Compositions and methods

Abstract

A method of producing a composition containing a nucleic acid-lipid particle, the method comprising the steps (a) to (d): (a) preparing a first mixture which is a lipid mixture comprising a cationically ionizable lipid in a water-soluble organic solvent; (b) preparing a second mixture in aqueous solution, the second mixture comprising (i) a nucleic acid and (ii) malate buffer; (c) mixing the first mixture with the second mixture to produce an intermediate composition comprising the nucleic acid-lipid particle; and (d) further processing of the intermediate composition by buffer exchange, wherein the buffer used in the buffer exchange comprises tris(hydroxymethyl)-aminomethane (Tris) and / or a pharmaceutically acceptable salt thereof, to produce the composition comprising the nucleic acid-lipid particle; is provided.

Description

[0001] COMPOSITIONS AND METHODS

[0002] Technical Field

[0003] The present disclosure relates generally to a method for producing a composition comprising a nucleic acid-lipid particle, to a composition produced by the method, and its use in medicine.

[0004] Background

[0005] Compositions comprising nucleic acid particles, such as lipid nanoparticle (LNP) formulations, are typically produced using an ethanolic-aqueous mixing protocol where a negatively charged nucleic acid, such as RNA, is mixed with cationically ionizable lipids, typically also in the presence of helper and structural lipids (e.g. cholesterol, phospholipids, and grafted lipids such as PEG-conjugated lipids). Assembly of the complexes is achieved through rapid mixing of the two liquid phases leading to nanoprecipitation and driven by electrostatic and hydrophobic interactions of lipids with the nucleic acid.

[0006] Conventional methods of preparing lipid nanoparticles often encounter issues related to instability upon ageing at frozen or liquid conditions, leading to particle aggregation, leakage and / or degradation of encapsulated cargo, and reduced efficacy. Additionally, maintaining the stability of lipid nanoparticles during storage, particularly in frozen and liquid states, has proven to be a significant challenge. Therefore, there is a need for an innovative approach to improve the stability of lipid nanoparticles and enhance their storage potential.

[0007] Summary

[0008] In a first aspect, there is provided a method of producing a composition containing a nucleic acid-lipid particle, the method comprising the steps (a) to (d):

[0009] (a) preparing a first mixture which is a lipid mixture comprising a cationically ionizable lipid in a water-soluble organic solvent;

[0010] (b) preparing a second mixture in aqueous solution, the second mixture comprising (i) a nucleic acid and (ii) malate buffer;

[0011] (c) mixing the first mixture with the second mixture to produce an intermediate composition comprising the nucleic acid-lipid particle; and

[0012] (d) further processing of the intermediate composition by buffer exchange, wherein the buffer used in the buffer exchange comprises tris(hydroxymethyl)aminomethane (Tris) and / or a pharmaceutically acceptable salt thereof, to produce the composition comprising the nucleic acid-lipid particle.

[0013] In a second aspect, the present disclosure provides a composition comprising a nucleic acid-lipid particle, obtainable by the method of the first aspect.

[0014] In a third aspect, the present disclosure provides a pharmaceutical composition containing the composition of the second aspect and a pharmaceutical carrier.

[0015] In a fourth aspect, the present disclosure provides a composition of the second aspect for use in medicine.

[0016] In a fifth aspect, the present disclosure provides a composition of the second aspect for use in a prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for use in inducing an immune response.

[0017] In a sixth aspect, the present disclosure provides a composition of the second aspect for use in treating cancer.

[0018] In a seventh aspect, the present disclosure provides use of a composition of the second aspect in the manufacture of a medicament for use in a prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for use in inducing an immune response.

[0019] In an eighth aspect, the present disclosure provides use of a composition of the second aspect in the manufacture of a medicament for use in treating cancer.

[0020] In a ninth aspect, the present disclosure provides a method of prophylactic and / or therapeutic treatment of a disease involving an antigen and / or method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition of the second aspect.

[0021] In a tenth aspect, the present disclosure provides a method of prophylactic and / or therapeutic treatment of cancer in a subject in need thereof, comprising administering to the subject a composition of the second aspect.

[0022] Advantages and Surprising Findings

[0023] It has surprisingly been found by the present inventors that using a malate acidification buffer during the formation of compositions comprising nucleic acid- lipid particles, particularly although not exclusively lipid nanoparticles, followed by buffer exchange of the composition into a Tris buffer, leads to superior results compared with other buffer systems and combinations. Firstly, the sequential use of the malate acidification buffer during mixing and the Tris buffer for the final composition, significantly improves the colloidal and chemical stability of the compositions during their formation and subsequent storage in both frozen and liquid states. Secondly, the improved stability achieved through the sequential malate / Tris buffer system extends the storage potential of lipid nanoparticle formulations at - 20 °C. This is particularly beneficial for long-term storage, transportation, and distribution of lipid nanoparticles, thereby facilitating their widespread application. Thirdly, the sequential malate / Tris buffer system can be integrated into various manufacturing processes for producing nucleic acid-lipid particles, allowing its utilization with different lipid compositions, encapsulated cargoes, and preparation methods. This versatility ensures the broad applicability of the invention across diverse fields of research and industry. Fourthly, the components of the sequential malate / Tris buffer system are compatible with lipid nanoparticles, ensuring minimal interference with their functionality. Furthermore, the chosen components are known to be safe for use in biomedical and pharmaceutical applications. Brief Description of the Figures

[0024] Figure 1. Box-Behnken design space with the ranges of logD buffer, logD acidifier and buffer concentration

[0025] Figure 2. Response surface intersection of LogD acidifier vs. logD buffer at a buffer concentration of 12.5 mM

[0026] Figure 3. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNPs with various RNA constructs

[0027] Figure 4. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl with various flow rate ratios and in-line dilution buffers.

[0028] Figure 5. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate concentrations (10 - 50 mM) LNPs size and stability with varying pH of LNPs before purification

[0029] Figure 6. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate concentrations (10-300 mM) LNP size and stability with various buffer preparations of malate acidifier

[0030] Figure 7. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate concentrations (values in mM, 0 - 10 mM)

[0031] Figure 8. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate concentrations and pH values

[0032] Figure 9. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate pH values and buffer compositions

[0033] Figure 10. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl with various intermediate LNP pH values

[0034] Figure 11. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various malate concentrations for in-line dilution

[0035] Figure 12. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl with water or malate at various pH values for in-line dilution

[0036] Figure 13. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various flow rate ratios and production concentrations

[0037] Figure 14. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various manufacturing concentrations and malate concentrations

[0038] Figure 15. Freeze-thaw stability over 5 cycles at -20 °C for large-scale manufactured LNP-Abl at various flow rates Figure 16. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl for various production concentrations, process steps and dialysis temperatures

[0039] Figure 17. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl at various dialysis temperatures

[0040] Figure 18. Freeze-thaw stability over 5 cycles at -20 °C for small-scale manufactured LNP-Abl with high malate concentrations

[0041] Figure 19. Freeze-thaw stability over 5 cycles at -20 °C for large-scale manufactured LNP-Abl with high malate concentrations

[0042] Figure 20. Freeze-thaw stability over 5 cycles at -20 °C for large-scale LNP-Abl manufactured with lower malate concentrations

[0043] Figure 21. Freeze-thaw stability over 5 cycles at -20 °C for LNP-Ab2 manufacturing with lower malate concentrations

[0044] Figure 22. Freeze-thaw stability over 5 cycles at -20 °C for large-scale or pilot scale manufactured LNP-Ab2 with various LNP concentrations

[0045] Detailed Description

[0046] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0047] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are 25 explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Bey er / W alter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0048] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0049] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0050] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Definitions

[0051] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0052] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of or "consisting of. Likewise, at each occurrence in the present application, the term "consisting essentially of may be replaced with the term "consisting of .

[0053] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0054] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0055] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. For example, with respect to a pH value, the term “about” may in preferred instances indicate deviation from the indicated numerical value by up to 0.3. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0056] The expression "substantially free of X", as used herein, means that the composition described herein is free of X in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Specific meanings of the term “substantially free” in relation to certain components of the composition are defined herein.

[0057] "Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0058] "Physiological conditions" as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and / or a temperature of about 37°C.

[0059] As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0060] As used in the present disclosure, "mol % of the lipid mixture" is defined as the ratio of the number of moles of that particular lipid component to the total number of moles of all lipids in the lipid mixture, multiplied by 100. In this context, in some embodiments, the term "total lipid" and / or “total lipid mixture” includes lipids and lipid-like material.

[0061] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2- dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, ndecyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n- tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n- nonadecyl, n-icosyl, n-triacontyl, n-tetracontyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).

[0062] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carboncarbon double bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carboncarbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1 -propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5- heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7- nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6- decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10- undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6- dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like.

[0063] The term “carboxylic acid” or “carboxylate” means a compound containing the functional group -CO2H. The term “carboxylate” is used when the group is deprotonated. Depending on the pH, the carboxylic acid may be protonated or deprotonated.

[0064] The term “dicarboxylic acid” or “di carb oxy late” means a compound containing two functional groups -CO2H. The term “di carb oxy late” is used when the group is deprotonated. Depending on the pH, the dicarboxylic acid may be fully protonated, mono-deprotonated, or di -deprotonated.

[0065] The term "ester" as used herein means a compound having the functional group -C(O)O- (including its isomerically arranged structure -OC(O)-, unless it is specified to the contrary).

[0066] “Halo” means fluoro (-F), chloro (-C1), bromo (-Br) or iodo (-1). “Amine” means the group -NR2, wherein each R is H or an organic group, such as an alkyl or alkenyl group (as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. When both groups R are hydrogen, the amine group is a primary amine group. When one R is hydrogen and the other R is other than hydrogen, the amine group is a secondary amine group. When both groups R are other than hydrogen, the amine group is a tertiary amine group.

[0067] A “quaternary ammonium” salt is a compound containing a group -N+R3, wherein each R is an organic group, such as an alkyl or alkenyl group (as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. In contrast to some amines as defined above which are protonated only at certain pH, a quaternary ammonium salt carries a constitutive positive charge (as defined herein) at all pH.

[0068] “Carbohydrate” means a compound having the empirical formula Cm(H20)n where m may or may not be different from n. The term “carbohydrate residue” or “carbohydrate moiety” defines a residue attached to another atom, where one hydrogen atom of the carbohydrate is replaced by a bond attached to the rest of the molecule. The carbohydrate moiety may be a monosaccharide moiety. The monosaccharide moiety may have the D- or L-configuration. Furthermore, the monosaccharide moiety may be an aldose or ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6, carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (i.e. it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose, and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0069] In another embodiment, the monosaccharide moiety is a pentose moiety (i.e. it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0070] In another embodiment, the carbohydrate may be a higher saccharide (i.e. a di-, or oligosaccharide) comprising more than one monosaccharide moiety joined together by glycoside bonds. When the monosaccharide moieties are hexose moieties, the glycoside bonds may be l-a,l'-a glycoside bonds, l,2'-gly coside bonds (which maybe l-a2’ or 1 '-P-2' glycoside bonds), l,3'-glycoside bonds (which may be l-a-3' or 1-P- 3 '-glycoside bonds), l,4'-gly coside bonds (which may be l-a-4' or l-P-4'-gly coside bonds), l,6'-gly coside bonds (which may be l-a-6' or l-P-6'-gly coside bonds), or any combination thereof. In one embodiment, the higher saccharide comprises 2 monosaccharide units (i.e. is a di saccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose and trehalulose.

[0071] The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between the CHI and CH2 domains of the heavy chain and is highly flexible. Disulphide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present disclosure is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(l):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). In general, CDRs described herein are Kabat defined. In some embodiments, an immunoglobulin is an antibody.

[0072] Throughout this document, a reference to a heavy chain (HC) or a light chain (LC) does not necessarily imply the presence of an entire heavy chain (HC) or a light chain (LC) but is used as shorthand to indicate the presence of at least a relevant or distinguishing portion of a heavy chain (HC) or a light chain (LC). For example, if a (Fab)-(scFv)2-based bispecific antibody has two chains and one comprises a variable region of a heavy chain (VH) derived from a parental immunoglobulin as well as a scFv, and the other chain comprises a variable region of a light chain (VL) derived from a parental immunoglobulin as well as a scFv, the two chains may respectively be referred to as the heavy chain (HC) and the light chain (LC). This can be the case even though neither of the chains in fact comprises a heavy or light chain, and both chains comprise a scFv, meaning that they both comprise elements derived from a parental heavy and a parental light chain.

[0073] The term “antibody” (Ab) in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to bind, preferably specifically bind to an antigen. In some embodiments, binding takes place under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term “antigen-binding region”, "binding region" or "binding domain", as used herein, refers to the region or domain which interacts with the antigen and typically comprises both a VH region and a VL region. The term antibody when used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g. three or more, different antigen-binding regions. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. As indicated above, the term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, z.e., retain the ability to specifically bind to the antigen, and antibody derivatives, i.e., constructs that are derived from an antibody. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full- length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(l l):484-90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l): l l l-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0074] The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain (VH and VL) of a traditional two chain antibody have been joined to form one chain. Optionally, a linker (usually a peptide) is inserted between the two chains to allow for proper folding and creation of an active binding site.

[0075] An antibody can possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgGl, than to other isotypes. Thus, e.g. an IgGl antibody of the disclosure may be a sequence variant of a naturally-occurring IgGl antibody, including variations in the constant regions.

[0076] In various embodiments, an antibody is an IgGl antibody, more particularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, K, 1), an IgG2a antibody (e.g. IgG2a, K, 1), an IgG2b antibody (e.g. IgG2b, K, 1), an IgG3 antibody (e.g. IgG3, K, 1) or an IgG4 antibody (e.g. IgG4, K, 1).

[0077] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

[0078] The term “chimeric antibody” as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. The terms “variable region” or “variable domain” as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin. Chimeric antibodies may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. The chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody according to the present disclosure may be performed by other methods than described herein.

[0079] The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non- human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non- human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0080] The term “human antibody” as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B- lymphocytes or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies can thus e.g. be generated using transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Accordingly, in some embodiments, a human antibody is obtained from a transgenic animal, such as a mouse or a rat, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced in the animal, but the final antibody sequence is the result of said human germline immunoglobulin sequences being further modified by somatic hypermutations and affinity maturation by the endogenous animal antibody machinery, see e.g. Mendez et al. 1997 Nat Genet. 15(2): 146-56. The term “full-length” when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g., the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody.

[0081] When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.

[0082] As used herein, the term “binding” or "capable of binding" in the context of the binding of a binding agent, e.g., an antibody, to a predetermined antigen or epitope typically refers to a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO'10M or less, or about 10'11M or even less, for instance, when determined using Bio-Layer Interferometry (BLI), when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the binding agent as the analyte or, when determined using a quartz crystal microbalance system using target-expressing cells as “ligand”. In some embodiments, the binding agent binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the binding agent, so that when the KD of the binding agent is very low (that is, the binding agent is highly specific), then the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.

[0083] The term “kd” (sec-1), as used herein, refers to the dissociation rate constant of a particular binding agent-antigen interaction. Said value is also referred to as the koir value. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular binding agent-antigen interaction.

[0084] The present disclosure also envisions binding agents comprising functional variants of the VL regions, VH regions, or one or more CDRs described herein. A functional variant of a VL, VH, or CDR used in the context of a binding agent still allows the binding agent to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or the specificity / selectivity of the “reference” or “parent” binding agent and in some cases, such a binding agent may be associated with greater affinity, selectivity and / or specificity than the parent binding agent. Such functional variants typically retain significant sequence identity to the parent sequence.

