven to transfer the water as a vapor to a titration vessel with an anolyte solution (Hydranal coulomat A for oven, Sigma-Aldrich Belgium). The moisture content was calculated from a standard plot (water standard oven 1 %, Apura, Merck), based on the sample weight and the amount of water evaporated into the titration vessel. Three blank vials were analyzed to subtract the background moisture from the freeze-dried cake samples. All sample manipulations were performed in a nitrogen glove box (Jacomex, Belgium) with relative humidity and oxygen conditions below 1 ppm.Determination of the glass transition temperature (Ta) by Differential Scanning Calorimeter (DSC)The glass transition of the dry product (Tg) was measured using a DSC 250 equipment (TA Instruments) essentially according to manufacturer’s recommendations. By having separate heating sources for the sample and a reference material, the DSC is capable of calculating theheat flow to the sample material. When the sample goes through a physical or chemical change in response to a change in temperature, it will give off / absorb a small amount of heat to / from the environment. The instrument senses this and applies or removes heat to keep the sample at the same temperature as the reference. Since the instrument is measuring heat flow instead of just the temperature difference between the sample and reference, it is now possible to calculate some of the thermodynamic values for the transition.In a glove box, under nitrogen atmosphere a smashed lyophilizate buffer-only (i.e. no mRNA- LNP) control was placed in an aluminium cupule and the cupule was sealed. The recipe applied was the following: cooled from 40°C to -20°C at -1 °C / minEquilibrated at -20°C for 5 min;Heated to 120°C at 10°C / min.The inflexion point of the heat transfer curve was reported as the T g value.LNP size and distribution analysis by DLSDynamic light scattering (DLS; Zetasizer Nano S and ZS, Malvern Panalytical) was performed to determine the hydrodynamic size and polydispersity index of the LNPs. The liquid final bulk or reconstituted composition was diluted to a mRNA-LNP content of 1.5pg / mL in its native buffer 20mM Tris, 5mM NaCI, 7.5%w / w Sucrose, pH 8 and transferred in a plastic microcuvette (ZEN0040, Malvern) for measurement. Measurement was performed at 25°C after 90 seconds of equilibration time. The refractive index of polystyrene latex was used, 1.59, and a custom dispersant viscosity was created to account for 7.5%w / w sucrose, 1 .1108 cP. Its refractive index is 1.340. 3 Measurements were made af fixed position 4.65, and the reported value is the average of 3 individual dilutions and acquisition of one sample. mRNA content and encapsulation by RiboGreenThe RiboGreen kit (CAT#R11490, Thermofisher Scientific) and Triton (Sigma-Aldrich,CAT#T8787) w. The standard mRNA from the kit was used for the calibration curve. The liquid final bulk or reconstituted compositions were diluted to a mRNA-LNP content of 1 ,5pg / mL in its native buffer without sucrose (20mM Tris, 5mM NaCI, pH 8) in triplicates.The standard protocol of the kit is followed. The plate was read using fluorescent mode at 485nm - emission 528nm with the Synergy microplate reader (H4 hybrid Reader, Agilent). The ratio between free mRNA in the test samples and free mRNA in sample lysed with Triton was calculated to obtain mRNA encapsulation percentage.mRNA transfection in-vitro potency assayIn vitro relative potency (IVRP) was assessed using flow cytometry by measuring the expression of the protein of interest in BHK-21 cells transfected with the mRNA-LNP.More specifically, the mRNA construct (eGFP-nanoLuc, see FIG. 1 and SEQ ID NO: 11) was designed to ensure high expression of a green fluorescent protein (eGFP). BHK-21 cells were seeded in 96-well collagen coated plate (#354407, Corning) at 40,000 cells per well. Beads and cakes were reconstituted in water for injection at 60pg / ml. Cells were then transfected with seven serial dilutions of samples and reference material (stock solution: 60pg / ml) in duplicates with starting concentration of 12ng / well and 1.9-fold dilutions steps and then incubated for 20h at 37°C; 5% CO2. The following day, cells were trypsinized and fixed for 15min at 4°C with cytofix / cytoperm (BDB554714, BD Biosciences) before acquisition with MacsQuant VYB (Miltenyi Biotec). Percentage of singlets and GFP positive cells was determined by using FlowJo software and results were analysed using the PLA approach using PLA3.0 software (BioAssay). The potency for each sample was then determined by the calculated shift between sample and reference material curves using the principle of parallel line analysis (4 parameters curve). Reference material was a standardized batch of eGFP-nLuciferase final liquid-bulk formulation, CDW 39375). This reference batch was added in every microplate as internal reference and samples’ relative potency is expressed compared to this 100% reference value.Example 2: Results from Example 1The experiment and batch nomenclature is as follow (see Table 1). Cakes and Beads were both made from the final liquid bulk after 1 freeze-thaw.Table 1LXB512 is RV94 cationic lipidTests performed on freeze-dried product:Moisture content by Karl-Fisher:The