WO2024089229A1 - Formulations améliorées comprenant des transporteurs à base de lipides encapsulant de l'arn - Google Patents
Formulations améliorées comprenant des transporteurs à base de lipides encapsulant de l'arn Download PDFInfo
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- WO2024089229A1 WO2024089229A1 PCT/EP2023/080036 EP2023080036W WO2024089229A1 WO 2024089229 A1 WO2024089229 A1 WO 2024089229A1 EP 2023080036 W EP2023080036 W EP 2023080036W WO 2024089229 A1 WO2024089229 A1 WO 2024089229A1
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- rna
- pharmaceutical composition
- lipid
- glycerol
- sucrose
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Classifications
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- RNA-based therapeutics include RNA molecules encoding antigens for use as vaccines.
- RNA molecules for replacement therapies, e.g. providing missing proteins such as growth factors or enzymes to patients.
- noncoding RNAs suitable for genome editing e.g. CRISPR/Cas9 guide RNAs
- RNA-based therapeutics with the use in immunotherapy, gene therapy and vaccination belong to the most promising and quickly developing therapeutic modalities in modem medicine.
- RNA is typically delivered by lipid-based carrier systems including liposomes and lipid nanoparticles. These lipid-based carriers typically comprise the RNA and can improve intracellular delivery and effectiveness of the RNA.
- compositions comprising lipid-based carriers comprising RNA are its instability. It is generally known that the physio-chemical stability of RNA molecules is extremely low. RNA is susceptible to hydrolysis by ubiquitous ribonucleases or e.g. by divalent cations and is typically rapidly degraded, e.g. already after a few hours or days. Rapid degradation occurs even in the absence of RNases, e.g. when RNA is stored in solution at room temperature or at -20 Q C for a few hours or days even when formulated in lipid-carriers. Moreover, degradation of the lipid-based carriers and/or the RNA is more pronounced at low concentrations.
- compositions of lipid carriers encapsulating RNA are typically stored frozen at -60°C or even -80°C. Such storage conditions, however, do not sufficiently prevent a loss of function over time. Additionally, applying such conditions is very cost-intensive, especially for shipping and storage.
- the underlying object is therefore to provide a storage-stable pharmaceutical composition comprising lipid-based carriers comprising RNA that displays advantageous temperature stability in liquid form (e.g. at 5°C) and/or at -20°C or -40°C.
- a further object is to provide a storage-stable pharmaceutical composition comprising lipid-based carriers comprising RNA that displays advantageous stability at very low concentration, and advantageous stability upon freeze-thaw step.
- a further object is to provide a storage-stable pharmaceutical composition comprising lipid-based carriers comprising RNA that does not exceed a certain tonicity to guarantee a well tolerable administration (e.g. via intramuscular injection).
- the objects of the invention are inter alia solved by providing a pharmaceutical composition of lipid-based carriers comprising RNA, wherein the lipid-based carriers are formulated in an improved and optimized buffer system.
- Lipid-based carrier formulations comprising or encapsulating RNA typically require storage at very low temperatures in frozen form, e.g. at -60°C or even at -80°C. At these very low temperatures, the quality characteristics of the lipid-based carriers, e.g. the LNPs, and the RNA are typically well preserved. However, in the fridge or at freezing temperature - 20°C/-40°C, such formulations are typically less stable and can only be stored for a few days without quality loss.
- the inventors identified buffer systems for stabilizing compositions comprising lipid-based carriers comprising or encapsulating RNA.
- compositions comprising lipid-based carriers comprising or encapsulating RNA that are formulated or contained in the optimized buffer allow for a long-term storage at -20°C/-40°C and, additionally at 5°C.
- these lipid-based carriers contained in the improved buffer can be stored at or diluted down to low concentrations which is desirable if e.g. only one dose of a medicament is to be administered, e.g. by using pre-filled syringe.
- the present invention is inter alia directed to pharmaceutical compositions or vaccines comprising lipid- based carriers, wherein the lipid-based earners comprise RNA and are contained in a buffer system that comprises at least one polyol component (e.g. glycerol), at least one sugar component (e.g. sucrose), and at least one buffer agent (e.g. Tris).
- a buffer system that comprises at least one polyol component (e.g. glycerol), at least one sugar component (e.g. sucrose), and at least one buffer agent (e.g. Tris).