[0085] Exemplary variants include those which differ from VH and / or VL and / or CDR regions of the parent sequences mainly by conservative substitutions; for instance, up to 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0086] Functional variants of sequences described herein such as VL regions, or VH regions, or sequences having a certain degree of homology or identity to sequences described herein such as VL regions, or VH regions preferably comprise modifications or variations in the non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0087] A binding agent comprising variants of heavy and / or light chain variable regions sequences as described herein, e.g., comprising modifications in the CDRs and / or a certain degree of identity as described herein, may compete for binding to an antigen with another binding agent, e.g., a binding agent comprising heavy and light chain variable regions as described herein, or may have the specificity for an antigen of another binding agent, e.g., a binding agent comprising heavy and light chain variable regions as described herein. The term “specificity” as used herein is intended to have the following meaning unless contradicted by context. Two binding agents have the “same specificity” if they bind to the same antigen and the same epitope.

[0088] The term “competes” and “competition” may refer to the competition between a first binding agent and a second binding agent to the same antigen. It is well known to a person skilled in the art how to test for competition of binding agents such as antibodies for binding to a target antigen. An example of such a method is a so-called cross-competition assay, which may e.g. be performed as an ELISA or by flowcytometry. Alternatively, competition may be determined using biolayer interferometry.

[0089] Binding agents which compete for binding to a target antigen may bind different epitopes on the antigen, wherein the epitopes are so close to each other that a first binding agent binding to one epitope prevents binding of a second binding agent to the other epitope. In other situations, however, two different binding agents may bind the same epitope on the antigen and would compete for binding in a competition binding assay. Such binding agents binding to the same epitope are considered to have the same specificity herein. Thus, in some embodiments, binding agents binding to the same epitope are considered to bind to the same amino acids on the target molecule. That binding agents bind to the same epitope on a target antigen may be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to a person skilled in the art. Preferably, binding agents or binding domains binding to different epitopes are not competing with each other for binding to their respective epitopes.

[0090] As described above, various formats of antibodies have been described in the art. The binding agent of the disclosure can in principle comprise sequences of an antibody of any isotype. Exemplary isotypes are IgGl, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. In some embodiments, the sequences of a binding agent described herein such as CHI and CL are derived from an antibody of the IgGl isotype, for instance an IgGlK antibody. Preferably, each of the antigen-binding regions or domains comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said variable regions each comprise three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively. Furthermore, preferably, the binding agent described herein comprises a heavy chain constant regions (CH), and a light chain constant regions (CL).

[0091] The term “binding agent” in the context of the present disclosure refers to any agent capable of binding to one or more desired antigens. The term "binding agent" includes antibodies, antibody fragments, or any other binding protein, or any combination thereof. In some embodiments, the binding protein comprises antibody fragments such as Fab and scFv.

[0092] Naturally occurring antibodies are generally monospecific, i.e. they bind to a single antigen. The binding agents described herein may be at least bispecific or multispecific such as trispecific, tetraspecific and so on. In some embodiments, a binding agent described herein is be an artificial protein that is composed of fragments of two different antibodies (said fragments of two different antibodies forming three binding domains).

[0093] As used herein, a bispecific binding agent, in particular a bispecific protein, is a molecule that has two different binding specificities and thus may bind to two epitopes. Particularly, the term "bispecific binding agent " as used herein includes an antibody-derived molecule comprising three antigen-binding sites, a first binding site having affinity for a first epitope and a second and third binding site having binding affinity for a second epitope distinct from the first.

[0094] The term “bispecific” in the context of the present disclosure refers to an agent comprising two different antigen-binding regions binding to different epitopes, in particular different epitopes on different antigens.

[0095] "Multispecific binding agents" are molecules which have more than two different binding specificities. In some embodiments, a binding agent described herein is at least trivalent. As used herein, "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen binding sites or binding domains in a binding agent. Antigen binding sites binding to the same antigen may recognize the same epitope or different epitopes.

[0096] As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a nucleic acid encodes a polypeptide if transcription and / or translation of the nucleic acid produces the polypeptide in a cell or other biological system.

[0097] As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. In some embodiments, the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0098] The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'- UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5' end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3' end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence. Buffers

[0099] In some aspects, the methods of the present disclosure employ buffers.

[0100] As is known to the person skilled in the art, a buffer solution is a solution comprising, a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it.

[0101] As will be apparent to those skilled in the art, depending on the pH, the buffer will be present as a mixture of the neutral (including zwitterionic) molecule and salts thereof in which the buffer molecule is protonated or deprotonated. In view of this, all references in this specification to a particular buffer molecule include all possible salts thereof where acidic moieties are deprotonated and / or basic moieties are protonated.

[0102] In one embodiment, the buffer used in the methods of the present disclosure (e.g., in step (b)) is an acidic buffer. In one embodiment, the acidic buffer has a pKa of between about 2.5 and about 6.0. In one embodiment, the acidic buffer has a pKa of between about 3.0 and about 5.5. In one embodiment, the acidic buffer has a pKa of between about 4.0 and about 5.0.

[0103] In one embodiment, the buffer used in the methods of the present disclosure (e.g., in step (d)) is a neutral buffer. In one embodiment, the neutral buffer has a pKa of between about 6.0 and about 8.0. In one embodiment, the neutral buffer has a pKa of between about 6.5 and about 8.5. In one embodiment, the neutral buffer has a pKa of between about 7.5 and about 8.5.

[0104] It will be apparent to those skilled in the art that the buffer will include a mixture of the undissociated acid or base and salts of the acid or base with a suitable counter-ion. Any pharmaceutically acceptable counter-ion may be used provided that it is nontoxic and does not materially affect the efficacy of the composition. When the buffer contains an acidic moiety, the counter-ion may be any suitable inorganic or organic ion. Examples of suitable inorganic cations include alkali metal ions, including Li+, Na+, K+, Rb+, and Cs+, alkaline earth metal ions such as Mg2+and Ca2+, transition metal ions such as Fe2+, Ti2+, and Zn2+, p-block metal ions such as Al3+, and ammonium ion NH4+. Examples of suitable organic cations include primary, secondary, tertiary ammonium ions derived from protonation of a suitable primary, secondary, or tertiary amine as described above (e.g. diethylamine or triethylamine), quaternary ammonium ions as described above, and amino acids such as arginine, glycine, or lysine.

[0105] When the buffer comprises a basic moiety, the counter-ion may be any pharmaceutically acceptable counter-ion derived from any pharmaceutically acceptable acid. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrofluoride / fluoride, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinafoate salts.

[0106] Malate buffer

[0107] In one embodiment, the buffer used in the methods of the present disclosure is a malate buffer.

[0108] Malic acid is a dicarboxylic acid with a pKai of 3.51 and a pKa2 of 5.03 at 20°C. It will therefore be immediately apparent to those skilled in the art that, depending on the pH, a malate buffer will comprise a mixture of undissociated malic acid, malate mono-anion (in which either one of the carboxylic moieties may be deprotonated), and malate di-anion (in which both carboxylic acid moieties are deprotonated) in varying proportions.

[0109] As will be understood by those skilled in the art, the malate buffer also contains pharmaceutically acceptable inorganic or organic cations, which may be any of those as defined and exemplified above. Preferred cations include inorganic cations such as alkali metal ions, including Li+, Na+, K+, Rb+, and Cs+, and especially preferred is Na+.

[0110] In one embodiment, the malate buffer is a malic acid / sodium malate buffer. As will be immediately apparent to those skilled in the art, depending on the pH, the malic acid / sodium malate buffer will comprise a mixture of malic acid, monosodium malate, and disodium malate in varying proportions.

[0111] In one embodiment, the concentration of malate in the malate buffer is about 0.1 to about 1000 mM. In one embodiment, the concentration of malate in the malate buffer is about 1 to about 500 mM. In one embodiment, the concentration of malate in the malate buffer is about 10 to about 300 mM. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate dianion, the concentration being the total concentration of all of these malate species in the solution.

[0112] In one embodiment, the pH of the malate buffer is about 2.5 to about 5.5. In one embodiment, the pH of the malate buffer is about 3.0 to about 5.0. In one embodiment, the pH of the malate buffer is about 4.0 to about 5.0. In one embodiment, the pH of the malate buffer is about 3.5 to about 4.5. In one embodiment, the pH of the malate buffer is about 3.8 to about 4.2. In one embodiment, the pH of the malate buffer is about 3.9 to about 4.1. In one embodiment, the pH of the malate buffer is about 4.0.

[0113] In one embodiment, the malate buffer has a malate concentration of about 10 to about 40 mM and a pH of about 3.5 to about 5.5. In one embodiment, the malate buffer has a malate concentration of about 20 to about 30 mM and a pH of about 4.0 to about 5.0. In one embodiment, the malate buffer has a malate concentration of about 25 mM and a pH of about 4.0. In one embodiment, the malate buffer has a malate concentration of about 25 mM and a pH of about 4.5. In one embodiment, the malate buffer has a malate concentration of about 25 mM and a pH of about 5.0. Example malate buffers used in the present disclosure and combinations of malic acid and sodium salt used for buffer preparation are given below.

[0114] Tris buffer

[0115] In one embodiment, the buffer used in the methods of the present disclosure is a tris(hydroxymethyl)aminomethane (Tris, tromethamine) buffer.

[0116] As is known to the person skilled in the art, Tris is a primary amine whose conjugate acid has a pKaof 8.10 at 25°C. It will therefore be immediately apparent to those skilled in the art that, depending on the pH, a Tris buffer will comprise a mixture of the neutral, unprotonated amine and the conjugate acid (in which the amino group is protonated) in varying proportions.

[0117] As will be understood by those skilled in the art, the Tris buffer also contains pharmaceutically acceptable inorganic or organic anions, which may be any of those as defined and exemplified above. Suitable Tris buffers include Tris-HCl (Tris hydrochloride), Tris-EDTA (TE), Tris-buffered saline (TBS), Tris-acetate, Tris- acetate-EDTA (TAE), Tris-borate, and Tris-borate-EDTA (TBE). Preferred counterions include those derived from organic acids, more preferably the acetate.

[0118] In one embodiment, the concentration of Tris in the Tris buffer is about 1 to about 500 mM. In one embodiment, the concentration of Tris in the Tris buffer is about 2 to about 250 mM. In one embodiment, the concentration of Tris in the Tris buffer is about 5 to about 100 mM. In one embodiment, the concentration of Tris in the Tris buffer is about 10 mM to about 50 mM. In one embodiment, the concentration of Tris in the Tris buffer is about 10 mM. In one embodiment, the concentration of Tris in the Tris buffer is about 50 mM. In this context, it will be understood by the person skilled in the art that the concentration expressed is the total concentration of both neutral Tris and its conjugate acid in the solution.

[0119] In one embodiment, the pH of the Tris buffer is about 6.5 to about 8.5. In one embodiment, the pH of the Tris buffer is about 7.0 to about 8.0. In one embodiment, the pH of the Tris buffer is about 7.2 to about 7.6. In one embodiment, the pH of the Tris buffer is about 7.3 to about 7.5. In one embodiment, the pH of the Tris buffer is about 7.4. In one embodiment, the pH of the Tris buffer is about 7.5 to about 8.5. In one embodiment, the pH of the Tris buffer is about 7.8 to about 8.2. In one embodiment, the pH of the Tris buffer is about 7.9 to about 8.1. In one embodiment, the pH of the Tris buffer is about 8.0.

[0120] Water

[0121] In some embodiments, the methods of the present invention use water as the aqueous phase, and / or use aqueous solutions comprising water. In some embodiments, as described herein, certain steps of the present invention, in particular the dilution step (c’), also uses water as the aqueous phase (e.g., as the diluent).

[0122] In some embodiments, the aqueous phase comprises water, as defined generally herein. In some embodiments, the aqueous phase consists essentially (as defined generally herein, in either its broadest aspect or a preferred aspect) of water, as defined generally herein. In some embodiments, the aqueous phase consists of water, as defined generally herein.

[0123] In one embodiment, the water is substantially free (as defined generally herein, in either its broadest aspect or a preferred aspect) of impurities.

[0124] In one embodiment, the water is substantially free (as defined generally herein, in either its broadest aspect or a preferred aspect) of ribonuclease (RNAse, as defined generally herein, in either its broadest aspect or a preferred aspect). In one embodiment, the water contains less than 10 pg / mL of RNAse, such as less than 1 pg / mL of RNAse. In one embodiment, the water contains less than 0.5 pg / mL. In one embodiment, the water is RNAse-free, i.e., does not contain RNAse.

[0125] In one embodiment, the water has a pH of from about 5.0 to about 7.0. In one embodiment, the water has a pH of from about 5.2 to about 6.5. In one embodiment, the water has a pH of from about 5.3 to about 6.0. In one embodiment, the water has a pH of from about 5.4 to about 5.7. In one embodiment, the water has a pH of from about 5.4 to about 5.6. In one embodiment, the water has a pH of from about 5.45 to about 5.55. In one embodiment, the water has a pH of about 5.5. In one embodiment, the water has a pH of from about 6.0 to about 8.0. In one embodiment, the water has a pH of from about 6.5 to about 7.5. In one embodiment, the water has a pH of from about 6.6 to about 7.4. In one embodiment, the water has a pH of from about 6.7 to about 7.3. In one embodiment, the water has a pH of from about 6.8 to about 7.2. In one embodiment, the water has a pH of from about 6.9 to about 7.1. In one embodiment, the water has a pH of from about 6.95 to about 7.05. In one embodiment, the water has a pH of from about 6.96 to about 7.04. In one embodiment, the water has a pH of from about 6.97 to about 7.03. In one embodiment, the water has a pH of from about 6.98 to about 7.02. In one embodiment, the water has a pH of from about 6.99 to about 7.01. In one embodiment, the water has a pH of about 7.0.

[0126] In one embodiment, the water comprises purified water. As is known to the person skilled in the art, purified water is water that has been mechanically filtered or processed to remove impurities. Typical processes used to purify water include distillation, deionization, vapour compression, reverse osmosis, carbon filtering, microfiltration, ultrafiltration, ultraviolet oxidation, or electrodeionization. Combinations of a number of these processes can be used to produce ultrapure water, as defined generally herein.

[0127] Typical impurities that may need to be removed in order to purify water include inorganic ions (typically monitored as electrical conductivity or resistivity or specific tests), organic compounds (typically monitored as TOC or by specific tests), bacteria (typically monitored by total viable counts or epifluorescence), endotoxins and nucleases (typically monitored by LAL or specific enzyme tests), particulates (typically controlled by filtration) and gases (typically managed by degassing when required).

[0128] In one embodiment, the water comprises distilled water. In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of distilled water. In one embodiment, the water consists of distilled water. As is known to the person skilled in the art, distillation involves boiling the water and then condensing the vapour into a clean container, leaving solid contaminants behind.

[0129] In one embodiment, the water comprises double distilled water. In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of double distilled water. In one embodiment, the water consists of double distilled water. As is known to the person skilled in the art, double-distilled water is prepared by slow boiling the uncontaminated condensed water vapour from a prior slow boiling.

[0130] In one embodiment, the water comprises deionized water. In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of deionized water. In one embodiment, the water consists of deionized water. As is known to the person skilled in the art, deionized water is water that has had substantially all of its mineral ions removed. Deionization is a chemical process that typically uses ion-exchange resins, which exchange hydrogen and hydroxide ions for other dissolved ions, and then recombine to form water. Three types of deionization are generally known in the art: co-current, counter-current, and mixed bed. Co- current deionization is a downflow process where both input water and regeneration chemicals enter at the top of an ion-exchange column and exit at the bottom. Countercurrent deionization comes in two forms, each requiring engineered internals. The first type comprises upflow columns where input water enters from the bottom and regenerants enter from the top of the ion exchange column. The second type comprises upflow regeneration where water enters from the top and regenerants enter from the bottom. Mixed bed deionization is a 40 / 60 mixture of cation and anion resin combined in a single ion-exchange column. In one embodiment, the water comprises ultrapure water. In one embodiment, the water comprises Type 1 ultrapure water (such as e.g., MilliQ water). In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of ultrapure water. In one embodiment, the water consists of ultrapure water. As is known to the person skilled in the art, ultrapure water is water of such high purity that its trace contaminants are typically measured in parts per billion (ppb) or parts per trillion (ppt).