moisture content in the cakes and beads depending on the freeze-drying cycle is reported in Table 2 and 3.Table 2 and Table 3: Results of moisture content (n=1) in the cakes and beads after lyophilisation.Table 2: 22NPT28 (DSPC / Cholesterol / DMG-PEG / LXB512)Table 3: 22NPT35 (DSPC / Cholesterol / DMPE-PEGZ LXB512)As can be seen from the data the primary drying temperature increase has a significant impact on the moisture content of the cakes: the higher the primary drying temperature, the higher the residual moisture. A hypothesis for the residual moisture increases with primary drying temperature would be the micro-collapse of the cake structure due to Tg passing during aggressive primary drying.All analysed beads samples %RM were between 0.15 and 0.23% RM. Overall %RM was low compared to cakes was not impacted by the cycle’s primary drying temperature. Drying appeared more advanced with beads being less sensitive to high temperature drying compared to cakes.Glass transition temperature:The glass transition temperature of cakes and beads depending on the freeze-drying cycle is reported in Table 4 and 5. Tg analysis was performed on the same vials as %RM analysis except for the results marked with * (tests failed and required a retest).Table 4 and 5: Glass transition temperature (Tg) (n=1) of cakes and beads after freeze-dryingTable 4: 22NPT28 (DSPC / Cholesterol / DMG-PEGZ LXB512)Table 5: 22NPT35 (DSPC / Cholesterol / DMPE-PEGZ LXB512)A parallel with the %RM can be made because the increase in the moisture content of the cake due to higher primary drying temperature led to a decrease in the Tg of the cakes, while the beads Tg remained between 58 and 64°C independently of the drying protocol used. The Tg of the cakes and beads was similar when using the conservative drying cycle with primary drying at -29°C. Samples having a %RM below 0.6% demonstrate comparable Tg, while reaching a %RM of 1% or above led to a significant drop in Tg value.Tests performed on the reconstituted compositionsLNP size and distribution analysis:The DLS analysis of LNPs size and polydispersity index (Pdl) of the reconstituted cakes and beads are presented in Table 6 and 7Table 6 and Table 7: Reconstituted cakes and beads particles’ size and polydispersity index (n=3)Table 6: 22NPT28 (DSPCZCholesterolZDMG-PEGZ LXB512)Table 7: 22NPT35 (DSPC / Cholesterol / DMPE-PEGZ LXB512)An overall particle size increase was observed after drying independently of the drying recipe or form, except for beads of DEL5010.Regarding cakes, aggressive primary drying at 15°C leads to a large particle size increase for DMG-PEG formulation compared to both conservative primary drying at -29°C and to liquid final bulk. This increase is less prominent for DMPE-PEG formulations. Regarding beads, the particles’ size increase was significantly more limited compared to cakes and did not seem to be linked to the formulation but rather to the primary drying temperature. For primary drying at -29°C, particles’ size increase was about 0 to +12 nm compared to liquid final bulk although the replicate runs were variable. For primary drying at 0°C, particles’ size increasewas +10 nm. For primary drying at 15°C, particles’ size increase was between +14 and +20nm compared to liquid final bulk for both formulations.Comparing cakes and beads for the same lyophilisation run, beads demonstrated a lower particle size and slightly higher Pdl, (except for DEL5085 where particles’ size is equivalent for cake and beads). The Pdl difference between cakes and beads was limited (maximum +0.06) and all PDI values remained below 0.20. mRNA content and encapsulation:The RiboGreen analysis of mRNA encapsulation and total content of the reconstituted cakes and beads are presented in Table 8 and 9. Table 8 and Table 9: Reconstituted cakes and beads mRNA encapsulation rate and total content (n=3)Table 8: 22NPT28 (DSPC / Cholesterol / DMG-PEGZ LXB512)Table 9: 22NPT35 (DSPC / Cholesterol / DMPE-PEGZ LXB512)For a I analysed samples, the total mRNA contents were consistent with the content of the respective liquid final bulks, within less than 5% variability compared to the starting material. The different freeze-drying cycles or different forms (e.g. cakes v beads) has no impact on total mRNA content. There was an overall mRNA encapsulation loss due to drying.For cakes, the higher the primary drying temperature was, the larger the encapsulation loss. This observation was more pronounced for the DMPE-PEG formulation compared to the DMG-PEG.For beads, the mRNA encapsulation values were all within narrow range of 69% to 77% for all formulations and freeze-drying cycles. For DMPE-PEG formulation, beads drastically limited the encapsulation loss due to aggressive primary drying compared to cakes. mRNA transfection in-vitro relative potency assay:The in-vitro relative potency (IVRP) analysis of the reconstituted cakes and beads are presented in Table 10 and 11.Table 10 and Table 11 Reconstituted cakes and beads mRNA-LNP in-vitro potency compared to reference batch (n=3)Table 10: 22NPT28 (DSPC / Cholesterol / DMG-PEGZ LXB512)Table 11 : 22NPT35 (DSPC / Cholesterol / DMPE-PEGZ LXB512)For primary drying at -29°C with DMG-PEG formulation, no potency loss was observed after drying