- the improved formulations advantageously stabilize the lipid-based carriers comprising the RNA during storage.
- Further aspects inter alia relate to methods of manufacturing, methods for stabilization, and various uses. Also provided are methods of treatment and medical uses.
- the present invention provides a pharmaceutical composition
- lipid-based carriers wherein the lipid-based earners comprise or encapsulate RNA and are contained in a buffer system that comprises a) at least one polyol component, preferably glycerol; b) at least one sugar component, preferably sucrose; c) at least one buffer agent, preferably T ris.
- the buffer system comprises a) at least 50mM glycerol; b) at least 50mM of at least one sugar component, preferably sucrose; c) at least one buffer agent, preferably T ris.
- the added total concentration of glycerol and the at least one sugar component is preferably in a range of 10OmM to 400mM, preferably in a range of 200mM to 300mM.
- the lipid-based carriers are contained in a buffer system that comprises a) 50mM to 200mM glycerol; b) 50mM to 200mM sucrose (as sugar component); c) at least 10mM T ris as buffer agent, preferably more than 20mM, e.g. 30mM.
- the lipid-based carriers encapsulate RNA and are contained in a buffer system that comprises 100mM glycerol, 150mM sucrose, and 10mM, preferably more than 20mM, e.g. 30mM Tris as buffer agent.
- the lipid-based carriers encapsulate RNA and are contained in a buffer system that comprises 150mM glycerol, 10OmM sucrose, and 10mM, preferably more than 20mM, e.g. 30mM T ris as buffer agent.
- the lipid-based carriers are selected from lipid nanoparticles.
- the present invention provides vaccine comprising the pharmaceutical composition of the first aspect.
- the present invention provides a syringe, for injection containing the pharmaceutical composition of the first aspect or the vaccine of the second aspect.
- the present invention provides a kit or kit of parts comprising the pharmaceutical composition of the first aspect, the vaccine of the second aspect, or the syringe of the third aspect.
- the present invention provides first, second and further medical uses of the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe of the third aspect, or the kit or kit of parts of the fourth aspect.
- the present invention provides a method of treating or preventing a disease, disorder or condition wherein the method comprises applying or administering to a subject in need thereof the pharmaceutical composition of the first aspect, and/or the vaccine of the second aspect, and/or the syringe for injection of the third aspect, and/or the kit or kit of the fourth aspect.
- the seventh aspect relates to a buffer system for preparing, storing, diluting, or freezing of lipid-based carriers comprising RNA, wherein the buffer preferably comprises 50mM to 200mM glycerol, 50mM to 200mM sucrose (as sugar component), Tris as buffer agent at preferably more than 20mM, and wherein the added total concentration for both glycerol and sucrose is preferably in a range of 10OmM to 400mM.
- the invention provides the use of the buffer system for storing, diluting, stabilizing, producing, or freezing lipid-based carriers comprising or encapsulating RNA and/or for reducing and/or preventing LEPs in compositions comprising lipid-based carriers comprising or encapsulating RNA, preferably wherein the lipid-based carriers comprising or encapsulating RNA
- the invention provides the use of glycerol for preserving quality attributes of lipid-based carriers comprising or encapsulating RNA at -20°C or -40°C, wherein the quality attributes are selected from RNA encapsulation, Z-average particle size, PDI and/or RNA integrity
- the invention provides the use of T ris for reducing or preventing LEPs or for preserving Z-average particle size and/or PDI of lipid-based carriers comprising or encapsulating RNA.
- a further aspect relates to a method of manufacturing the pharmaceutical composition, the vaccine, or the syringe.
- Another aspect relates to a method of stabilizing a pharmaceutical composition or vaccine comprising lipid-based carriers comprising or encapsulating RNA.
- Cationisable means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also in non-aqueous environments where no pH value can be determined, a cationisable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationisable or polycationisable compound, in particular the pKa of the respective cationisable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged.
- the fraction of cationisable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art.
- a compound or moiety is cationisable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e. under physiological conditions, particularly under physiological salt conditions of the cell in vivo.
- the cationisable compound or moiety is predominantly neutral at physiological pH values, e.g. about 7.0-7.4, but becomes positively charged at lower pH values.
- the preferred range of pKa for the cationisable compound or moiety is about 5 to about 7.