[0131] In one embodiment, the water comprises Water for Injection (WFI). In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of Water for Injection (WFI). In one embodiment, the water consists of Water for Injection (WFI). As is known to the person skilled in the art, Water for Injection (WFI) is sterile, hypotonic, non-pyrogenic, and contains no bacteriostatic or antimicrobial agents. Water for injection is described in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169 and in US Pharmacopoeia (USP) monograph no. 1231 “Water for Pharmaceutical Purposes”, incorporated herein by reference.

[0132] In one embodiment, the water comprises water having an equivalent or higher level of purity as, or complying with the standard of, Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169 and / or in US Pharmacopoeia (USP) monograph no. 1231. In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of water having an equivalent or higher level of purity as, or complying with the standard of, Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169, and / or in US Pharmacopoeia (USP) monograph no. 1231. In one embodiment, the water consists of water having an equivalent or higher level of purity as, or complying with the standard of, Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169, and / or in US Pharmacopoeia (USP) monograph no. 1231.

[0133] In one embodiment, the water comprises water that has been produced by distillation or an equivalent purification process (such as reverse osmosis, coupled with electrodeionisation, ultrafiltration or nanofiltration) to Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169, and / or in US Pharmacopoeia (USP) monograph no. 1231. In one embodiment, the water consists essentially (as defined above, either in its broadest aspect or a preferred aspect) of water that has been produced by distillation or an equivalent purification process (such as reverse osmosis, coupled with electro-deionisation, ultrafiltration or nanofiltration) to Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169, and / or in US Pharmacopoeia (USP) monograph no. 1231. In one embodiment, the water is water that has been produced by distillation or an equivalent purification process (such as reverse osmosis, coupled with electro-deionisation, ultrafiltration or nanofiltration) to Water for Injection (WFI), as defined in the European Pharmacopoeia (Ph. Eur.) 11.4, monograph no. 0169, and / or in US Pharmacopoeia (USP) monograph no. 1231.

[0134] Cryoprotectants

[0135] In one embodiment, the composition containing nucleic acid-lipid particles according to the present disclosure contains a cryoprotectant. In this specification, the term “cryoprotectant” when used in its broadest sense means any substance capable of protecting a composition from damage caused by freezing temperatures, such as those below 0°C. Examples of cryoprotectants include glycols (i.e. alcohols containing at least two hydroxy groups, such as glycerol and propylene glycol) and carbohydrates, as defined and exemplified herein.

[0136] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, lactose and glucose, or a mixture of any thereof. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose and glucose, or a mixture of any thereof. Preferably, the cryoprotectant is sucrose.

[0137] In one embodiment, the cryoprotectant is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 3% to about 25% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 8% to about 22% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the cryoprotectant is present in a concentration of 8% to about 12% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 10% (w / v). In one embodiment, the cryoprotectant present in a concentration of about 15% to about 25% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 18% to about 12% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 20% (w / v).

[0138] Payload

[0139] The lipid particle compositions of the present application contain a payload. The payload is a nucleic acid, as defined and exemplified herein. In some embodiments, the nucleic acid is RNA, such as mRNA. The nucleic acid (e.g., mRNA) payload may encode any polypeptide, such as any therapeutic polypeptide. The buffer conditions described herein provide enhanced stability of the lipid particles composition for any nucleic acid (e.g., mRNA) payload, e.g., irrespective of the nucleic acid sequence. In some embodiments, the nucleic acid (e.g. RNA) encodes a binding agent, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific binding agent, as defined and exemplified herein. In some embodiments, the nucleic acid (e.g. RNA) encodes an antibody, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific antibody, as defined and exemplified herein. In some embodiments, the nucleic acid comprises one or more (such as two) RNA (e.g., mRNA) species. In some embodiments, the lipid particle composition comprises two RNA (e.g., mRNA) species, each encoding one chain of an antibody, as described herein. In some embodiments, the lipid particle composition comprises as a payload a first RNA molecule and a second RNA molecule, as described herein.

[0140] Nucleic Acid

[0141] The lipid particle compositions of the present application contain a nucleic acid. Preferably the lipid particle compositions of the present application contain RNA, such as mRNA. Typically, the lipid particle compositions described herein comprise lipid particles that encapsulate the nucleic acid. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA.

[0142] A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0143] RNA

[0144] In some embodiments of all aspects of the disclosure, the nucleic acid is RNA (such as mRNA). According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acids adenosine (A), uridine (U), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The nucleic acid may be mRNA, saRNA, taRNA, or mixtures thereof. The nucleic acid is preferably mRNA. In some instances, the nucleic acid is not siRNA.

[0145] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may capable of or configured to express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and capable of or configured for expressing a pharmaceutically active peptide or protein. Alternatively, the RNA can be non-coding RNA such as antisense- RNA, micro RNA (miRNA) or siRNA. mRNA

[0146] In preferred embodiments of all aspects of the disclosure, the nucleic acid is mRNA. According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. An mRNA may comprise a 5’cap, a protein coding region, and a 3'-poly-A tail. The 3’poly-A tail may typically comprise 100-150 A nucleotides, which may be interrupted by one or more non-A sequences. An mRNA may comprise a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region and a 3' untranslated region (3'-UTR). Typically, an mRNA may comprise a 5’cap, a 5'-UTR, a protein coding region, a 3'- UTR and a poly- A tail. mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0147] In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0148] In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The term "in vitro transcription" or "IVT" as used herein means that the transcription (z.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™).

[0149] For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription. The RNA (such as mRNA) may be modified. The RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly). One or more uridine in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. The RNA may comprise a modified nucleoside in place of at least one uridine. Preferably, the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside). The modified nucleoside may be independently selected from pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (\| / ) or Nl-methyl-pseudouridine (mly).

[0150] In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0151] DNA

[0152] In some embodiments of all aspects of the disclosure, the nucleic acid is DNA. Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" represents "deoxy"). In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0153] Nucleic acids encoding binding agents and antibodies

[0154] In some embodiments, the nucleic acid (e.g. RNA) encodes a binding agent, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific binding agent, as defined and exemplified herein. In some embodiments, the nucleic acid (e.g. RNA) encodes an antibody, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific antibody, as defined and exemplified herein.

[0155] Anti-claudin-18.2 antibody

[0156] In some embodiments, the antibody is encoded on a first nucleic acid molecule and a second nucleic acid molecule. For example, the heavy chain of an antibody may be encoded on a first nucleic acid molecule (e.g. a first RNA molecule), and the light chain of an antibody may be encoded by a second nucleic acid molecule (e.g. a second RNA molecule).

[0157] In some embodiments, the nucleic acid encodes an antibody that specifically binds to Claudin-18.2 (CLDN-18.2). In some embodiments, the antibody specifically binds to a first extracellular domain (ECD1) of a CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an epitope of ECD 1 that is exposed in cancer cells.

[0158] Exemplary sequences of CLDN-18.2 (SEQ ID NO: 1) and the splice variant

[0159] CLDN18.1 (SEQ ID NO: 2) are shown below:

[0160] In some embodiments, such an antibody agent may have a binding affinity e.g., as measured by a dissociation constant) for a CLDN-18.2 polypeptide, e.g., an epitope of ECD1 of a CLDN-18.2 polypeptide) of at least about 10'4M, at least about 10'5M, at least about 10'6M, at least about 10'7M, at least about 10'8M, at least about 10'9M, or lower. Those skilled in the art will be familiar with a variety of technologies for measuring binding affinity and / or dissociation constants in accordance with the present disclosure, including, e.g., but not limited to ELIS As, gel-shift assays, pulldown assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), bio-layer interferometry, grating-coupled interferometry, and spectroscopic assays.

[0161] In some embodiments, an antibody targeting CLDN-18.2 may bind specifically to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide. In some embodiments, an antibody targeting CLDN-18.2 does not bind to any other claudin family member including the closely related splice variant 1 of Claudin-18 (CLDN18.1) that is predominantly expressed in tissues, e.g., lung.

[0162] In some embodiments, an antibody agent targeting CLDN-18.2 may be any one of CLDN-18.2-targeting antibodies described in W02007 / 059997, WO2008 / 145338, and W02013 / 174510, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.

[0163] In some embodiments, an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues (GYTFTSYW) (SEQ ID NO: 5); (ii) CDR2 represented by amino acid residues (IYPSDSYT) (SEQ ID NO: 6); and (iii) CDR3 represented by amino acid residues (TRSWRGNSFDY) (SEQ ID NO: 7); and / or (b) a variable light chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of (i) CDR1 represented by amino acid residues (QSLLNSGNQKNY) (SEQ ID NO: 8); (ii) CDR2 represented by amino acid residues (WAS) (SEQ ID NO: 9); and (iii) CDR3 represented by amino acid residues (QNDYSYPFT) (SEQ ID NO: 10).

[0164] In some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising an amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO: 3 as set forth below (wherein the underlined amino acid sequence corresponds to a secretion signal sequence); and a light chain consisting of or comprising an amino acid represented by amino acid residues 27-246 of SEQ ID NO: 4 as set forth below (wherein the underlined amino acid sequence corresponds to a secretion signal sequence).

[0165] In some embodiments, the payload comprises (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 11, or a nucleotide sequence having at least 80%, at least 85%, at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 11, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 12, or a nucleotide sequence having at least 80%, at least 85%, at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 12.

[0166] In some embodiments, the payload comprises (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 85%, at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.

[0167] In some embodiments, a provided RNA can comprise a nucleotide sequence that encodes a 5’UTR of interest and / or a 3’ UTR of interest. One of skill in the art will appreciate that untranslated regions (e.g., 3’ UTR and / or 5’ UTR) of a mRNA sequence can contribute to mRNA stability, mRNA localization, and / or translational efficiency.

[0168] In some embodiments, the payload comprises (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the RNA, e.g., each RNA, comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 13 or 15, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 13 or 15 and / or a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 14 or 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 14 or 16.

[0169] In some embodiments, the RNA, e.g., each RNA, comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 13 or 15, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 13 or 15 and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 14 or 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 14 or 16.

[0170] In some embodiments, the RNA, e.g., each RNA, comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 13, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 14.

[0171] In some embodiments, the RNA, e.g., each RNA, comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 15, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 16.

[0172] In some embodiments, the coding region under (i) comprises the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 11, and the coding region under (ii) comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 12.

[0173] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

[0174] In some embodiments, the RNA under (i) is a first RNA molecule and the RNA under (ii) is a second RNA molecule.

[0175] In some embodiments, the RNA, e.g., each RNA, comprises a poly-A sequence. Preferably, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 18.

[0176] In some embodiments, the payload comprises (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 19 or 21, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 19 or 21, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 20 or 22, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20 or 22. In some embodiments, the payload comprises (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 19, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the payload comprises (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 21, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 22.

[0177]

[0178]

[0179]

[0180]

[0181] Anti-CD3 / CLDN6 bispecific antibody

[0182] In some embodiments, the nucleic acid (e.g. RNA) encodes a bispecific binding agent. In some embodiments, the binding agent is bispecific for CD3 and CLDN6, i.e., it is capable of binding to an epitope of CD3 and an epitope of CLDN6.

[0183] The term "claudin 6" or "CLDN6" preferably relates to human CLDN6, and, in particular, to a protein comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24 or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80 or 29 to 81, more preferably amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24. Said first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0184] The CD3 (cluster of differentiation 3) complex is a T cell-specific antigen. A T cellspecific antigen is an antigen on the surface of T cells.

[0185] The human CD3 epsilon is indicated in GenBank Accession No. NM 000733 and comprises SEQ ID NO: 25. As used herein, "CD3" includes human CD3 and denotes an antigen that is expressed on human T cells as part of the multimolecular T-cell receptor complex.

[0186] In some embodiments, the binding agent described herein recognizes the epsilonchain of CD3, in particular, it recognizes an epitope that corresponds to the first 27 N- terminal amino acids of CD3 epsilon or functional fragments of this 27 amino acid stretch.

[0187] In some embodiments, a binding agent described herein has at least one antigen binding site or binding domain for CD3 and at least two antigen binding sites or binding domains for CLDN6.

[0188] In some embodiments, the binding agent described herein is in the format of a Fab- SCFV2 construct, i.e., a Fab fragment specific for CD3 is provided with two scFv fragments specific for CLDN6 at the C-terminus of the constant regions of the Fab fragment.

[0189] In some embodiments, the binding agent is a dimer composed of two polypeptide chains preferably bound together by a disulfide bridge, in which the first polypeptide comprises an scFv linked to an additional VH domain through a CHI polypeptide chain, and the second polypeptide comprises an scFv linked to an additional VL domain through a CL polypeptide chain. The disulfide bridge is preferably formed between a Cys residue in the CHI and a Cys residue in the CL, such that the additional VH of the first polypeptide associates with the additional VL of the second polypeptide in an antigen-binding configuration, such that the binding agent as a whole includes three antigen-binding domains. Thus, in some embodiments, the binding agent comprises the heavy chain (Fd fragment) and light chain (L) of a Fab fragment which are able to heterodimerize and upon which scFv binding domains are incorporated (preferably at the C-terminus of Fd / L). In some embodiments, the VH and VL domains in the scFv moieties are connected by peptide linkers and / or the Fab chains and the scFv are connected by peptide linkers.

[0190] In some embodiments of the disclosure, a binding agent described herein does not comprise a full-length antibody. In some embodiments of the disclosure, a binding agent described herein does not comprise CH2 and CH3 domains of an antibody. In some embodiments of the disclosure, a binding agent described herein does not comprise a Fc region. In some embodiments of the disclosure, a binding agent described herein does not comprise Fc sequences which are able of exerting effectorfunctions.

[0191] In some embodiments, the two polypeptide chains are encoded by two RNA molecules.

[0192] In some embodiments, a binding agent targeting CD3 comprises a variable heavy chain domain comprising a CDR1 comprising the amino acid sequence GYTFTRYT (SEQ ID NO: 33) or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT (SEQ ID NO: 34) or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYSLDY (SEQ ID NO: 35) or a functional variant thereof.

[0193] In some embodiments, a binding agent targeting CD3 comprises a variable light chain domain comprising CDR1 comprising the amino acid sequence SSVSY (SEQ ID NO: 37) or a functional variant thereof, a CDR2 comprising the amino acid sequence DTS (SEQ ID NO: 38) or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQWSSNPLT (SEQ ID NO: 39) or a functional variant thereof.

[0194] In some embodiments, a binding agent targeting CLDN6 comprises a variable heavy chain domain comprising a CDR1 comprising the amino acid sequence GYSFTGYT (SEQ ID NO: 40) or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT (SEQ ID NO: 41) or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY (SEQ ID NO: 42) or a functional variant thereof. In some embodiments, a binding agent targeting CLDN6 comprises a variable light chain domain comprising a CDR1 comprising the amino acid sequence SSVSY (SEQ ID NO: 43) or a functional variant thereof, a CDR2 comprising the amino acid sequence STS (SEQ ID NO: 44) or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT (SEQ ID NO: 45) or a functional variant thereof.

[0195] In some embodiments, the payload comprises (i) a first RNA encoding a first polypeptide chain comprising a variable region of a heavy chain (VH) derived from an immunoglobulin with specificity for CD3 (VH(CD3)), a variable region of a heavy chain (VH) derived from an immunoglobulin with specificity for CLDN6 (VH(CLDN6)) and a variable region of a light chain (VL) derived from an immunoglobulin with specificity for CLDN6 (VL(CLDN6)); and (ii) a second RNA encoding a second polypeptide chain comprising a variable region of a light chain (VL) derived from an immunoglobulin with specificity for CD3 (VL(CD3)), a variable region of a heavy chain (VH) derived from an immunoglobulin with specificity for CLDN6 (VH(CLDN6)) and a variable region of a light chain (VL) derived from an immunoglobulin with specificity for CLDN6 (VL(CLDN6)).