the beads compared to liquid final bulk. For the corresponding cakes, some potency loss was observed. For primary drying at 15°C of DMG-PEG formulation, potency loss (compared to liquid final bulk) is significantly more limited in the lyophilised beads compared to cakes.For DMPE-PEG formulation, the two drying cycles led to significant potency loss which can be explained by the fact that both cycles have an aggressive primary drying temperature >0°C. However, the potency loss observed with lyophilised cakes was significantly greater than with the corresponding lyophilised beads.All lyophilised beads demonstrated higher potency than their respective corresponding cake sample (see percentage increase in potency shown in Table 12 below):Table 12: Percentage increase in potency of Beads versus Cakes:Conclusions:In this study 2 formulations with 2 different pegylated lipids were freeze-dried in different forms, cakes and beads, using different primary drying temperatures.On the dry product (i.e., lyophilised composition) characteristics, beads conserved their low %RM (<0.2%RM) and high Tg (around 60°C) even when dried using aggressive primary drying cycles. Cakes and beads appeared equivalent when using conservative primary drying,On the reconstituted vaccine quality, beads had more constant characteristics when comparing drying runs, but also in comparison with the liquid bulk before drying.LNPs’ size and Pdl but also mRNA content and encapsulation showed limited variation and avoid problematic drastic increase for size / Pdl or drop for mRNA.Most noticeably however the IVRP assay showed limited loss of in-vitro potency for beads compared to cakes and on two occasions (DEL5010 and DEL5012 beads) no loss in potency was observed compared to the final liquid bulk (i.e. pre-lyophilisation). The potency loss was especially limited for DMG-PEG formulation beads with both conservative and aggressive primary drying.Example 3:In this study, a Moderna-like formulation, which comprises lipids comprising 50 mole% 9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 38.5 mole% cholesterol, 10 mole% distearoylphosphatidylcholine (DSPC), and 1.5 mole% 1 ,2- dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), were compared to the RV94 formulations of Example 2, which comprises lipids comprising 48 mole% 2-(5-((4-((1 ,4- dimethylpiperidine-4- carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1 ,3-diyl dioctanoate (a.k.a. RV94 and LXB-512), 40 mole% cholesterol, 10 mole% DSPC, and 2 mole% 1 ,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2k). Ribogreen analysis of encapsulation and percentage of eGFP-positive cells expressed from mRNA delivered with the lyophilized LNP were determined with the methods of Examples 1 and 2.FIG. 6 shows the lyophilisation cycle for the conditions in Example 3, and thereby in FIGS. 7 and 8. The graph (top) plots temperature (dotted lines) and pressure (solid lines) which are labelled with datapoints as set forth in columns 4 and 5 in the table (bottom).FIG. 7 depicts the average mass percentage of mRNA encapsulation in lipid nanoparticles (LNPs) was the same for cake lyophilization and bead lyophilization when comparing the Moderna-like formulation and the formulation comprising RV94.FIG. 8 shows that bead lyophilisation unexpectedly increased the percent of baby hamster kidney-21 (BHK-21) cells expressing enhanced green fluorescent protein (eGFP) encoded in mRNA delivered with the formulations of FIG. 7 when compared to the same measure but with cake lyophilization of the same formulations.Conclusion:The results here, with a different ionizable lipid and different formulation, recapitulate the unexpected results observed in Example 2.In this study, the RV94 formulation with DMPE-PEG2k from Examples 1-3 were repeated with 4%, 7.5%, 15%, or 20% weight by volume of sucrose. FIG. 9 shows the lyophilisation cycle for the conditions in FIGS. 10-13. The graph (top) plots temperature (dotted lines) and pressure (solid lines) which are labelled with datapoints as set forth in columns 4 and 5 in the table (bottom).For mean lipid nanoparticle diameter and polydispersity index (PDI), a Malvern zetasizer was used with one analytical replicate of 1 vial per batch in individual cuvette. A common dilution buffer was used for diameter and PDI analysis to allow comparison of the samples with different native sucrose rates. The mass percent of encapsulation of mRNA in the LNP and the percent of cells expressing eGFP are the same as in Examples 1-3.FIG. 10 depicts the average mass percentage of mRNA encapsulation in LNPs with 4%, 7.5%, 15%, and 20 % weight by volume of sucrose for formulations comprising RV94, cholesterol, DSPC, and DMPE-PEG2k. Bead lyophilization was less affected by varying the amount of sucrose than cake lyophilization was affected by the amount of sucrose. Unexpectedly, the average mRNA encapsulation for bead lyophilization with 4%, 7.5%, 15%, or 20% weight by volume of sucrose was higher than the same sucrose amounts for cake lyophilization. TO conditions are measurements from samples stored at -70°C after lyophilization until testing for encapsulation whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization until reconstitution and testing for encapsulation. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.FIG. 11 shows that bead lyophilization unexpectedly increased the percent of BHK-21 cells expressing enhanced green fluorescent protein (eGFP) encoded in mRNA delivered with the formulations of FIG. 10 when compared to the same measure but with cake lyophilization of the same formulations. TO conditions are measurements from samples stored at -70°C after lyophilization until reconstitution and testing for eGFP expression whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization before testing for eGFP expression. Error bars are standard deviation. Unlyophilized conditions are labeled “liquid bulk,” and these conditions were stored at -70°C between formulation and measurement.FIG. 12 shows the LNPs’ mean diameter for the formulations in FIG. 10. With 4% weight by volume sucrose, lyophilization in cakes or beads increased the mean diameter. But with 7.5% and 15% weight by volume sucrose, the LNP diameter was less affected across cake and bead lyophilization conditions when compared to non-lyophilized conditions (“bulk liquid”). However, as FIG. 11 demonstrates, the increase in mean LNP diameter does not negatively affect the potency of the formulations. TO conditions are measurements from samples stored at -70°C after lyophilization untilreconstitution and testing for diameter whereas T7 conditions were stored at room temperature (25°C) for seven days after lyophilization before testing for diameter. Error bars are standard deviation.FIG. 13 shows the LNPs’ polydispersity index for the formulations in FIG. 10. With 7.5% and 15% weight by volume sucrose, the PDI was largely unchanged for lyobeads or for cake lyophilization, except with the condition stored at room temperature for seven days after lyophilization.Conclusion:The sucrose conditions are adaptable and effective over a broad range of percent weight by volume without reducing the potency of the mRNA encapsulated in the LNP or the expression of the genes encoded in the mRNA.INTERPRETATION OF SEQUENCE LISTINGIn the XML-format electronic copy of the Sequence Listing for RNA, it is to be understood that any “U” or “u” (uridine) depicted as “T” or “t” (thymidine) therein may be replaced with any of the uridine- substitutable modified nucleotides or thymidine-modified nucleotides noted above, including a N1- methylpseudouridine, pseudouridine, N1-ethylpseudouridine, and that the percentage of “u” which is depicted as “t”, is replaced with uridine-substitutable modified nucleotides or thymidine-modified nucleotides corresponds with those described in the embodiments above. For example, a mole percentage of the N1 -methylpseudouridines to the total of the N1 -methylpseudouridines and the uridines of 50% contemplates and supports substitution of 50% of the “u” with “NI MT” For example, a mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines of 25% contemplates and supports substitution of 25% of the “u” with “NI MT” For example, a mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines of 75% contemplates and supports substitution of 75% of the “u” with “NI MT” In the paper copy and XML-format electronic copy of the Sequence Listing, it is to be understood that any “A” or “a” (adenosine) depicted therein may be replaced with any of the adenosine-substitutable modified nucleotides noted above, and that the percentage “A” or “a” replaced with adenosine-substitutable modified nucleotides corresponds with those described in the embodiments above. In the paper copy and XML-format electronic copy of the Sequence Listing for DNA, it is to be understood that any “T” or “t” (thymidine) depicted therein may be replaced with any of the thymidine-substitutable modified nucleotides noted above, and that the percentage “T” or “t” replaced with thymidine-substitutable modified nucleotides corresponds with those described in the embodiments above. In the paper copy and XML-format electronic copy of the Sequence Listing, it is to be understood that any “C” or “c” (cytosine) depicted therein may be replaced with any of the cytosine-substitutable modified nucleotides noted above, and that the percentage “C” or “c” replaced cytosine-substitutable modifiednucleotides corresponds with those described in the embodiments above. In the paper copy and XML- format electronic copy of the Sequence Listing, it is to be understood that any “G” or “g” (guanosine) depicted therein may be replaced with any of the guanosine -substitutable modified nucleotides noted above, and that the percentage “G” or “g” replaced guanosine-substitutable modified nucleotides corresponds with those described in the embodiments above.Where the present disclosure refers to a sequence by reference to a UniProt, Genbank, NationalCenter for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) reference sequence, accession number (No.), or accession code, the sequence referred to is the current version at the filing date of the earliest effective filing date. Herein a “reference sequence” and “accession code” is referred to as “accession code” for the sole purpose of simplicity and without regard to whether the original Uniprot,GenBank, or NCBI listing recites “reference sequence,” “accession no.,” or “accession code.”