- Coding sequence/codinq region The terms “coding sequence” or “coding region” and the abbreviation “cds” as used herein refer to a sequence of several nucleotide triplets that may be translated into a peptide or protein.
- a cds in the context of the present invention may be an RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon and preferably terminates with a stop codon.
- Delta change values relate to the change (or difference) of a respective value over the time of storage compared to the value of day 0 (that is, the day when the storage period starts).
- a “delta change 28d” value is calculated by subtracting the respective parameter value (e.g. RNA integrity, RNA encapsulation, etc.) measured at day 28 of storage with the respective value at day 0. Accordingly, if the RNA integrity value measured at day 0 is 80%, and the RNA integrity value measured after a storage of 28 days is 70%, the delta change 28 days value is 10%.
- Derived from The term “derived from” as used throughout the present specification in the context of a nucleic acid, i.e.
- nucleic acid “derived from” (another) nucleic acid means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived.
- sequence identity is typically calculated for the same types of nucleic acids, i.e. for DNA sequences or for RNA sequences.
- RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence).
- sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined.
- a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g.
- amino acid sequences e.g. antigenic peptides or proteins
- the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g. at least 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.
- fragment' as used throughout the present specification in the context of an RNA sequence or an amino acid sequence may typically be a shorter portion of a full-length sequence of e.g. an RNA sequence or an amino acid sequence.
- a fragment consists of a sequence that is identical to the corresponding stretch within the full- length sequence.
- a typical fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e. full- length) molecule from which the fragment is derived (e.g. a virus protein).
- fragment as used throughout the present specification in the context of proteins or peptides may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence, N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original protein. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
- heterologous refers to a sequence (e.g. RNA, amino acid) has to be understood as a sequence that is derived from another gene, another allele, or e.g. another species or virus.
- Two sequences are typically understood to be “heterologous” if they are not derivable from the same gene or from the same allele. I.e., although heterologous sequences may be derivable from the same organism or virus, in nature, they do not occur in the same nucleic acid or protein.
- Identity refers to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, e.g. RNA sequences or amino acid (aa) sequences as defined herein, preferably the aa sequences encoded by the RNA as defined herein or the aa sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence.
- a position in the first sequence is occupied by the same residue as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using an algorithm, e.g. BLAST.
- RNA species In the context of the invention, the term “RNA species” is not restricted to mean one single molecule but is understood to comprise an ensemble of essentially identical RNA molecules. Accordingly, it may relate to a plurality of essentially identical RNA molecules.
- variant in the context of an RNA sequence refers to a variant of an RNA sequence derived from another nucleic acid sequence.
- a variant of an RNA sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
- a variant of an RNA sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from.
- the variant is a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
- a “variant” of an RNA sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides of such sequence.
- the term “variant’ in the context of proteins or peptides is e.g. intended to refer to a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(sysubstitution(s), such as one or more substituted, inserted and/or deleted amino acid(s).
- these fragments and/or variants have the same, or a comparable specific antigenic property (immunogenic variants, antigenic variants).
- Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra).
- CD spectra circular dichroism spectra
- a “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide.
- a variant of a protein comprises a functional variant of the protein, which means, in the context of the invention, that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity as the protein it is derived from.
- sequences e.g. amino acid sequences or nucleic acid sequences (RNA, DNA) are explicitly incorporated herein by reference.
- the present invention provides a pharmaceutical composition comprising lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in an optimized buffer system.
- the lipid-based carriers that comprise or encapsulate RNA are formulated in an optimized buffer system as further specified herein.
- the lipid-based carriers are dispersed in the buffer system.
- the optimized buffer system is an aqueous phase buffer.
- the lipid-based carriers comprising RNA are contained in a buffer system that comprises a) at least one polyol component, preferably glycerol; b) at least one sugar component; c) at least one buffer agent;
- the at least one polyol component is selected from glycerol.
- the preferred polyol component glycerol may be substituted by a functionally equivalent polyol component.
- Suitable functionally equivalent polyols may be selected from a diol, a triol, or a sugar alcohol.
- glycerol may be substituted by any one of the compounds selected from 1 ,2-propanediol, 1 ,3-propanediol, (+/-)-2-methyl-2,4- pentanediol, 1 ,6-hexanediol, 1 ,2-butanediol, 2,3-butenediol, 1 ,4-butanediol, ethylene glycol, or diethylene glycol, or any combination thereof. Accordingly, embodiments of the invention relating to specific concentrations, amounts, or ratios of the preferred component “glycerol” may likewise be applicable to other functionally equivalent polyol compounds as defined herein.