[0196] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form a binding domain with specificity for CD3 and two binding domains with specificity for CLDN6. In some embodiments, the VH(CD3) of the first polypeptide chain and the VL(CD3) of the second polypeptide chain interact to form a binding domain with specificity for CD3, the VH(CLDN6) and the VL(CLDN6) of the first polypeptide chain interact to form a binding domain with specificity for CLDN6, and the VH(CLDN6) and the VL(CLDN6) of the second polypeptide chain interact to form a binding domain with specificity for CLDN6.

[0197] In some embodiments, the first and the second polypeptide chains comprise a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof and a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

[0198] In some embodiments, the immunoglobulin is IgGl, preferably the IgGl is human IgGl.

[0199] In some embodiments, the VH, the VL, and the CHI on the first polypeptide chain are arranged, from N-terminus to C-terminus, in the order VH(CD3)-CH1-VH(CLDN6)-VL(CLDN6), or VH(CD3)-CH1-VL(CLDN6)-VH(CLDN6).

[0200] In some embodiments, the CHI is connected to the VH(CLDN6) or VL(CLDN6) by a peptide linker. Preferably, the peptide linker comprises the amino acid sequence SGPGGGRS(G4S)2 (SEQ ID NO: 31) or a functional variant thereof.

[0201] In some embodiments, the VH, the VL, and the CL on the second polypeptide chain are arranged, from N-terminus to C-terminus, in the order VL(CD3)-CL-VH(CLDN6)-VL(CLDN6), or VL(CD3)-CL-VL(CLDN6)-VH(CLDN6).

[0202] In some embodiments, the CL is connected to the VH(CLDN6) or VL(CLDN6) by a peptide linker. Preferably, the peptide linker comprises the amino acid sequence DVPGGS (SEQ ID NO: 32) or a functional variant thereof. In some embodiments, the VH(CLDN6) and the VL(CLDN6) are connected to one another by a peptide linker. Preferably, the peptide linker comprises the amino acid sequence (G4S)Xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. More preferably, the peptide linker comprises the amino acid sequence (G4S)4 (SEQ ID NO: 28) or a functional variant thereof.

[0203] In some embodiments, the CHI on the first polypeptide chain interacts with the CL on the second polypeptide chain.

[0204] In some embodiments, the VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 46.

[0205] In some embodiments, the VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 47. In some embodiments, the VH(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 46.

[0206] In some embodiments, the VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 46 and preferably a serine residue in position +15 relative to CDR1 and / or a serine residue in position -3 relative to CDR2. In some embodiments, the VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 46, the VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 47, the VH(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 46, and the VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 46 and preferably the VL(CLDN6) comprises a serine residue in position +15 relative to CDR1 and / or a serine residue in position -3 relative to CDR2.

[0207] In some embodiments, the VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 46 or a functional variant thereof, the VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 47 or a functional variant thereof, the VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 46 or a functional variant thereof, and / or the VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 46 or a functional variant thereof.

[0208] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46 or a functional variant thereof.

[0209] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47 or a functional variant thereof.

[0210] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46 or a functional variant thereof and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47 or a functional variant thereof.

[0211] In some embodiments, at least one RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 48, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, each RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 48, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 48.

[0212] In some embodiments, at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17.

[0213] In some embodiments, each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17.

[0214] In some embodiments, at least one RNA comprises a poly-A sequence. In some embodiments, each RNA comprises a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. Preferably, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 18.

[0215] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 46; and / or the first RNA comprises the nucleotide sequence of SEQ ID NO: 49, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 49.

[0216] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 47; and / or the second RNA comprises the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 50.

[0217] In some embodiments, the payload comprises: (i) a first RNA encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 46; and

[0218] (ii) a second RNA encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 47.

[0219] In some embodiments, the first RNA comprises the nucleotide sequence of SEQ ID NO: 49, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 49.

[0220] In some embodiments, the second RNA comprises the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 50.

[0221]

[0222]

[0223] Nucleic Acid-Lipid Particle and Compositions containing it

[0224] The present disclosure further provides a composition comprising a lipid particle comprising a lipid mixture, as defined herein, and a nucleic acid. In one embodiment, there is provided a composition comprising a lipid particle obtained or obtainable by the methods defined herein. The lipid particles are loaded with a payload which is a nucleic acid, as defined herein. Such particles are also referred to herein as “nucleic acid-lipid particles” or “loaded lipid-particles”. When the nucleic acid is RNA, such particles are also referred to herein as “RNA-lipid particles”.

[0225] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In some embodiments, the nucleic acid (e.g. RNA) encodes a binding agent, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific binding agent, as defined and exemplified herein. In some embodiments, the nucleic acid (e.g. RNA) encodes an antibody, as defined and exemplified herein. In some embodiments, the binding agent is a bispecific antibody, as defined and exemplified herein.

[0226] In one embodiment of the present disclosure, the nucleic acid-lipid particle is a lipid nanoparticle (LNP). The function of the LNP is to stabilise and encapsulate the nucleic acid to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol. The LNPs and / or their lipid components may have adjuvant activity.

[0227] In the present disclosure, LNPs may be understood as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. LNPs thus typically comprise a central complex of the nucleic acid, such as RNA (e.g. mRNA) and lipid embedded in a disordered, non- lamellar phase made of lipid.

[0228] Nucleic acid-lipid nanoparticles are obtainable from combining a nucleic acid with lipids according to the methods defined herein. The lipids used for LNP formation typically do not form lamellar (bilayer) phases in water under physiological conditions. The LNPs typically do not comprise or encapsulate an aqueous core, in contrast to liposomes. The LNPs typically comprise a lipidic (or oily) core.

[0229] In some embodiments, the nucleic acid-lipid nanoparticles described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm. In some embodiments, the average diameter of the nucleic acid-lipid nanoparticles is about 30 nm to about 150 nm, about 40 nm to about 120 nm, about 50 nm to about 100 nm, or about 60 nm to about 90 nm. In some embodiments, the average diameter of the nucleic acid-lipid nanoparticles is about 40 nm to about 120 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using an appropriate algorithm (e.g., the so-called cumulant algorithm for monodisperse samples), which provides as results the so-called Z- average with the dimension of a length, and the poly dispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0230] In some embodiments, the average diameter of the nucleic acid-lipid nanoparticles is from 50 nm to about 110 nm. In some embodiments, the average diameter of the nucleic acid-lipid nanoparticles is from 60 nm to about 100 nm. In some embodiments, the average diameter of the nucleic acid-lipid nanoparticles is from 70 nm to about 90 nm.

[0231] The composition of the present disclosure comprises an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the nucleic acid-lipid particles, such as nucleic acid-lipid nanoparticles, comprising the dispersed phase. Carrying out the method of the present disclosure results in an aqueous dispersion containing nucleic acid-lipid particles, as described and exemplified herein.

[0232] In this specification the term “dispersion” in its broadest sense takes its usual meaning in chemistry as a system in which distributed particles of one material (the “dispersed phase”) are dispersed in a phase of another material (the “continuous phase” or the “mobile phase”). In one embodiment, the mobile phase is a solution, typically an aqueous solution. The term “solution” as used herein is a homogeneous mixture comprising a solvent which is typically water and solutes which can be salts, buffers, tonifiers and the like, as long as these materials are molecularly distributed within the solvent. The mobile phase may comprise solutes, as described further herein.

[0233] In one embodiment, the composition containing the nucleic acid-lipid particles contains Tris buffer, as defined herein. In one embodiment, the concentration of Tris in the composition is about 1 to about 500 mM. In one embodiment, the concentration of Tris in the composition is about 2 to about 250 mM. In one embodiment, the concentration of Tris in the composition is about 5 to about 100 mM. In one embodiment, the concentration of Tris in the composition is about 10 mM to about 50 mM. In one embodiment, the concentration of Tris in the composition is about 5 mM to about 25 mM. In one embodiment, the concentration of Tris in the composition is about 5 mM to about 15 mM. In one embodiment, the concentration of Tris in the composition is about 8 mM to about 12 mM. In this context, it will be understood by the person skilled in the art that the concentration expressed is the total concentration of both neutral Tris and its conjugate acid in the composition.

[0234] In one embodiment, the intermediate composition containing the nucleic acid-lipid particles resulting from step (c) as defined herein contains malate buffer, as defined herein. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c) as defined herein is from about 50 to about 400 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c) as defined herein is from about 100 to about 200 mM. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate di-anion, the concentration being the total concentration of all of these malate species in the solution.

[0235] In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 1 to about 200 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 2 to about 100 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 5 to about 50 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 10 to about 40 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 20 to about 30 mM. In one embodiment, the concentration of malate in the intermediate composition resulting from step (c’) as defined herein, when carried out, is about 25 mM. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate di-anion, the concentration being the total concentration of all of these malate species in the solution.

[0236] In one embodiment, the final composition containing the nucleic acid-lipid particles contains only a residual amount of malate buffer, for example as remaining from the earlier manufacturing steps. In one embodiment, the final composition containing the nucleic acid-lipid particles is substantially free of malate buffer. In one embodiment, the nucleic acid-lipid particles in the final composition contain a residual amount of malate buffer. In one embodiment, the final composition containing the nucleic acid- lipid particles comprises less than 100 mM malate buffer, such as less than 50 mM, less than 25 mM, less than 10 mM, less than 7.5 mM, less than 5 mM, less than 2.5 mM, less than 2 mM or less than 1 mM. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 25 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 10 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 5 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 2 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than or equal to 1 mM malate buffer. In one embodiment, the final composition containing the nucleic acid- lipid particles comprises less than or equal to 200 pg / L malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 100 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 50 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 20 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 10 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 5 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 2 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises less than 1 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 0.1 nM to about 10 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 0.5 nM to about 1 mM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 1 nM to about 50 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 1 nM to about 20 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 1 nM to about 10 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 1 nM to about 5 pM malate buffer. In one embodiment, the final composition containing the nucleic acid- lipid particles comprises from about 1 nM to about 2 pM malate buffer. In one embodiment, the final composition containing the nucleic acid-lipid particles comprises from about 1 nM to about 1 pM malate buffer. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate di-anion, the concentration being the total concentration of all of these malate species in the solution.

[0237] The level of malate or malate buffer may be quantified using assays and methods known in the art, for example using colorimetric assays based on enzymatic reactions followed by read-out measuring absorbance or fluorescence, such as the “ab83391 Malate Assay Kit” from Abeam, or similar (see, e.g., Shapiro & Silanikove, 2011, Food Chemistry, 129(2):608-613). Alternatively, for higher sensitivity, the level of malate or malate buffer may be quantified using gas chromatography (GC) with a flame ionization detector (FID), GC-mass spectrometry (MS), or nuclear magnetic resonance (NMR), using methods well known to the skilled person (see, e.g., Gout, E., et al., 1993, J. Biol. Chem., 268(6):3986-3992). In the compositions of the invention it may be necessary to disrupt the nucleic acid-lipid particles (for example using detergent or organic solvents) in order to quantify the residual malate.

[0238] In one embodiment, the composition comprising the nucleic acid-lipid particles has a pH of about 6.0 to about 8.0. In one embodiment, the composition comprising the nucleic acid-lipid particles has a pH of about 7.0 to about 8.0. In one embodiment, the composition comprising the nucleic acid-lipid particles has a pH of about 7.0 to about 7.5. In one embodiment, the composition comprising the nucleic acid-lipid particles has a pH of about 7.1 to about 7.4.

[0239] In one embodiment, the composition has a pH of about 7.0 to about 8.0 and comprises:

[0240] (a) the nucleic acid-lipid particle; and

[0241] (b) Tris buffer, at a concentration of about 5 to about 100 mM.

[0242] Method of Forming Nucleic Acid-Lipid Particle Composition

[0243] The present disclosure also provides a method of producing a composition containing a nucleic acid-lipid particle as defined herein.

[0244] The method according to the present disclosure comprises the steps (a) to (d):

[0245] (a) preparing a first mixture which is a lipid mixture comprising a cationically ionizable lipid in a water-soluble organic solvent;

[0246] (b) preparing a second mixture in aqueous solution, the second mixture comprising (i) a nucleic acid and (ii) malate buffer;

[0247] (c) mixing the first mixture with the second mixture to produce an intermediate composition comprising the nucleic acid-lipid particle; and

[0248] (d) further processing of the intermediate composition by buffer exchange, wherein the buffer used in the buffer exchange comprises Tris and / or a pharmaceutically acceptable salt thereof, to produce the composition comprising the nucleic acid-lipid particle. In one embodiment, the method also comprises the step (c’) of diluting the intermediate composition as defined herein.

[0249] In one embodiment, the method also comprises one or more further processing steps (e) as defined herein.

[0250] Each of the above steps (a) to (d) are defined in more detail herein.

[0251] Scale

[0252] The methods of the present invention may be carried out at a variety of scales. Typically, the scale at which the methods are carried out is defined by the input of nucleic acid, such as RNA, in particular mRNA, into the relevant step. As detailed below, this step is typically step (b).

[0253] In one embodiment, the method of the present invention is carried out using 1 to 100 mg nucleic acid. In one embodiment, the method of the present invention is carried out using 2 to 50 mg nucleic acid. In one embodiment, the method of the present invention is carried out using 5 to 20 mg nucleic acid.

[0254] In one embodiment, the method of the present invention is carried out using 100 mg to 1 g nucleic acid. In one embodiment, the method of the present invention is carried out using 200 to 500 mg nucleic acid.

[0255] In one embodiment, the method of the present invention is carried out using 1 g to 10 g nucleic acid. In one embodiment, the method of the present invention is carried out using 2 to 5 g nucleic acid.

[0256] In one embodiment, the method of the present invention is carried out using 10 g to 10 kg nucleic acid. In one embodiment, the method of the present invention is carried out using 20 g to 5 kg nucleic acid. In one embodiment, the method of the present invention is carried out using 50 g to 2 kg nucleic acid. In one embodiment, the method of the present invention is carried out using 100 g to 1 kg nucleic acid. In one embodiment, the method of the present invention is carried out using 200 g to 500 g nucleic acid. In one embodiment, the method of the present invention is carried out using 10 g to 100 g nucleic acid. In one embodiment, the method of the present invention is carried out using 10 g to 50 g nucleic acid.

[0257] Step (a) - Preparation of first mixture comprising lipids

[0258] In a first step, the present disclosure of the method comprises producing a first lipid mixture containing a cationically ionizable lipid, as defined herein, and optionally one or more additional lipids, as defined herein. Typically, the lipid mixture is prepared in a water-soluble organic solvent, as described below.

[0259] A number of methods of making such lipid mixtures are known in the art and the skilled person would be readily capable of selecting a suitable method and applying this to create the first mixture of the present disclosure. In one embodiment, the first lipid mixture produced in step (a) of the method of the present disclosure is substantially free of nucleic acids.

[0260] Typically, the lipid mixture is prepared in a water-soluble organic solvent. In the methods of the disclosure, the organic solvent (e.g., the water-soluble organic solvent) may be selected from the lists of Class 2 and Class 3 solvents, as described in the FDA’s “Q3C - Tables and List Guidance for Industry”, June 2017, Revision 3 (see, e.g., https: / / www.fda.gov / media / 71737 / download). When the organic solvent is a water-soluble organic solvent, examples include Cl -4 alcohols (e.g. isopropanol or ethanol), ketones (e.g. acetone), or mixtures thereof. The organic solvent (e.g., the water-soluble organic solvent) is preferably ethanol or isopropanol, more preferably ethanol.

[0261] Step (b) - Preparation of second mixture containing nucleic acid and malate buffer

[0262] Step (b) comprises preparing a second mixture comprising (i) a nucleic acid, as defined and exemplified herein; and (ii) malate buffer, as defined and exemplified herein. Typically, step (b) is carried out in aqueous solution. A number of methods of preparing such mixtures are known in the art and the skilled person would be readily capable of selecting a suitable method and applying this to create the second mixture of the present disclosure.