Claims

CLAIMSWhat is claimed is:

1. A lyophilised pharmaceutical composition said lyophilised pharmaceutical composition comprising lipid nanoparticles (LNPs) encapsulating a nucleic acid payload wherein the lyophilised pharmaceutical composition is in the form of a plurality of lyophilised beads (LyoBeads).

2. The lyophilised pharmaceutical composition of claim 1 wherein the LyoBeads are spherical shaped bodies, optionally wherein the LyoBeads are between 0.1 and 25 mm in diameter, such as between 0.5 and 20 mm, between 1 and 15 mm or between 2 and 10 mm.

3. The lyophilised pharmaceutical composition of claim 1 or claim 2 wherein the nucleic acid is ribonucleic acid (RNA), optionally wherein the RNA is messenger RNA (mRNA), optionally wherein the mRNA encodes for at least one protein.

4. The pharmaceutical composition of any preceding claim wherein the LNPs comprise a cationic lipid, a neutral lipid, and a polyethylene glycol-conjugated (PEGylated) lipid.

5. The pharmaceutical composition of any preceding claim wherein the LyoBeads are contained within a vessel(s).

6. The lyophilised pharmaceutical composition of any preceding claim wherein said lyophilised pharmaceutical composition is lyophilised from an aqueous pre-lyophilisation composition comprising the LNPs encapsulating the nucleic acid payload.

7. The lyophilised pharmaceutical composition of any preceding claim wherein said lyophilised pharmaceutical composition is reconstituted to form an aqueous post-reconstitution composition, optionally wherein said lyophilised pharmaceutical composition is reconstituted in a sterile reconstitution solution.