- Buffers comprising glycerol show advantageous effects.
- lipid-based carriers comprising RNA are well preserved in particular at temperatures below 0°C (e.g. at -20°C or at -40°C) during storage in a glycerol comprising buffer.
- these advantageous effects inter alia include an improved preservation of the RNA encapsulation, the Z-average size of the lipid-based carriers, the RNA integrity, and the PDI.
- the lipid-based carriers comprising RNA are contained in a buffer system that comprises a) glycerol, preferably at least 50mM glycerol; b) at least one sugar component; c) at least one buffer agent.
- the concentration of glycerol is less than 250mM, less than 225mM, less than 200mM, less than 175mM. In preferred embodiments, the concentration of glycerol is less than 250mM.
- the concentration of glycerol is at least 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM. More preferably, the concentration of glycerol is at least 50mM or at least 10OmM.
- the concentration of glycerol ranges from 10mM to 300mM, 50mM to 300mM, 50mM to 250mM, 50mM to 200mM, 75mM to 200mM, 100mM to 200mM, 100mM to 150mM.
- the concentration of glycerol ranges from 50mM to 250mM. More preferably, the concentration of glycerol ranges from 50mM to 200mM. Even more preferably, the concentration of glycerol ranges from 100mM to 200mM. Even more preferably, the concentration of glycerol ranges from 10OmM to 150mM.
- the concentration of glycerol is at about 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 220mM, 240mM, 260mM, 280mM, 300mM, 320mM, 340mM, 360mM, 380mM, 400mM, 420mM, 440mM, 460mM, 480mM, 500mM.
- the concentration of glycerol is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM. In more preferred embodiments, the concentration of glycerol is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM.
- the concentration of glycerol is at about 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 175mM.
- the concentration of glycerol is at about 100mM.
- the concentration of glycerol is at about 150mM.
- the at least one sugar component e.g. sucrose
- a sugar component in the context of the invention has to be understood as any type of monosaccharide or disaccharide.
- sugar component does not encompass polyols or sugar alcohols such as glycerol.
- Buffers comprising at least one sugar component have the advantageous effect that lipid-based carriers comprising RNA are well preserved in particular at temperatures below 0°C (e.g. at -20°C or at -40°C) during storage.
- these advantageous effects inter alia include an improved preservation of the Z-average particle size of the lipid-based carriers, the RNA integrity, and the PDI.
- the at least one sugar component is selected from sucrose, trehalose, maltose, lactose, dextrose, sorbitol, xylitol, trehalose, sucrose, raffinose, dextran, inulin, or any combination thereof.
- the at least one sugar component is selected from a disaccharide.
- the at least one sugar component comprises or consists of sucrose. In some embodiments, the at least one sugar component comprises sucrose and does not comprise trehalose.
- the at least one sugar component is selected from sucrose.
- the lipid-based carriers comprising RNA are contained in a buffer system that comprises a) glycerol, preferably at least 50mM glycerol; b) at least one sugar component, preferably at least 50mM of at least one sugar component; c) at least one buffer agent.
- the concentration of the at least one sugar component is at least 10mM, 25mM, 50mM, 75mM, 10OmM, 125mM, 150mM. More preferably, the concentration of the at least one sugar component is at least 100mM.
- the concentration of the at least one sugar component is less than 250mM, less than 225mM, less than 200mM, less than 175mM. In preferred embodiments, the concentration of the at least one sugar component is less than 250mM.
- the concentration of the at least one sugar component ranges from 10mM to 300mM, 50mM to 300mM, 50mM to 250mM, 50mM to 200mM, 75mM to 200mM, 100mM to 200mM, 75mM to 175mM, 100mM to 150mM.
- the concentration of the at least one sugar component ranges from 50mM to 250mM. More preferably, the concentration of the at least one sugar component ranges from 50mM to 200mM. Even more preferably, the concentration of the at least one sugar component ranges from 10OmM to 200mM. Even more preferably, the concentration of the at least one sugar component ranges from 10OmM to 150mM.