[0263] In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 0.1 to about 1000 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 0.3 to about 300 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 1 to about 200 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 1 to about 100 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 5 to about 50 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 10 to about 40 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 20 to about 30 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 25 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 1 to about 20 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 5 to about 15 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 50 to about 100 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 70 to about 80 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 50 to about 150 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 80 to about 120 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 200 to about 400 mM. In one embodiment, the concentration of malate in the malate buffer used in step (b) is about 250 to about 350 mM. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate di-anion, the concentration being the total concentration of all of these malate species in the solution.

[0264] In one embodiment, the concentration of the nucleic acid used in step (b) is about 0.01 to about 5 mg / mL. In one embodiment, the concentration of the nucleic acid used in step (b) is about 0.02 to about 2 mg / mL. In one embodiment, the concentration of the nucleic acid used in step (b) is about 0.05 to about 1 mg / mL. In one embodiment, the concentration of the nucleic acid used in step (b) is about 0.1 to about 1 mg / mL. In one embodiment, the concentration of the nucleic acid used in step (b) is about 0.1 to about 0.5 mg / mL.

[0265] In one embodiment, step (b) is carried out at a pH of about 2.5 to about 5.5. In one embodiment, step (b) is carried out at a pH of about 3.0 to about 5.0. In one embodiment, step (b) is carried out at a pH of about 3.5 to about 4.5. In one embodiment, step (b) is carried out at a pH of about 4.0 to about 5.0. In one embodiment, step (b) is carried out at a pH of about 4.0 to about 4.5.

[0266] Step (c) - Mixing first and second mixtures to produce intermediate composition

[0267] Step (c) of the method of the present disclosure comprises mixing the first mixture with the second mixture to produce an intermediate composition comprising the nucleic acid-lipid particle. The intermediate composition is further processed in step (d), and optionally also in intermediate step (c’), to produce the final composition containing the nucleic acid-lipid particle. During this step, the lipids present in the lipid mixture encapsulate the nucleic acid to form the nucleic acid-lipid particle.

[0268] A number of suitable mixing methods are known in the art and the skilled person would be readily capable of selecting a suitable method and applying this to create the intermediate composition comprising the nucleic acid-lipid particle.

[0269] In one embodiment, step (c) of the method according to the present disclosure comprises injecting the first mixture, as described and exemplified herein, in a water- soluble organic solvent, as described herein (such as a Cl -4 alcohol, preferably ethanol) into an aqueous phase containing the second mixture, to produce the intermediate composition.

[0270] In one embodiment, the mixing is carried out using a T-mixer or Y-mixer. In some embodiments, the flow rate during mixing may be from about 30 mL / min to about 800 mL / min, optionally from about 40 mL / min to about 700 mL / min, optionally from about 60 mL / min to about 480 mL / min.

[0271] In some embodiments, the volume ratio of the first mixture to the second mixture may be from about 1 : 1 to about 1 :5, optionally about 1 :2 to 1 :4, preferably about 1 :3.

[0272] Step (c ) - Dilution of the intermediate composition

[0273] Optional step (c’) of the method of the present disclosure comprises dilution of the intermediate composition comprising nucleic acid-lipid particles. Typically, this step is carried out in order to reduce the concentration of the water-soluble organic solvent, such as ethanol, in the composition to stabilise the particle colloid.

[0274] In one embodiment, step (c’) occurs directly after step (c) in an in-line fashion. In this embodiment, the dilution step (c’) of the intermediate composition comprising nucleic acid-lipid particles is carried out as soon as this intermediate composition is formed. By doing this the hold time before starting step (d) is increased.

[0275] Typically, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition before dilution step (c’) ranges from about 10% to about 40%. In one embodiment, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition before dilution step (c’) ranges from about 20% to about 30%. In one embodiment, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition before dilution step (c’) is about 25%.

[0276] Typically, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition after dilution step (c’) ranges from about 2% to about 30%. In one embodiment, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition after dilution step (c’) ranges from about 10 to about 25%. In one embodiment, the concentration of the water-soluble organic solvent (e.g. ethanol) in the intermediate composition after dilution step (c’) is from about 15% to about 20%. The diluent is not especially limited provided it is capable of mixing with the intermediate composition containing nucleic acid-lipid particles resulting from step (c) to reduce the concentration of the water-soluble organic solvent therein. Typical examples include water, salts, and buffers, as defined and exemplified herein.

[0277] In some embodiments, the flow rate ratio of first mixture, the second mixture, and the diluent is about 1 :2:3. In some embodiments, the flow rate ratio of first mixture, the second mixture, and the diluent is about 1 :3:3. In some embodiments, the flow rate ratio of first mixture, the second mixture, and the diluent is about 1 :2:2. In some embodiments, the flow rate ratio of first mixture, the second mixture, and the diluent is about 1 :3 :2. In some embodiments, the flow rate ratio of first mixture, the second mixture, and the diluent is about 1 :4:2.

[0278] In one embodiment, the diluent used in step (c’) is malate buffer, as defined and exemplified herein. In one embodiment, the concentration of malate in the malate buffer used in step (c’) is about 5 to about 50 mM. In one embodiment, the concentration of malate in the malate buffer used in step (c’) is about 10 to about 40 mM. In one embodiment, the concentration of malate in the malate buffer used in step (c’) is about 20 to about 30 mM. In one embodiment, the concentration of malate in the malate buffer used in step (c’) is about 25 mM. In this context, it will be understood by the person skilled in the art that the term “malate” includes undissociated malic acid, malate mono-anion (with either carboxylic acid moiety deprotonated), and malate di-anion, the concentration being the total concentration of all of these malate species in the solution.

[0279] In one embodiment, the diluent used in step (c’) is Tris buffer, as defined and exemplified herein. In one embodiment, the concentration of Tris in the Tris buffer used in step (c’) is about 5 to about 100 mM. In one embodiment, the concentration of Tris in the Tris buffer used in step (c’) is about 10 mM to about 50 mM. In this context, it will be understood by the person skilled in the art that the concentration expressed is the total concentration of both neutral Tris and its conjugate acid in the solution. In one embodiment, the diluent used in step (c’) is water, as defined generally herein. In one embodiment, the diluent used in step (c’) is ultrapure water. In one embodiment, the diluent used in step (c’) is Type 1 ultrapure water (such as e.g., MilliQ water). In one embodiment, the diluent used in step (c’) is Water for Injection (WFI).

[0280] Step (d) - Further processing - buffer exchange

[0281] Step (d) of the method of the present disclosure comprises further processing of the intermediate composition containing nucleic acid-lipid particles resulting from step (c) and optionally step (c’) to produce the final composition comprising the nucleic acid-lipid particle.

[0282] Step (d) takes place by buffer exchange, wherein buffers present in the intermediate composition are exchanged with buffers introduced in step (d).

[0283] In one embodiment, the further processing step (d) comprises a dialysis or filtration step. In one embodiment, the further processing step (d) comprises tangential flow filtration.

[0284] The buffer used in step (d) comprises tris(hydroxymethyl)aminomethane (Tris) and / or a pharmaceutically acceptable salt thereof. In one embodiment, the buffer used in step (d) is a Tris buffer. As will be immediately apparent to those skilled in the art that, depending on the pH, the Tris buffer used in step (d) will comprise a mixture of the neutral, unprotonated amine and the conjugate acid (in which the amino group is protonated) in varying proportions.

[0285] The Tris buffer may also contain pharmaceutically acceptable inorganic or organic anions, which may be any of those as defined and exemplified above. Preferred counter-ions include those derived from organic acids, more preferably the acetate. Suitable Tris buffers include Tris-HCl (Tris hydrochloride), Tris-EDTA (TE), Trisbuffered saline (TBS), Tris-acetate, Tris-acetate-EDTA (TAE), Tris-borate, and Tris- borate-EDTA (TBE). In one embodiment, the concentration of Tris in the Tris buffer used in step (d) is about 5 to about 100 mM. In one embodiment, the concentration of Tris in the Tris buffer used in step (d) is about 10 mM to about 50 mM. In this context, it will be understood by the person skilled in the art that the concentration expressed is the total concentration of both neutral Tris and its conjugate acid in the solution.

[0286] Step (e) - Optional further processing steps for nucleic acid-lipid particle composition

[0287] The method of the present disclosure optionally includes carrying out further processing steps (e) on the composition comprising the nucleic acid-lipid particle produced according to step (d).

[0288] In one embodiment, further processing step (e) comprises adding a cryoprotectant to the lipid particle. The cryoprotectant may be any of the cryoprotectants defined and exemplified above. In one embodiment, the cryoprotectant used in step (e) is a carbohydrate, such as a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose and glucose, or a mixture of any thereof, preferably sucrose. The cryoprotectant may be in solution, optionally in a Tris buffer solution. The cryoprotectant may be in the same buffer as used in step (d) comprising Tris, as further defined above.

[0289] In one embodiment, further processing step (e) comprises sterile filtration of the nucleic acid-lipid particle. Typically, the sterile filtration uses a 0.22 pm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0290] In one embodiment, the nucleic acid-lipid particles are not subjected to any further processing steps. In one embodiment, further processing step (e) comprises adding a cryoprotectant (as defined above), followed by sterile filtration (as defined above) of the composition comprising the nucleic acid-lipid particle.

[0291] Lipids

[0292] The compositions of the disclosure contain a mixture of lipids. The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and also one or more hydrophilic moieties or groups. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. Lipids may comprise a polar portion and an apolar (or non-polar) portion.

[0293] Cationic and Cationically Ionizable Lipids

[0294] The lipid mixtures present in the compositions disclosed herein also contain a cationically ionizable lipid or a mixture of any thereof. In one embodiment the aqueous dispersions and nucleic acid-lipid particles of the present disclosure comprise a cationically ionizable lipid.

[0295] In one embodiment, the lipid is a cationically ionizable lipid. As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which, depending on whether it is protonated or deprotonated, has a net positive charge or is neutral, z.e., a lipid which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral.

[0296] In one embodiment the aqueous dispersions and nucleic acid-lipid particles of the present disclosure do not comprise a cationic lipid. As used herein, the term “cationic lipid” means a lipid or lipid-like material, as defined herein, having a constitutive positive charge. In this context a “constitutive charge” means that the cationic lipid carries the positive charge at all physiological pH. The cationic lipids carrying constitutive charged cationic moieties are typically quaternary ammonium salts (as defined above) or salts of organic bases, such as nitrogen-containing bases. Typically, such organic bases are strong bases (i.e. bases which are completely protonated when dissolved in a solvent, such as but not limited to an aqueous solvent, such that the concentration of the unprotonated species is too low to be measured).

[0297] In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is capable of being protonated, preferably under physiological or slightly acidic conditions. In one embodiment, the cationically ionizable lipid comprises ((3- hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) (ALC-0366). In one embodiment, the cationically ionizable lipid consists essentially of ((3- hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) (ALC-0366). In one embodiment, the cationically ionizable lipid is or consists of ((3- hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366). ALC-0366 has the following structure:

[0298] In one embodiment, the cationically ionizable lipid is present in an amount of 20 to 70 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationically ionizable lipid is present in an amount of 30 to 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationically ionizable lipid is present in an amount of 40 to 50 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture component of both the first mixture, the intermediate composition comprising the nucleic acid-lipid particle and the final composition comprising the nucleic acid-lipid particle.

[0299] Additional Lipids

[0300] The lipid mixtures present in the compositions disclosed herein may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise a neutral or zwitterionic lipid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a steroid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid (such as a steroid), as defined and exemplified below. Neutral Lipid

[0301] In some embodiments, the lipid mixtures present in the compositions disclosed herein may also additionally comprise a neutral lipid. The neutral lipid is preferably a neutral phospholipid. In one embodiment, the phospholipid may be zwitterionic (i.e. it carries both a positive and a negative charge, so that it is neutral at a pH ranging around neutral).

[0302] Examples of suitable phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero- 3 -phosphocholine (OChemsPC), l-hexadecyl-sn-10-glycero-3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)- sn-glycero-3 -phosphocholine (DOPG), 1 ,2-dipalmitoyl-sn-glycero-3 -phospho-( 1 '-rac- glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0303] In some embodiments, the neutral or zwitterionic lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof. Preferably, the neutral or zwitterionic lipid is DSPC.

[0304] Thus, in some embodiments, the lipid mixtures described herein comprise a cationically ionizable lipid (as defined herein) and a phospholipid. In some embodiments, the lipid mixtures described herein comprise a cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof. The term “lipid mixture” in this context applies to the lipid mixture component of both the first mixture, the intermediate composition comprising the nucleic acid-lipid particle and the final composition comprising the nucleic acid-lipid particle.

[0305] In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 25 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 30 mol % to about 35 mol % of the total lipids present in the lipid mixture.

[0306] In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.

[0307] In each of the above embodiments, the term “lipid mixture” in this context applies to the lipid mixture component of both the first mixture, the intermediate composition comprising the nucleic acid-lipid particle and the final composition comprising the nucleic acid-lipid particle.

[0308] Steroid

[0309] In some embodiments, the lipid mixture present in the compositions disclosed herein also comprise a steroid. In one embodiment, the steroid comprises a sterol. In one embodiment, the steroid is cholesterol.

[0310] Thus, in some embodiments, the lipid mixtures present in the compositions described herein comprise a cationically ionizable lipid (as defined herein) and cholesterol. In some embodiments, the lipid mixtures present in the compositions described herein comprise a cationically ionizable lipid (as defined herein), a phospholipid (as defined herein) and cholesterol.

[0311] In one embodiment, the steroid is present in an amount ranging from about 10 mol % to about 65 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 20 mol % to about 60 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 30 mol % to about 50 mol % of the total lipids present in the lipid mixture.

[0312] In each of the above embodiments, the term “lipid mixture” in this context applies to the lipid mixture component of both the first mixture, the intermediate composition comprising the nucleic acid-lipid particle and the final composition comprising the nucleic acid-lipid particle.

[0313] Grafted Lipids

[0314] The lipid mixtures present in the compositions disclosed herein may further comprise a grafted lipid. In the present specification the term “grafted lipid” in its broadest sense means a lipid or lipid-like material, as defined above (either in a broadest aspect or a preferred aspect) conjugated to a polymer, as defined below (either in a broadest aspect or a preferred aspect”).

[0315] A "polymer" as used herein, is given its ordinary meaning, z.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, z.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0316] In one embodiment, the grafted lipid is capable of acting as a stealth lipid. In this specification the term “stealth lipid” means a stealth polymer (as defined below) conjugated to a lipid (as defined herein). In this specification the term “stealth polymer” means a polymer (as defined above) having the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge. In some embodiments, a stealth polymer is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a stealth polymer can reduce its association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0317] In one embodiment, the grafted lipid is a polyethylene-glycol conjugated lipid (also known as a PEG-lipid or PEGylated lipid). The term "PEGylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.

[0318] PEGylated lipids are known in the art. The PEG-lipid may comprise 5-1000, 5-500, 5- 100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100, or 10-50, ethylene glycol repeating units, which may be consecutive.

[0319] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein each of R12and R13is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0320] In some embodiments of this formula, each of R12and R13is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

[0321] In some embodiments of this formula, R12and R13are identical. In some embodiments, each of R12and R13is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 16 carbon atoms.

[0322] In some embodiments of this formula, R12and R13are different. In some embodiments, one of R12and R13is a straight alkyl chain containing 12 carbon atoms and the other of R12and R13is a straight alkyl chain containing 14 carbon atoms.

[0323] In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

[0324] In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g / mol, such as from about 1000 to about 5000 g / mol, from about 1500 to about 4000 g / mol, or from about 2000 to about 3000 g / mol. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

[0325] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monom ethoxy -poly ethyleneglycol)- 2, 3 -dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG- PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3'- di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoylxy)propyl-N-(co methoxy(polyethoxy)ethyl)carbamate, and the like. In some embodiments of this formula, the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g / mol, such as from about 1000 to about 5000 g / mol, from about 1500 to about 4000 g / mol, or from about 1700 to about 3000 g / mol, or from about 1800 to about 2200 g / mol. In one embodiment, the PEG portion of the pegylated lipid has an average molecular weight of about 2000 g / mol.