8. The lyophilised pharmaceutical composition of claim 7 wherein the potency of the nucleic acid in the aqueous post-reconstitution composition is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, such as at least 99% compared to the potency of the nucleic acid in the aqueous pre-lyophilisation composition (i.e., final liquid bulk).

9. The lyophilised pharmaceutical composition of claim 7 wherein the potency of the nucleic acid is increased in the aqueous post-reconstitution composition compared to a comparator composition reconstituted from a comparator lyophilizate.

10. The lyophilised pharmaceutical composition of claim 9 wherein the potency of the nucleic acid is increased in the aqueous post-reconstitution composition by more than 5%, more than 7.5%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% compared to a comparator composition reconstituted from a comparator lyophilizate.

11. The lyophilised pharmaceutical composition of claims 8-10 wherein potency is calculated by measuring the expression of a protein encoded for by the nucleic acid following transfection into a eukaryotic cell.

12. The lyophilised pharmaceutical composition of claims 9-11 wherein the comparator lyophilizate is not in the form of LyoBeads.

13. The lyophilised pharmaceutical composition of claims 9-12 wherein the comparator lyophilizate is identical with the exception that the comparator lyophilize is in the form of a lyophilised cake.

14. The lyophilised pharmaceutical composition of claims 9-13 wherein the comparator lyophilizate is produced by the steps of a) taking a volume of the aqueous pre-lyophilisation composition disclosed in claim 6 and subjecting the entirety of said volume to freezing conditions such that the entire volume of said aqueous pre-lyophilisation composition freezes into a single frozen mass; and b) drying the single frozen mass to form a lyophilised cake using an identical drying protocol to the drying protocol used to produce the LyoBeads.

15. A vaccine comprising the lyophilised pharmaceutical composition of any of claims 1-14.

16. A vaccine reconstituted from the lyophilised pharmaceutical composition of any of claims 1-14.

17. A multicomponent vaccine comprising: a first lyophilised pharmaceutical composition according to claims 1-14 (“First LyoBead”) and a second lyophilised pharmaceutical composition according to claims 1-14 (“Second LyoBead”) wherein the first LyoBead comprises first LNPs encapsulating a first nucleic acid payload and the second LyoBead comprises second LNPs encapsulating a second nucleic acid payload, wherein the first and second nucleic acid payloads encode protein immunogens that are different from one another.

18. A process for preparing the lyophilised pharmaceutical composition of claims 1-14 said process comprising: i) introducing an aqueous pre-lyophilisation composition comprising the LNPs encapsulating the nucleic acid payload into a dispensing tip, ii) positioning the dispensing tip above a cryogenic liquid or a cryogenically cooledsurface, iii) dispensing single droplet aliquot(s) of the aqueous pre-lyophilisation composition into the cryogenic liquid or onto the cryogenically cooled surface in a manner such that the single droplet aliquot(s) freezes to form a frozen droplet(s); and iv) drying the frozen droplet(s) under conditions sufficient to produce a lyophilised bead(s) (LyoBead(s)).

19. The process of claim 18 wherein the cryogenic liquid is liquid nitrogen, liquid helium, liquid oxygen, or liquid carbon dioxide.

20. The process of claims 18-19 wherein between step iii) and step iv) the frozen droplet(s) are transferred into a vessel(s) followed by said vessel(s) being transferred to a lyophilisation chamber that has been pre-cooled to a temperature sufficient to maintain the frozen droplet(s) in their frozen state.21 . The process of claims 18-20 wherein during step iv) the frozen droplet(s) is subjected to a primary drying step, and after the primary drying step, is subjected to secondary drying step, both primary and secondary drying steps taking place within a lyophilisation chamber22. A lyophilised pharmaceutical composition obtained from the process of claims 18-21.

23. A vaccine comprising the lyophilised pharmaceutical composition of claim 22.

24. A vaccine reconstituted from the lyophilised pharmaceutical composition of claim 22.

25. The pharmaceutical composition of claims 1 -14 or claim 22 or the vaccine of claims 15-17 or claims 23-24 for use in the treatment or prevention of disease in a subject, optionally wherein the subject is a human subject.

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