- the concentration of the at least one sugar component is about 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 220mM, 240mM, 260mM, 280mM, 300mM, 320mM, 340mM, 360mM, 380mM, 400mM, 420mM, 440mM, 460mM, 480mM, 500mM.
- the concentration of the at least one sugar component is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM. In more preferred embodiments, the concentration of the at least one sugar component is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM.
- the concentration of the at least one sugar component is at about 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 175mM.
- the concentration of the at least one sugar component is at about 150mM. In other preferred embodiments, the concentration of the at least one sugar component is at about 10OmM.
- the at least one sugar component of the buffer (e.g. sucrose) and the glycerol component have different effects on the lipid-based carriers that comprise RNA (see e.g. Table A).
- the at least one sugar component is suitable for preserving the RNA integrity.
- the at glycerol component is suitable for preserving the integrity of the lipid-based carriers (e.g. Z-average particle size, encapsulation, PDI).
- the concentration of the at least one sugar component e.g. sucrose
- the concentration glycerol may be increased.
- the added total concentration of glycerol and the at least one sugar component does not exceed 500mM, 450mM, 400mM, 350mM, 300mM.
- the added total concentration of glycerol and the at least one sugar component does not exceed 400mM, 350mM, 300mM.
- glycerol and the at least one sugar component as defined herein are desired for pharmaceutical compositions that are configured for injection into subjects (e.g. intramuscular injection or intraocular injection), e.g. human subjects.
- Exceeding values of 400mM would increase the tonicity of the composition and hence cause pain upon injection, e.g. upon intramuscular injection.
- one objection of the invention is to provide a well tolerable pharmaceutical composition for injection that is storage stable, which requires to balance the respective amounts of the components of the buffer system.
- RNA integrity in particular when the composition is stored in liquid form (e.g. at 5°C or25°C). Therefore, the added total concentration of glycerol and the at least one sugar component should not exceed a certain value or should be in a range specified herein to be well tolerable upon administration and/or to avoid or reduce RNA degradation upon storage of the composition in liquid form.
- the lipid-based carriers comprising RNA are contained in a buffer system that comprises a) glycerol, preferably at least 50mM glycerol; b) at least one sugar component, preferably at least 50mM of at least one sugar component; c) at least one buffer agent, wherein the added total concentration of glycerol and the at least one sugar component is preferably in a range of 100mM to 400mM.
- the added total concentration of glycerol and the at least one sugar component is in a range of 100mM to 400mM, 150mM to 400mM, 200mM to 400mM, 150mM to 350mM, 200mM to 350mM, 200mM to 300mM.
- the added total concentration of glycerol and the at least one sugar component is at about 100mM, 150mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, 375mM, 400mM.
- the added total concentration of glycerol and the at least one sugar component is at about 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, more preferably at about 250mM.
- the molar ratio of glycerol to the at least one sugar component is ranging from 1 :10 to 10:1 , or 1 :2 to 2:1. In preferred embodiments, the molar ratio of glycerol to the at least one sugar component (e.g. sucrose) is ranging from 1 :1 .5 to 1 .5:1 . In preferred embodiments, the molar ratio of glycerol to the at least one sugar component is at about 1 to 1 .5 (e.g. in embodiments where the RNA has a length of more than 3000 nucleotides). In other preferred embodiments, the molar ratio of glycerol to the at least one sugar component is at about 1.5 to 1 (e.g. in embodiments where the RNA has a length of less than 3000 nucleotides).
- the buffer system comprises 50mM to 250mM glycerol and 50mM to 250mM sugar component (e.g. sucrose). In more preferred embodiments, the buffer comprises 50mM to 200mM glycerol and 50mM to 200mM sugar component (e.g. sucrose). In even more preferred embodiments, the buffer comprises 10OmM to 200mM glycerol and 10OmM to 200mM sugar component (e.g. sucrose). In even more preferred embodiments, the buffer comprises 100mM to 150mM glycerol and 100mM to 150mM sugar component (e.g. sucrose). In embodiments, the buffer system comprises:
- 150mM glycerol and 50mM to 150mM sugar component e.g. sucrose
- the sugar component for each combination is selected from sucrose.
- the buffer system comprises 100mM glycerol and 100mM to 200mM sugar component (e.g. sucrose) or 150mM glycerol and 50mM to 150mM sugar component (e.g. sucrose).