[0326] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is or comprises 2- [(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the pegylated lipid has the following structure: pharmaceutically acceptable salt thereof, where n’ is an integer from about 45 to about 50.

[0327] Other examples of grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipids and poly(2- methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipids.

[0328] In one embodiment, the grafted lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N- methylglycine) portion, the polysarcosine portion having the repeating unit shown below: wherein x refers to the number of sarcosine units. The polysarcosine may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 sarcosine units, preferably from 15 to 50 sarcosine units, more preferably from 20 to 30 sarcosine units, even more preferably 21 to 25 sarcosine units.

[0329] In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (Ce-30 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group. In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (C12-20 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group. In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (C14 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group.

[0330] In one especially preferred embodiment, the grafted lipid is n-tetradecyl poly(sarcosine)23 (C14-pSar 23), having the following structure: 23.

[0331] In one especially preferred embodiment, the grafted lipid is n-tetradecyl poly(sarcosine)23 acetate (C14-pSar 23 Ac), having the following structure: where n is 23.

[0332] In one embodiment, the grafted lipid is an amphiphilic oligoethylene glycol (OEG)- conjugated lipid. Examples of amphiphilic oligoethylene glycol (OEG)-conjugated lipids include poly(aminoethyl-ethylene glycol acetyl) (pAEEA) and / or poly(methylaminoethyl-ethylene glycol acetyl) (pmAEEA). The terms “pAEEA” and “pmAEAA” means a polymer having the repeating unit shown below: pAEEA pmAEEA wherein x refers to the total number of pAEEA and / or pmAEEA units in the polymer. The total number of pAEEA and / or pmAEEA repeating units in the polymer may comprise from 1 to 100, from 5 to 50, from 5 to 25, from 7 to 14, preferably from 10 to 20, more preferably 12 to 16.

[0333] The lipid portion of the (pAEEA)-conjugated lipid may be any of those defined above in relation to lipids, either in a broadest aspect or a preferred aspect. In one embodiment, the lipid portion is a tocopherol or tocotrienol residue. In one embodiment, the lipid portion is a-tocopherol.

[0334] The grafted lipid may be a PEG-conjugated lipid, pSar-conjugated lipid, pAEEA- conjugated lipid, or pmAEEA-conjugated lipid. The grafted lipid may be selected from the group consisting of: DSPE-pAEEAw-AC, a-tocopherol-pAEEAw-AC, DMG-pAEEAs-AC, PEG2000-DMG, or ALC-0159. Preferably, the grafted lipid is ALC-0159.

[0335] In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture component of both the aqueous dispersion containing intermediate lipid particles and the final nucleic acid-lipid particles.

[0336] Preferred Combinations

[0337] In one embodiment, the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); and distearoylphosphatidylcholine (DSPC). In one embodiment, the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); and cholesterol.

[0338] In one embodiment, the lipid mixture comprises:

[0339] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); and 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0340] In one embodiment, the lipid mixture comprises:

[0341] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); distearoylphosphatidylcholine (DSPC); and cholesterol.

[0342] In one embodiment, the lipid mixture comprises:

[0343] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); distearoylphosphatidylcholine (DSPC); and

[0344] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0345] In one embodiment, the lipid mixture comprises:

[0346] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); cholesterol; and

[0347] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0348] In one embodiment, the lipid mixture comprises:

[0349] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366); di stearoylphosphatidylcholine (D SPC); cholesterol; and

[0350] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0351] In one embodiment, the lipid mixture comprises:

[0352] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; and distearoylphosphatidylcholine (DSPC), in an amount of 5 to 15 wt.% of the total lipids present in the lipid mixture.

[0353] In one embodiment, the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; and cholesterol, in an amount of 20 to 60 wt.% of the total lipids present in the lipid mixture.

[0354] In one embodiment, the lipid mixture comprises:

[0355] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; and 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 0.5 to 5 wt.% of the total lipids present in the lipid mixture.

[0356] In one embodiment, the lipid mixture comprises:

[0357] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 5 to 15 wt.% of the total lipids present in the lipid mixture; and cholesterol, in an amount of 20 to 60 wt.% of the total lipids present in the lipid mixture.

[0358] In one embodiment, the lipid mixture comprises:

[0359] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 5 to 15 wt.% of the total lipids present in the lipid mixture; and

[0360] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 0.5 to 5 wt.% of the total lipids present in the lipid mixture.

[0361] In one embodiment, the lipid mixture comprises:

[0362] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; cholesterol, in an amount of 20 to 60 wt.% of the total lipids present in the lipid mixture; and

[0363] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 0.5 to 5 wt.% of the total lipids present in the lipid mixture.

[0364] In one embodiment, the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 5 to 15 wt.% of the total lipids present in the lipid mixture; cholesterol, in an amount of 20 to 60 wt.% of the total lipids present in the lipid mixture; and

[0365] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 0.5 to 5 wt.% of the total lipids present in the lipid mixture.

[0366] In one embodiment, the lipid mixture comprises:

[0367] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; and distearoylphosphatidylcholine (DSPC), in an amount of 10 wt.% of the total lipids present in the lipid mixture.

[0368] In one embodiment, the lipid mixture comprises:

[0369] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; and cholesterol, in an amount of 40.7 wt.% of the total lipids present in the lipid mixture.

[0370] In one embodiment, the lipid mixture comprises:

[0371] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; and 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 1.8 wt.% of the total lipids present in the lipid mixture.

[0372] In one embodiment, the lipid mixture comprises: ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 10 wt.% of the total lipids present in the lipid mixture; and cholesterol, in an amount of 40.7 wt.% of the total lipids present in the lipid mixture.

[0373] In one embodiment, the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 10 wt.% of the total lipids present in the lipid mixture; and

[0374] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 1.8 wt.% of the total lipids present in the lipid mixture.

[0375] In one embodiment, the lipid mixture comprises:

[0376] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; cholesterol, in an amount of 40.7 wt.% of the total lipids present in the lipid mixture; and

[0377] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 1.8 wt.% of the total lipids present in the lipid mixture.

[0378] In one embodiment, the lipid mixture comprises:

[0379] ((3 -hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 10 wt.% of the total lipids present in the lipid mixture; cholesterol, in an amount of 40.7 wt.% of the total lipids present in the lipid mixture; and

[0380] 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 1.8 wt.% of the total lipids present in the lipid mixture.

[0381] In each of the above embodiments, the term “lipid mixture” in this context applies to the lipid mixture component of both the first mixture, the intermediate composition comprising the nucleic acid-lipid particle and the final composition comprising the nucleic acid-lipid particle.

[0382] Pharmaceutical Compositions

[0383] The nucleic acid-lipid particle compositions described herein are useful as or for preparing pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

[0384] The nucleic acid-lipid particle compositions described herein may be administered in the form of any suitable pharmaceutical composition.

[0385] The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. In some embodiments, the therapeutically effective agent is or comprises the active ingredient, as described herein. In the context of the present disclosure, the pharmaceutical composition comprises a nucleic acid as described herein. In some embodiments, the therapeutically effective agent is or comprises a nucleic acid, as described in the present disclosure, which comprises a nucleic acid sequence (e.g., an ORF) encoding one or more polypeptides, e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein.

[0386] The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (z.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses. In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

[0387] In some embodiments, the pharmaceutical composition is in frozen form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature ranging from about -90°C to about -10°C, such as from about -90°C to about -40°C or from about -40°C to about -25°C, or from about -25°C to about - 10°C, or a temperature of about -20°C.

[0388] In some embodiments of the pharmaceutical compositions in frozen form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at -20°C.

[0389] In some embodiments, the pharmaceutical composition is in liquid form and can be stored at a temperature ranging from about 0°C to about 20°C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature ranging from about 1°C to about 15°C, such as from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C.

[0390] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0391] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition. The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the particles or pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0392] In particular embodiments, a pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, z.e., a pharmaceutical composition which can be used for inducing an immune response) is formulated as a single-dose in a container, e.g., a vial. In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in the vial is equal in volume. In some embodiments, a first dose is a different volume than a subsequent dose.

[0393] A "stable" multi-dose formulation preferably exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunologic response when administered to a subject. The pharmaceutical compositions of the present disclosure may contain buffers (in particular, derived from the nucleic acid (such as RNA) compositions with which the pharmaceutical compositions have been prepared), preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure, in particular the ready-to-use pharmaceutical compositions, comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0394] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0395] The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavouring agents, or colorants.

[0396] The term "diluent" relates a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol and water.

[0397] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxy ethylene / polyoxy-propylene copolymers.

[0398] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0399] In one embodiment, the compositions described herein, such as the pharmaceutical compositions or ready -to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly or intratumourally. In certain embodiments, the (pharmaceutical) composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the (pharmaceutical) compositions, in particular the ready -to- use pharmaceutical compositions, are formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) compositions, in particular the ready -to-use pharmaceutical compositions, are formulated for intramuscular administration.

[0400] Medical Uses and Methods of Treatment

[0401] The nucleic acid-lipid particle compositions and pharmaceutical compositions comprising them as described herein may be used in the therapeutic or prophylactic treatment of various diseases, in particular diseases in which provision of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, provision of an antigen or epitope which is derived from a virus may be useful in the treatment or prevention of a viral disease caused by said virus. Provision of a tumour antigen or epitope may be useful in the treatment of a cancer disease wherein cancer cells express said tumour antigen. Provision of a functional protein or enzyme may be useful in the treatment of genetic disorder characterized by a dysfunctional protein, for example in lysosomal storage diseases (e.g. mucopolysaccharidoses) or factor deficiencies. Provision of a cytokine or a cytokine-fusion may be useful to modulate tumour microenvironment. Therefore, in one aspect there is disclosed the nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in medicine.

[0402] In one embodiment, there is provided a nucleic acid-lipid particle, or pharmaceutical composition as defined herein for use in delivery of a nucleic acid (such as an mRNA) to a cell. In one embodiment, there is provided a nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in transfecting a cell with a nucleic acid (such as an mRNA). In one embodiment, there is provided a method of delivery of a nucleic acid (such as an mRNA) to a cell, the method comprising administering to the cell the nucleic acid-lipid particle, or pharmaceutical composition as defined herein. The cell may be any cell capable of receiving nucleic acid (such as an mRNA) to produce a therapeutic effect. In one embodiment, the cell is a liver cell. In one embodiment, the cell is a spleen cell.

[0403] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in a prophylactic and / or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein in the manufacture of a medicament for a prophylactic and / or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided a method of prophylactic and / or therapeutic treatment of a disease involving an antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

[0404] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in inducing an immune response. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for inducing an immune response.

[0405] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, for use in treating cancer. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for treating cancer. In one embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

[0406] The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively.

[0407] In some embodiments, the nucleic acid-lipid particle or a pharmaceutical composition described herein may be used in the therapeutic or prophylactic treatment of an infectious disease.

[0408] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0409] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

[0410] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer, infectious diseases) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

[0411] The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0412] In some embodiments of the disclosure, the aim is to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, to a subject, in particular a subject in need thereof. In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination.

[0413] A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Pharmaceutical compositions described herein are thus useful in a prophylactic and / or therapeutic treatment of a disease involving an antigen or epitope.

[0414] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons. Examples

[0415] Materials

[0416] Acidifiers and buffers

[0417] Lipids and mRNA

[0418] In these Examples, “Abl” represents the mRNA encoding the anti-claudin 18.2 antibody and which is synthesised according to WO2024 / 074634A1 and “Ab2” represents the mRNA encoding the bispecific anti-CD3 / CLDN6 antibody and which is synthesised according to WO2023 / 285560A1.

[0419] Methods

[0420] Small-scale manufacturing process

[0421] The small-scale manufacturing process was utilized for the manufacturing of LNPs with an input RNA amount up to 10 milligrams. LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA using syringe pumps (T-piece mixer, DI 0.5 mm) with a total flow rate of 150 mL / min The lipid mixture was composed of an ionizable lipid ALC 366, l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol and the stealth moiety ALC 0159 dissolved in ethanol at molar ratios of 47.5: 10:40.7: 1.8. The aqueous phase contained mRNA diluted in the desired acidic buffer (pH 4.0) was mixed with the ethanol phase at a volume ratio of 3: 1 (aqueous: ethanol), followed by (1 :2) in-line dilution with the identical acidic buffer (pH 4.0) via (T-piece mixer, DI 2.0 mm). The ratio of nitrogen present in the ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6 for each formulation. The obtained formulation was purified with dialysis being performed using 10 kDa molecular weight cut-off (MWCO) Slide-A-Lyzer G2 or G3 dialysis cassettes. After manufacturing, intermediate LNPs were transferred to the respective cassettes at the desired volume and dialyzed overnight (16-20 hours) against the respective buffer (1 :500 sample:buffer v / v ratio) at room temperature. After buffer exchange the bulk drug product was adjusted with the buffer to a RNA concentration of 0.1 mg / mL and diluted with 1.2 M sucrose solution as cryoprotectant to the final RNA concentration (After dialysis, LNPs were up-concentrated to 0.5 mg / mL if needed). Thereafter, the adjusted bulk drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -20°C.

[0422] Large-scale manufacturing process

[0423] The large-scale manufacturing process was utilized for the manufacturing of LNPs with an input RNA amount up to 300 milligrams. LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA using HPLC pumps (T-piece mixer, DI 0.5 mm) with a total flow rate of 240 mL / min. The lipid mixture was composed of an ionizable lipid ALC 366, 1,2-distearoyl-sn-glycero- 3 -phosphocholine (DSPC), cholesterol and the stealth moiety ALC 0159 dissolved in ethanol at molar ratios of 47.5: 10:40.7: 1.8. The aqueous phase contained mRNA diluted in the desired acidic buffer (pH 4.0) was mixed with the ethanol phase at a volume ratio of 3: 1 (aqueous: ethanol), followed by (1 :2) in-line dilution with the identical acidic buffer (pH 4.0) via (T-piece mixer, DI 2.0 mm). The ratio of nitrogen present in the ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6 for each formulation. The obtained formulation was purified with tangential flow filtration (TFF) using 100 kDa molecular weight cut-off (MWCO) hollow fiber. After manufacturing, intermediate LNPs were transferred to the TFF at the desired volume and purified against 10 Mm Tris buffer at room temperature. After buffer exchange the bulk drug product was adjusted with the buffer to an RNA concentration of 0.1, 0.5, and / or 1.0 mg / mL and diluted with 1.2 M sucrose solution as cryoprotectant to the final RNA concentration. Thereafter, the adjusted bulk drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -20°C.

[0424] Pilot-scale manufacturing process

[0425] The pilot-scale manufacturing process was utilized for the manufacturing of LNPs with an input RNA amount up to 3 grams. LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA using HPLC pumps (T-piece mixer, DI 0.5 mm) with a total flow rate of 720 mL / min. The lipid mixture was composed of an ionizable lipid ALC 366, l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol and the stealth moiety ALC 0159 dissolved in ethanol at molar ratios of 47.5: 10:40.7: 1.8. The aqueous phase contained mRNA diluted in the desired acidic buffer (pH 4.0) was mixed with the ethanol phase at a volume ratio of 3: 1 (aqueous: ethanol), followed by (1 :2) in-line dilution with the identical acidic buffer (pH 4.0) via (T-piece mixer, DI 2.0 mm). The ratio of nitrogen present in the ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6 for each formulation. The obtained formulation was purified with tangential flow filtration (TFF) using 100 kDa molecular weight cut-off (MWCO) hollow fiber. After manufacturing, intermediate LNPs were transferred to the TFF at the desired volume and purified against 10 mM Tris buffer at room temperature. After buffer exchange the bulk drug product was adjusted with the buffer to an RNA concentration of 0.1, 1.0 and / or 2.0 mg / mL and diluted with 1.2 M sucrose solution as cryoprotectant to the final RNA concentration. Thereafter, the adjusted bulk drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -20°C.