- 100mM glycerol and 100mM to 200mM sugar component e.g. sucrose
- 150mM glycerol and 50mM to 150mM sugar component e.g. sucrose
- the buffer system comprises:
- 150mM sugar component e.g. sucrose
- sugar component e.g. sucrose
- sugar component e.g. sucrose
- 225mM sugar component e.g. sucrose
- 50mM to 75mM glycerol 50mM to 75mM glycerol
- the sugar component for each combination is selected from sucrose.
- the buffer system comprises 10OmM sugar component and 10OmM to 200mM glycerol or 150mM sugar component and 50mM to 150mM glycerol.
- the buffer system comprises:
- 125mM glycerol and 125mM sugar component e.g. sucrose
- glycerol • 125mM glycerol and 150mM sugar component (e.g. sucrose);
- 150mM glycerol and 150mM sugar component e.g. sucrose
- the sugar component for each combination is selected from sucrose.
- the buffer system comprises 50mM to 200mM glycerol and 50mM to 200mM sugar component, wherein the added total concentration of glycerol and sugar component is preferably in a range of 200mM to 300mM, more preferably at about 250mM.
- the buffer system comprises:
- 125mM glycerol and 125mM sugar component e.g. sucrose
- the sugar component for each combination is selected from sucrose.
- the buffer system comprises 100mM to 200mM glycerol, preferably 100mM to 150mM glycerol, and 100mM to 200mM sugar component, preferably 100mM to 150mM sugar component, wherein the added total concentration of glycerol and sugar component is preferably in a range of 200mM to 300mM, more preferably at about 250mM.
- the buffer system comprises:
- the sugar component for each combination is selected from sucrose.
- the buffer system comprises 100mM glycerol and 150mM sugar component, preferably sucrose. In other preferred embodiments, the buffer system comprises 150mM glycerol and 100mM sugar component, preferably sucrose.
- the buffer system comprises
- the buffer system comprises 100mM glycerol and 150mM sucrose. In other preferred embodiment, the buffer system comprises 150mM glycerol and 10OmM sucrose.
- the at least one buffer agent is selected from a non-phosphate buffer agent.
- the at least one buffer agent is not selected from Na-Phosphate or K-Phosphate.
- Buffers comprising non-phosphate buffer agents have advantages over phosphate buffers as inter alia the Z-average size of the lipid-based carriers, the RNA encapsulation, and the PDI are better preserved in non-phosphate buffers and as the formation of LPEs is reduced in non-phosphate buffers.
- the at least one buffer agent is selected from Tris, Bis-tris-methane, triethanolamine (TEA), imidazole, histidine (e.g. histidine-HCI), citrate (e.g. sodium citrate), MES, MOPS, HEPES, sodium succinate, sodium malate, sodium carbonate.
- the at least one buffer agent is selected from a Good’s buffer.
- the at least one buffer agent is selected from bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris-methane or BTM) and its protonated form, triethanolamine (TEA) and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-l-ol and its protonated form, triethylamine and its protonated form, 2-[2- (diethylamino)ethoxy]ethan-l -ol and its protonated form, diethanolamine and its protonated form, N,N'-bis(2- hydroxyethyl)piperazine and its protonated form, N,N,N , ,N'-tetrakis(2-hydroxyethyl)ethylenediamine and its protonated form, and trimethylamine N-oxide and its protonated form.
- BTM bis(2-hydroxyethyl)amino-tris(
- the buffer substance is selected from bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris-methane or BTM) and its protonated form, triethanolamine (TEA) and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-l-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan- 1 -ol and its protonated form, and N,N'-bis(2-hydroxyethyl)piperazine and its protonated form.
- the at least one buffer agent comprises or is triethanolamine (TEA) or its protonated form.
- the at least one buffer agent comprises at least one amine group, e.g. a monoamine.
- the monoamine may be a primary amine, a secondary amine, or a tertiary amine.
- the at least one buffer agent is selected from Tris (Tris(hydroxymethyl)aminomethane; Trometamol).
- Tris is a buffer substance that comprises at least one amine group.
- the concentration of the at least one buffer agent is at least 10mM, 15mM, 20mM, 25mM, 30mM. In preferred embodiments, the concentration of the at least one buffer agent is at least 25mM or 30mM.
- the concentration of the at least one buffer agent is more than 10mM, more than 15mM, more than 20mM, more than 25mM. In preferred embodiments, the concentration of the at least one buffer agent is more than 20mM.
- the concentration of the at least one buffer agent is in a range from 10mM to 10OmM, 10mM to 75mM, 10mM to 50mM, 15mM to 50mM, 20mM to 50mM, 25mM to 50mM, 25mM to 40mM, 25mM to 35mM. In particularly preferred embodiments, the concentration of the at least one buffer agent is in a range from 20mM to 50mM, 20mM to 40mM, 20mM to 30mM, 25mM to 50mM, 25mM to 40mM, 25mM to 35mM.
- the concentration of the at least one buffer agent is at about 10mM, 11 mM, 12mM, 13mM, 14mM, 14mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21 mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31 mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41 mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, or 50mM.
- the concentration of the at least one buffer agent is at about 20mM, 21 mM, 21 mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31 mM, 32mM, 33mM, 34mM, 35mM.
- the concentration the at least one buffer agent is at about 10mM.
- the concentration of the at least one buffer agent is at about 20mM.
- the concentration of the at least one buffer agent is at about 30mM.
- the at least one buffer agent is suitably selected from T ris.
- Buffers comprising at least one amine group such as T ris as a buffer agent have the advantageous effect that lipid-based carriers comprising RNA are well preserved in frozen or liquid form during storage.
- these advantageous effects inter alia include an improved preservation of the Z-average size of the lipid-based carriers, an improved preservation of the PDI, and the prevention or reduction of LEPs.
- the pharmaceutical composition may comprise residues of a phosphate buffer (even in preferred embodiments where the lipid-based carriers are formulated in a phosphate-free buffer).
- the pharmaceutical composition comprising lipid-based carriers may be manufactured using a phosphate buffer and subsequently transferred to the buffer system of the invention by e.g. dilution and/or filtration.
- the pharmaceutical composition comprises T ris as the at least one buffer agent of the buffer system and the composition may additionally comprise residues of phosphate (if a phosphate buffer has been used e.g. in the manufacturing process of the lipid-based carriers), in particular less than 1 mM phosphate, preferably less than 0.5mM phosphate.
- the buffer system comprises Tris at a concentration of more than 20mM or 30mM.
- Tris at a concentration of more than 20mM or 30mM.
- concentration of Tris may be reduced, e.g. to a value of less than 30mM (e.g. 15mM, 20mM, 25mM), preferably to a value of about 10mM or about 20mM.
- glycerol preferably sucrose
- the at least one buffer agent e.g. Tris
- the lipid-based carriers encapsulate RNA are contained in a buffer system that comprises a) glycerol, preferably at a concentration of at least 50mM; b) sucrose, preferably at a concentration of at least 50mM; c) at least one buffer agent, preferably T ris.
- the buffer system comprises 50mM to 250mM glycerol, 50mM to 250mM sugar component, and preferably at least 10mM or more than 20mM buffer agent (e.g. 30mM), and the added total concentration for glycerol and sugar component is preferably in a range of 100mM to 400mM.
- the buffer system comprises 50mM to 200mM glycerol, 50mM to 200mM sugar component, and at least 10mM or more than 20mM buffer agent (e.g. 30mM), and the added total concentration for glycerol and sugar component is preferably in a range of 200mM to 300mM.
- the buffer system comprises 100mM to 200mM glycerol, preferably 100mM to 150mM glycerol, and 100mM to 200mM sugar component, preferably 100mM to 150mM sugar component, and at least 10mM or more than 20mM buffer agent (e.g. 30mM), and the added total concentration for glycerol and sugar component is preferably in a range of 200mM to 300mM.
- the buffer system comprises glycerol and the sugar component is selected from sucrose and the buffer agent is selected from Tris.
- the buffer system comprises a) at least 50mM glycerol; b) at least 50mM sucrose (as a sugar component) and c) Tris (as buffer agent) preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration of glycerol and the at least one sugar component is preferably in a range of 100mM to 400mM.
- the buffer system comprises a) 50mM to 250mM glycerol; b) 50mM to 250mM sucrose (as a sugar component) and c) Tris (as buffer agent) preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration of glycerol and the at least one sugar component is in a range of 100mM to 400mM.