[0426] Analysis of Lipid Nanoparticles size and polydispersity

[0427] Mean particle size and size distribution of LNPs in a sample of drug product was by dynamic light scattering (DLS). The method employs a particle sizer that uses backscatter at 173° to determine particle size. The results are reported as the Z-average size of the particles and the poly dispersity index. The poly dispersity values are used to describe the width of fitted log-normal distribution around the measured Z-average size and are generated using proprietary mathematical calculations within the particle sizing software. Results for size and poly dispersity are reported as nm and poly dispersity index (PDI) value, respectively. pH

[0428] The sample pH was determined according to Ph.Eur. 2.2.3. Results are reported as pH units.

[0429] Osmolality

[0430] The determination of osmolality was performed by freezing point depression according to Ph.Eur. 2.2.35. Results are reported as mOsmol / kg of water.

[0431] Analysis of mRNA encapsulation efficiency

[0432] The RNA content was determined by disrupting the LNPs with detergent TritonTM X- 100 and subsequently measuring the total RNA content based on the signal of the RNA- binding fluorescent dye RiboGreen® using a spectrofluorophotometer. RNA encapsulation was calculated by comparing the RiboGreen® signals of LNP samples in the absence (free RNA) and presence (total RNA) of Triton™ X-100. Results for RNA content and encapsulation were reported as mg / ml and percentage, respectively.

[0433] RNA integrity

[0434] RNA integrity was determined by capillary electrophoresis. RNA-LNPs treated with Triton / Ethanol / Tween20 were applied to a gel matrix contained in a capillary. The RNA and its derivatives, degradants and impurities were separated according to their sizes. The gel matrix contains a fluorescence dye which binds specifically to the RNA components which allows detection by a laser-induced fluorescence (LIF) detector. The excitation wavelength was set to 495 nm, the emission wavelength was 537 nm. The data were evaluated using the software Prosize. The integrity of the RNA was verified by comparing with the reference standard. Example 1: One-factor-at-a time exploration of acidifiers and buffers

[0435] Study design

[0436] In the composition of this example, various acidifiers and buffers were evaluated across a range of logD coefficients. To systematically rank acidifier and buffer types, it was sought to generalize their chemical properties such as pKa and lipophilicity into a single descriptor. Since acidifiers and buffers are used at a different pH value, the distribution coefficient of ionizable compounds, the logD value, was employed. For the buffers that could serve as final storage matrices, a non- exhaustive list of Good’s buffers was made and the relevant logD values at pH 7.5 determined by ACD / Labs software were obtained from the ChemSpider database (Table 1). In the case of acidifiers, citrate and acetate typically serve as golden standards within LNP manufacturing. Therefore, these compounds and structurally relevant and commercially accessible compounds were selected. Here, the relevant acidic logD values were only obtainable at pH 5.5, which was assumed to be closely comparable to the desired manufacturing pH of 4.0 (Table 2). The objective was to determine the initial interactions between acidifier and buffer choice during the formulation of the LNPs and the size increase over 5 freeze-thaw cycles at -20 °C.

[0437] Table 1: List of buffers and their appropriate logP and logD values at pH 7.5

[0438] Table 2: List of acidifiers and their appropriate logP and logD values at pH 5.5

[0439] Results LNPs were prepared using the small-scale manufacturing process described in the Methods section. Colloidal properties (size and PDI) were measured after dialysis and normalization of the concentration with sucrose, as well as after 5 FT cycles at -20 °C. Besides from the combination of acetate and Bis-Tris, all other combinations had acceptable colloidal properties after manufacturing (size < 100 nm, PDI < 0.2). Following FT cycles (FTCs), the acetate and Bis-tris combination showed a significant increase in particle size, whereas there was no clear trend visible for other buffers combined with the acetate acidifiers (Table 3). Regarding the acidifiers in combination with the TRIS buffer, the change in particle size was the lowest throughout all the samples, with minor differences between acidifiers. Altogether, the combination of acidifiers with logD values closer to 0 appear to show an important connection. This information was used for further development on which will be elaborated in Example 2.

[0440] Table 3: Combinations of acidifiers and buffers selected for OFAT experiment and the freeze-thaw stability over 5 cycles at -20 °C

[0441] ACES = N-(2-acetamido)-2-aminoethanesulfonic acid, BES = N,N-Bis-(2-hydroxyethyl)-2-amino-ethanesulfonic acid, N,N-Bis-(2-hydroxyethyl)-taurine, BIS-TRIS = Bis-(2-hydroxy-ethyl)-amino-iris(hydroxymethyl)-methane, MOPS = 3- (Morpholin-4-yl)propane-1 -sulfonic acid, nm = nanometer, PDI = polydispersity index, TRIS = 2-Amino-2- hydroxymethyl-propane-1 ,3-diol

[0442] Example 2: Design-of-Experiments based evaluation of acidifiers and buffers

[0443] Study design

[0444] In the composition of this example, a Design-of-Experiments (DoE) was used to give the unknown detailed effect of acidifiers. Here, the full range of acidifiers was considered, along with an abbreviated range of buffers and a third factor was also included, being buffer concentration. Altogether, from the three factors a low level (- 1), mid-point (0) and high-level value (+1) were determined (Table 4). With these levels, the Box-Behnken design space could be constructed, as is shown in Figure 1. Furthermore, validation experiments were performed to justify the ideal range and the preferred choice of acidifier, buffer and buffer concentration. Table 4: Level determination for DoE design

[0445] ACES = N-(2-acetamido)-2-aminoethanesulfonic acid, BES = N,N-Bis-(2-hydroxyethyl)-2-amino-ethanesulfonic acid, N,N-Bis-(2-hydroxyethyl)-taurine, TRIS = 2-Amino-2-hydroxymethyl-propane-1 ,3-diol Results

[0446] Out of the Box-Behnken design space, a total of 15 runs were performed, which consisted of the 12 edge points as well as a triplicate of the center point Table 5. Here, the respective buffers and acidifiers were chosen from their respective list, with the value being the closest to the computed theoretical values. All samples were manufactured using the small-scale manufacturing and purification process as described in the methods section, after which the samples were submitted to 5 FTCs at -20 °C. Out of the size measurements performed before and after FTCs, the A-size and A-PDI could be calculated and shown in Table 5.

[0447] Table 5. DoE design runs and colloidal stability over 5 freeze- thaw cycles at -20 °C. ACES = N-(2-acetamido)-2-aminoethanesulfonic acid, BES = N,N-Bis-(2-hydroxyethyl)-2-amino-ethanesulfonic acid, N,N-Bis-(2-hydroxyethyl)-taurine, FT = freeze-thaw, nm = nanometer, PDI = poly dispersity index, TRIS = 2-Amino-2- hydroxymethyl-propane-1 ,3-diol Table 6. Response factors and their model fitting values

[0448] FT = freeze-thaw, nm = nanometre, PDI = polydispersity index, TO = time zero, color indications have been given for the fit of the values corresponding to the desired criteria set.

[0449] From the response surface plots (one intersection at buffer concentration of 12.5 mM is shown in Figure 2) taking into consideration acceptance criteria of particle size < 120 nm, PDI < 0.2, delta PDI < 0.1 and delta size < 20 nm, areas were able to be determined which were meeting the initial criteria. From these areas, relevant combinations of buffer (TRIS and MOPS), their concentration (7.5 - 14 mM) and respective acidifiers (malate, citrate, oxalate), were selected. These combinations were produced, and the freeze-thaw stability was determined (Table 7). Here, it was found that all combinations with TRIS seem to follow the ranges provided by the DoE model, whereas those with MOPS buffer did not meet the set criteria. This finding supports that the use of TRIS buffer is preferred with an acidifier having a logD (pH 5.5) value in the middle range, being oxalate, malate or citrate.

[0450] Table 7. Downscale validation experiments with Freeze thaw stability at -20 °C

[0451] FT = freeze-thaw, MOPS =3-morpholinopropane-1 -sulfonic acid, nm = nanometer, PDI = polydispersity index, TO = time zero, TRIS = 2-Amino-2-hydroxymethyl-propane-1 ,3-diol

[0452] To determine if the freeze-thaw resistance that was observed for the batches manufactured with the malate acidifier were dependent on the TRIS concentration during purification and storage, an OFAT-experiment (Table 8) was performed. Here, the concentration of the TRIS buffer during dialysis varied between 7.5 - 45 mM and the effect on particle size, polydispersity, and the change thereof over 5 FTCs at -20 °C was determined. It is shown that the TRIS concentration has little to no impact on the colloidal stability of the formulation and all met the cut-off criteria.

[0453] Table 8. Freeze thaw stability at -20 °C with malate acidifier and varying TRIS concentrations

[0454] FT = freeze-thaw, nm = nanometre, PDI = polydispersity index, TO = time zero, TRIS = 2-Amino-2-hydroxymethyl- propane-1 ,3-diol

[0455] Since suitable areas meeting all the cut-off criteria were limited to the upper edge of the design space (logD of TRIS = -3.75), it was decided to evaluate if the combination of various acidifiers with TEA, possessing a logD value of -1.65 at pH 7.5, would be a suitable candidate. Here, it was found that combinations of citrate and malate acidifiers with the TEA buffer, are fitting to the all the cut-off criteria, whereas combinations of acetate and phosphate do not meet the criteria for TO PDI as well as FT5-FT0 delta size (Table 9). This reinforces the finding that acidifiers with a logD in the intermediate range are most fitting for maintaining a low size increase over 5FTCs. However, since the absolute values for combinations of malate and citrate with TEA (Amaiate-TEA = 18 nm, Acitrate-TEA = 13 nm, Table 9) buffer were not lower than those of malate and citrate in combination with TRIS (Amaiate-TRis = 7 nm, Acitrate-TRis = 5 nm, Table 7).

[0456] Table 9. Freeze-thaw stability at -20 °C of TEA buffer with various acidifiers

[0457] FT = freeze-thaw, nm = nanometre, PDI = polydispersity index, TO = time zero, TEA = 2-[bis(2-hydroxyethyl)- amino]ethan-1 -ol Example 3: Large-scale manufacturing and long-term stability

[0458] Study design

[0459] Given the initial results showing a clear preference of the acidifiers malate, oxalate and citrate in combination with TRIS for the stabilization over freeze-thaw cycles at -20 °C, the objective of the next example was to formulate the LNPs with large-scale manufacturing and perform long-term stability at -20 °C over 18 months to evaluate their stability profiles. Additionally, it was aimed to evaluate the stability profiles at different DP concentrations, being 0.1, 0.5 and 1.0 mg / mL.

[0460] Results

[0461] LNPs were formulated using the large-scale manufacturing process described in the Methods section. Here, the composition consisted of a 25 mM acidifier buffer (pH 4.0) and a 7.5 mM TRIS buffer (pH 7.4). After formulation, the samples were initially tested for stability over 5 FTCs, showing a slight preference for all storage concentrations for the formulations made with the malate acidifier and TRIS buffer (Table 10). The formulations were also kept at -20 °C for over 18 months, in which fresh vials were taken every 6 months, and analysis was performed. Here, also all LNPs manufactured with the malate acidifier showed a superior performance compared to their counterparts formulated with the oxalate or citrate acidifier at all three concentrations. These results demonstrate that stabilization of LNP formulations is the best with formulation acidifiers having an intermediate logD value, preferentially being malate, together with a TRIS buffer.

[0462] Table 10. Freeze-thaw stability at -20 °C of upscale manufactured batches with various acidifiers and TRIS buffer (7.5 mM, pH 7.4)

[0463] FT = freeze-thaw, nm = nanometre, PDI = polydispersity index, TO = time zero Table 11. Long-term colloidal stability at -20 °C. Size, PDI and encapsulation efficiency.

[0464] EE = encapsulation efficiency, FT = freeze-thaw, nm = nanometre, PDI = polydispersity index, TO = time zero

[0465] Example 4: Demonstration with various mRNA constructs

[0466] Study design

[0467] In the composition of this example, lipid nanoparticles were manufactured using the malate acidifier (25 mM pH 4.0) and process further using TRIS buffer (10 mM pH 7.4) according to the small-scale manufacturing process described in the Methods section using various mRNA constructs. The objectives were to demonstrate the colloidal stability of the LNPs with multiple RNA constructs over 5 freeze-thaw cycles at -20 °C.

[0468] Results

[0469] Similar particle sizes and poly dispersity were obtained for all LNPs (Table 12). All LNPs were evaluated at an RNA concentration of 0.1 mg / mL. Figure 3 shows the stability, where all samples have a size increase of 5 nm or less over 5 freeze-thaw cycles at -20 °C. The results demonstrate that the methods described herein can be practiced using various mRNA constructs.

[0470] Table 12. Physicochemical properties of LNPs manufactured with the malate acidifier and TRIS buffer using various mRNA constructs Example 5: Impact of process chemistry parameters such as concentration, pH and acidifier preparation

[0471] Study design

[0472] Given the clear preference for the use of a malate acidifier together with a TRIS buffer in the formulation of LNPs, as shown herein, experiments were performed to explore possible variants of the methods, focusing on chemistry and process parameters. In the composition of this example, lipid nanoparticles were manufactured according to the small-scale manufacturing process described in the Methods section, but various process parameters were varied such as the acidifier and buffer concentration, flow rate ratios, pH values and in-line dilution strategies. The objectives were to demonstrate the colloidal stability of the LNPs produced with various process parameters over 5 freezethaw cycles at -20 °C.

[0473] Results

[0474] The first experiment focused on the flow rate ratio and in-line dilution buffer type, being varied according to the description in Table 13. LNPs were evaluated at a storage concentration of 0.1 mg / mL RNA. Figure 4 displays the size increase over 5 FTCs, where all groups display similar sizes and minimal increase over 5 freeze-thaw cycles at -20 °C. This data supports that the methods can be practiced using multiple in-line dilution strategies and ratios.

[0475] Table 13. Experimental parameters and physiochemical parameters for LNPs produced with a variation of in-line dilution buffers and flow rate ratios

[0476] The next experiments focused on using various concentrations of the malate acidifier. In these experiments, the listed concentrations are those used for the dilution of the RNA phase as well as those for the in-line dilution, according to the small-scale manufacturing process described in the Methods section. LNPs were evaluated at a storage concentration of 0.1 mg / mL RNA. The first series evaluated malate concentrations between 10 - 50 mM (Table 14) Here, all LNPs showed similar physicochemical properties and Figure 5 also shows that the size increase over 5 freeze-thaw cycles at -20 °C is minimal (<5 nm). The second series evaluated malate concentrations between 10 and 300 mM (Table 15). Here, minor differences were observed in particle size where the lower concentrations led to smaller particles, but the size increase over 5 freeze-thaw cycles at -20 °C was minimal and similar for all LNPs (Figure 6, <5 nm).

[0477] Table 14. Experimental design and physicochemical characteristics of LNPs produced with various malate buffer concentrations

[0478] Table 15. Experimental design and physicochemical characteristics of LNPs produced with various malate buffer concentrations

[0479] The third series evaluated malate concentrations between 0 and 10 mM (Table 16). Here, it is shown that presence of the malate acidifier is needed to efficiently encapsulate mRNA into the LNPs, which is successful even as low as 1.25 mM. Figure 7 shows however that the size increase over 5 freeze-thaw cycles at -20 °C is higher for malate concentrations below 10 mM. The data from these three series supports that the methods can be practiced at various concentrations of malate buffer, but that the preferred range lies around a concentration of 10 mM or higher to maintain the stability at -20 °C.

[0480] Table 16. Experimental design and physicochemical characteristics of LNPs produced with various malate buffer concentrations

[0481] The next experiment focused on the variation of malate concentration and pH values. Here, the malate concentration was varied between 1-100 mM and the pH value was varied between 4, 5 and 6. All LNPs were processed further with 10 mM TRIS buffer pH7.4 and evaluated at a final storage concentration of 0.1 mg / mL RNA. Table 17 shows the physicochemical properties of the produced LNPs, displaying that the use of malate buffer with a pH value of 6 led to inefficient encapsulation of mRNA into LNPs at all given malate concentrations and were not evaluated for stability over freeze-thaw cycles. Figure 8 shows the size increase over 5 freeze-thaw cycles at -20 °C, in which the preferred conditions are shown to be 10 mM or higher at pH 4.0. For pH 5.0, the size increase over freeze-thaw cycles is shown to be higher. These results support that the methods are preferably carried out with a pH value lower than 5, and with malate concentrations that lie around 10 mM or higher.

[0482] Table 17. Experimental design and physicochemical characteristics of LNPs produced with various malate buffer concentrations and pH values

[0483] The next experiment focused on various compositions of the malate acidifier buffer with various pH values at 25 mM with a production concentration of 0.2 mg / mL RNA (Table 18). Figure 9 shows LNPs size and stability with varying pH of malate acidifier in production. All particles display similar sizes and minimal increase over 5 freezethaw cycles. This data supports that the observed advantages are independent of the malate acidifier buffer composition, and the malate buffer is useable within a range of malate acidifier pH values.

[0484] Table 18. Experimental design and physicochemical properties of LNPs manufactured with combinations of malic acid and sodium salt combinations for buffer preparation The next experiments focused on various in-line buffer chemistries, pH values and concentrations to manipulate the pH of the intermediate LNPs prior to dialysis (Table 19). Here, it is shown that the higher intermediate LNP pH values negatively impact the encapsulation efficiency. Ideally, the intermediate LNP pH value should be kept < 6.0 to maintain an EE% higher than 80%. Figure 10 shows LNPs size and stability with varying intermediate LNP pH values. All particles display similar sizes and minimal increase over 5 freeze-thaw cycles. This data supports that the methods can be practiced with various intermediate LNP pH values but that lower intermediate LNP pH values (< 5.0) are preferred to maintain high encapsulation efficiencies.

[0485] Table 19. Experimental design and physicochemical properties of LNPs manufactured with various In-line dilution buffers, concentration, and pH values.

[0486] When using malate as an acidifier, additional concentrations below 25 mM show that going as low as 1 mM is still sufficient to generate LNPs with small particle size and high encapsulation efficiency (Table 20) and maintain the stability over 5 freeze thaw cycles at -20 °C (Figure 11).

[0487] Table 20. Experimental design and physicochemical properties of LNPs manufactured with in-line dilution using malate buffer at pH 4.0

[0488] Furthermore, the in-line dilution using various pH values of malate buffer was compared to in-line dilution with water. Here, it is shown that all conditions were able to generate LNPs with particle sizes in the range of 60-75 nm and high encapsulation efficiencies above 80% (Table 21). However, regarding stability over 5 freeze-thaw cycles at -20 °C, a pH value around 4.0 showed optimal results (Figure 12). Table 21. Experimental design and physicochemical properties of LNPs manufactured with in-line dilution using malate buffer at pH 4.0

[0489] These data support that the methods can be practiced with various in-line dilution concentrations and pH values of the malate acidifier, but that a malate pH around 4.0 and a concentration between 5 - 20 mM is preferred to maintain the best -20 °C stability over 5 freeze-thaw cycles.

[0490] Example 6: Impact of process parameters such as flow rate, production concentration and temperature

[0491] Study design

[0492] Given the clear preference observed for the use of a malate acidifier together with a TRIS buffer in the formulation of LNPs, experiments were performed to explore further variants of the methods, focusing on process parameters, such as flow rates, flow rate ratios, productions concentrations, process temperatures, amount of process steps and scale of production. The objectives were to demonstrate the colloidal stability of the LNPs produced with various process parameters over 5 freeze-thaw cycles at -20 °C.

[0493] Results

[0494] The first experiment focused on the combination of various production concentrations (0.1, 0.2 and 0.3 mg / mL) and RNA:Lipid:In-line dilution flow rate ratios (2: 1 :2, 3: 1 :2, 4: 1 :2). In this experiment lipid nanoparticles were manufactured according to the small- scale manufacturing process described in the Methods section. Here, the initial small- scale process was performed using a 72.5 mM malate buffer (pH 4.0) for the RNA phase dilution and a 32.5 mM malate buffer (pH 4.0) for the in-line dilution at a production concentration of 0.2 mg / mL. For other production concentrations, the RNA phase and lipid concentrations were adjusted accordingly while keeping the malate concentrations in the RNA phase and in-line phase constant. All LNPs were evaluated at a final RNA concentration of 0.1 mg / mL. The parameters had minimal impact on physicochemical properties of the LNPs (Table 22). Figure 13 shows the size and PDI for all LNPs over 5 freeze-thaw cycles at -20 °C, where all LNPs display minimal increase over 5 freeze-thaw cycles (< 5 nm). This data supports that the methods can be practiced at with various flow rate ratios and production concentrations.

[0495] Table 22. Physicochemical properties of LNPs produced with various production concentrations and flow rate ratios

[0496] In a separate experiment, the manufacturing concentration was increased further to the total of 0.5 mg / mL at the ratio of 3: 1 :2. In this experiment LNPs were manufactured according to the small-scale manufacturing process described in the Methods section. Here, the initial small-scale process was performed using a 10.0 mM malate buffer (pH 4.0) for the RNA phase dilution and a 10.0 mM malate buffer (pH 4.0) for the in-line dilution at a production concentration of 0.2 mg / mL. For other production concentrations, the RNA phase and lipid concentrations were adjusted accordingly. The malate concentration in the RNA phase was adjusted to maintain a constant output of malate in the intermediate LNPs, while keeping the malate concentrations in-line phase constant. All LNPs were evaluated at a final RNA concentration of 0.1 mg / mL. The parameters had minimal impact on physicochemical properties of the LNPs (Table 23). It is seen that the manufacturing concentration and malate concentration during initial mixing can impact LNP particle size, but that in all cases RNA is encapsulated efficiently. Figure 14 shows the size and PDI for all LNPs over 5 freeze-thaw cycles at -20 °C, where all LNPs display minimal increase over 5 freeze-thaw cycles (< 10 nm). This data supports that the methods can be practiced at various production concentrations. Table 23. Physicochemical properties of LNPs produced with various flow rates

[0497] The next experiment focused on various production flow rates (60 to 720 mL / min, Table 24) In this experiment lipid nanoparticles were manufactured according to the large-scale manufacturing process described in the Methods section. In all cases a 25 mM malate buffer at pH 4.0 was used followed by a 10 mM TRIS buffer at pH 7.4 for further processing. Figure 15 shows flow rate impact on LNP size and stability. All particles display similar sizes and minimal increase over 5 freeze-thaw cycles (<5 nm). This data supports that the methods can be practiced at a large range of production flow rates.

[0498] Table 24. Physicochemical properties of LNPs produced with various flow rates

[0499] The next experiment focused on the amount of process steps, production concentrations and process temperature. In this experiment lipid nanoparticles were manufactured according to the small-scale manufacturing process described in the Methods section. Here, the initial small-scale process was performed using a 72.5 mM malate buffer (pH 4.0) for the RNA phase dilution and a 32.5 mM malate buffer (pH 4.0) for the in-line dilution. LNPs are produced at both 0.2 and 0.3 mg / mL and dialysis is either performed in a single step using only 10 mM TRIS pH 7.4 at 4 °C or room temperature (overnight), or in a two-step process using first 32.5 mM pH 4.0 malate at 4 °C or room temperature (4 hours), followed by 10 mM pH 7.4 TRIS 4 °C or room temperature (overnight) (Table 25). Figure 16 shows LNPs size and stability with varying purification steps and temperature with malate acidifier and TRIS buffer. Change of this process temperature and steps only affected particle size but all LNPs display similar sizes and minimal increase over 5 freeze-thaw cycles at -20 °C. These data support that the methods can be practiced with various steps and temperatures for further processing. Table 25. Physicochemical properties of LNPs produced with a variation of production concentration, process steps and process temperature

[0500] The next experiment focused on the further exploration of the process temperature for the dialysis step, using various buffer exchange temperature being 13, 20, 25, and 30 °C. In this experiment lipid nanoparticles were manufactured according to the small-scale manufacturing process described in the Methods section. Here, the process was performed using a 72.5 mM malate buffer (pH 4.0) for the RNA phase dilution and a 32.5 mM malate buffer (pH 4.0) for the in-line dilution, followed using a 10 mM TRIS buffer (pH 7.4) for further processing. Change of this process temperature affected initial particle size but not encapsulation efficiency (Table 26). Figure 17 shows that all LNPs display similar sizes and minimal increase over 5 freeze-thaw cycles at -20 °C (< 10 nm). This data supports that the methods can be practiced with various process steps and that further processing with TRIS can be performed at various temperatures.

[0501] Table 26. Physicochemical properties of LNPs manufactured with various process temperatures during dialysis

[0502] The next experiment focused on the influence of malate concentration and scale of the process. In this experiment lipid nanoparticles were manufactured according to the small-scale manufacturing process described in the Methods section. Here, the initial small-scale process was performed using a 72.5 mM malate buffer (pH 4.0) for the RNA phase dilution and a 32.5 mM malate buffer (pH 4.0) for the in-line dilution, followed using a 10 mM TRIS buffer (pH 7.4) for further processing (Table 27). Figure

[0503] 18 displays the minimal size increase over 5 freeze-thaw cycles at -20 °C (< 5 nm).

[0504] Table 27. Physicochemical properties of LNPs with higher malate concentrations in the small-scale process

[0505] The manufacturing was repeated on a larger scale according with the same malate and TRIS buffer concentrations as for the small-scale process, and the LNPs were finally evaluated at 3 concentrations being 0.1, 0.5 and 1.0 mg / mL RNA (Table 28). In this experiment lipid nanoparticles were manufactured according to the large-scale manufacturing process described in the Methods section. Figure 19 shows the minimal size increase over 5 freeze-thaw cycles at -20 °C for all LNP concentrations (< 10 nm).

[0506] Table 28. Physicochemical properties of LNPs with higher malate concentrations in the large-scale process

[0507] Next, the larger scale manufacturing was performed using a lower malate buffer concentration, being 10 mM malate buffer (pH 4.0) for the RNA phase dilution and a 10 mM malate buffer (pH 4.0) for the in-line dilution, followed using a 10 mM TRIS buffer (pH 7.4) for further processing (Table 29). Figure 20 shows the minimal size increase over 5 freeze-thaw cycles at -20 °C for all LNP concentrations (< 5 nm).

[0508] Table 29. Physicochemical properties of LNPs with lower malate concentrations in the large-scale process

[0509] The large-scale manufacturing with lower malate concentration was repeated using a different RNA, being Ab2 instead of Abl. The change of the construct showed to have no impact on the physicochemical properties of the LNPs (Table 30). Figure 21 also shows the size of the LNPs after manufacturing and repeated freeze-thaw cycles at - 20 °C. All particles display similar sizes and minimal increase over 5 freeze-thaw cycles (< 5nm). Table 30. Physicochemical properties of LNPs with 10 mM malate concentrations in the large-scale process

[0510] Finally, the scale of LNP manufacturing was further evaluated by increasing from 200 mg RNA input to 3 grams. In this experiment LNPs were manufactured according to the large-scale or the pilot scale manufacturing process described in the Methods section. In both cases, 10 mM malate buffer (pH 4.0) was used for the RNA phase dilution and a 10 mM malate buffer (pH 4.0) for the in-line dilution, followed using a 10 mM TRIS buffer (pH 7.4) for further processing. In the case of the large-scale batch, the LNPs were evaluated at a concentration of 0.1 mg / mL (A-l) and 1.0 mg / mL (A-2), whereas the LNPs from the pilot scale batch were evaluated at concentrations of 0.1 mg / mL (B-l), 1.0 mg / mL (B-2) and 2.0 mg / mL (B-3). All LNPs showed similar particles of approximately 85 nm and high encapsulation efficiencies (Table 31). Furthermore, Figure 22 shows the minimal size increase over 5 freeze-thaw cycles at - 20 °C for all LNP concentrations (< 5 nm).

[0511] Table 31. Physicochemical properties of LNPs with 10 mM malate concentrations in the large-scale and pilot scale processes

[0512] This series of experiments support that the methods can be practiced at various production scales, with various malate buffer concentrations and using various RNA constructs.

[0513] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biochemistry, molecular biology, biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. CLAIMS1. A method of producing a composition containing a nucleic acid-lipid particle, the method comprising the steps (a) to (d):(a) preparing a first mixture which is a lipid mixture comprising a cationically ionizable lipid in a water-soluble organic solvent;(b) preparing a second mixture in aqueous solution, the second mixture comprising (i) a nucleic acid and (ii) malate buffer;(c) mixing the first mixture with the second mixture to produce an intermediate composition comprising the nucleic acid-lipid particle; and(d) further processing of the intermediate composition by buffer exchange, wherein the buffer used in the buffer exchange comprises tris(hydroxymethyl)- aminomethane (Tris) and / or a pharmaceutically acceptable salt thereof, to produce the composition comprising the nucleic acid-lipid particle.

2. A method according to claim 1, wherein the nucleic acid is RNA, such as mRNA.

3. A method according to claim 1 or claim 2, wherein the nucleic acid encodes a binding agent, such as an antibody.

4. A method according to any preceding claim, wherein step (b) is carried out at a pH of 2.5 to 5.5.

5. A method according to any preceding claim, wherein the concentration of the malate anion in the malate buffer used in step (b) is 10 to 300 mM.

6. A method according to any preceding claim, further comprising the following step (c’) after step (c) but before step (d):(c’) dilution of the intermediate composition comprising the lipid particle.

7. A method according to claim 6, wherein the dilution step (c’) is carried out using a malate buffer solution.

8. A method according to any preceding claim, further comprising the following step (e) after step (d):(e) adding a cryoprotectant to the lipid particle; preferably wherein the cryoprotectant used in step (e) is a carbohydrate, such as a monosaccharide or disaccharide; more preferably selected from the group consisting of sucrose, trehalose and glucose, or a mixture of any thereof; most preferably sucrose.

9. A method according to any preceding claim, wherein the cationically ionizable lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) (ALC-0366).

10. A method according to any preceding claim, wherein the lipid mixture further comprises one or more additional lipids; such as(a) a neutral or zwitterionic lipid; preferably wherein the neutral or zwitterionic lipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); di ol eoy Iphosphati dy 1 ethanol amine (DOPE) ; l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG);N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); or a mixture of any thereof; more preferably distearoylphosphatidylcholine (DSPC); and / or(b) a steroid, preferably cholesterol; and / or(c) a grafted lipid; preferably a PEG-conjugated lipid; more preferably [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

11. A method according to any preceding claim, wherein the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2 -butyl octanoate) (ALC- 0366), preferably in an amount of 30 to 60 wt.% of the total lipids present in the lipid mixture;distearoylphosphatidylcholine (DSPC), preferably in an amount of 5 to 15 wt.% of the total lipids present in the lipid mixture; cholesterol, preferably in an amount of 20 to 60 wt.% of the total lipids present in the lipid mixture; and2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), preferably in an amount of 0.5 to 5 wt.% of the total lipids present in the lipid mixture.

12. A method according to claim 11, wherein the lipid mixture comprises: ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2 -butyl octanoate) (ALC- 0366), in an amount of 47.5 wt.% of the total lipids present in the lipid mixture; distearoylphosphatidylcholine (DSPC), in an amount of 10 wt.% of the total lipids present in the lipid mixture; cholesterol, in an amount of 40.7 wt.% of the total lipids present in the lipid mixture; and2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), in an amount of 1.8 wt.% of the total lipids present in the lipid mixture.

13. A composition comprising a nucleic acid-lipid particle, obtainable by the method of any preceding claim.

14. A composition according to claim 13, which has a pH of about 7.0 to about 8.0 and comprises:(a) the nucleic acid-lipid particle; and(b) Tris buffer, at a concentration of about 5 to about 100 mM.

15. A composition comprising a nucleic acid-lipid particle according to claim 13 or14, for use in medicine.

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