- the buffer system comprises a) 50mM to 200mM glycerol; b) 50mM to 200mM sucrose (as a sugar component) and c) Tris (as buffer agent) at a concentration at least 10mM, preferably more than 20mM;
- the buffer system comprises a) 50mM to 200mM glycerol; b) 50mM to 200mM sucrose (as a sugar component) and c) Tris (as buffer agent) at a concentration of at least 10mM or more than 20m M; and wherein the added total concentration for glycerol and sucrose is preferably in a range of 200mM to 300mM.
- the buffer system comprises a) 10OmM to 200mM glycerol; b) 10OmM to 200mM sucrose (as a sugar component) and c) Tris (as buffer agent) at a concentration of at least 10mM or more than 20mM, preferably 30mM; and wherein the added total concentration for glycerol and sucrose is in a range of 200mM to 300mM, preferably at about 250mM.
- the buffer system comprises a) 10OmM to 150mM glycerol; b) 10OmM to 150mM sucrose (as a sugar component) and c) Tris (as buffer agent) at a concentration of at least 10mM or more than 20mM, preferably 30mM; and wherein the added total concentration for glycerol and sucrose is in a range of 200mM to 300mM, preferably at about 250mM.
- the buffer system comprises
- the buffer system comprises
- the buffer system comprises
- 140mM glycerol 140mM sucrose, and at least 10mM or more than 20mM Tris, preferably 30mM Tris;
- the buffer system comprises 150mM glycerol, 100mM sucrose, and 10mM to 50mM Tris.
- the buffer comprises 150mM glycerol, 100mM sucrose, and 10mM, 20mM, 30mM, 40mM or 50mM T ris.
- the buffer comprises 150mM glycerol, 10OmM sucrose, and more than 20mM T ris, in particular 30mM Tris.
- the lipid-based carriers are contained in a buffer system that comprises a) 150mM glycerol; b) 10OmM sucrose; c) at least 10mM or more than 20mM T ris, preferably 30mM T ris.
- the buffer system comprises 100mM glycerol, 150mM sucrose, and 10mM to 50mM Tris. In embodiments, the buffer comprises 100mM glycerol, 150mM sucrose, and 10mM, 20mM, 30mM, 40mM or 50mM Tris. In preferred embodiments, the buffer system comprises 100mM glycerol, 150mM sucrose, and Tris at preferably more than 20mM, in particular 30mM.
- the lipid-based carriers are contained in a buffer system that comprises a) 10OmM glycerol; b) 150mM sucrose; c) at least 10mM or more than 20mM T ris, preferably 30mM T ris.
- the buffer system in which the lipid-based carriers are contained is essentially free of NaCI and/or essentially free of phosphate.
- a different buffer system may be used (e.g. Sucrose-PBS) the pharmaceutical composition may comprise NaCI and/or phosphate.
- phosphate and/or NaCI may be present in the pharmaceutical composition in embodiments where the lipid- based carriers comprising RNA have been manufactured using a PBS (e.g. Sucrose-PBS) buffer followed by a step of dilution with the inventive buffer system to obtain the pharmaceutical composition.
- the buffer system / the pharmaceutical composition comprises less than 50mM NaCI, preferably less than 25mM NaCI, more preferably less than 5mM NaCI.
- the buffer system / the pharmaceutical composition comprises less than 20mM phosphate, preferably less than 10mM phosphate, more preferably less than 1 mM phosphate.
- the buffer system / the pharmaceutical composition has a pH in a range of about pH 7.0 to about pH 8.0, preferably a pH of about 7.4
- the buffer system / the pharmaceutical composition has an osmolality of less than 450 mOsmol/kg, preferably less than 400 mOsmol/kg. Not exceeding a certain osmolality is desired for pharmaceutical compositions that are configured for injection into subjects (e.g. intramuscular injection or intraocular injection), e.g. human subjects. Exceeding values of 400mOsmol/kg would increase the tonicity of the composition and hence cause pain upon injection, e.g. upon intramuscular injection. Providing a pharmaceutical composition that is in the herein defined osmolality range suitable for injection into subjects is one objection of the invention, as the components of the buffer should not exceed a certain total amount as defined herein,
- the buffer system / the pharmaceutical composition has an osmolality of 100 mOsmol/kg to 400 mOsmol/kg, 150 mOsmol/kg to 400 mOsmol/kg, 200mOsmol/kg to 400mOsmol/kg, 300mOsmol/kg to 400mOsmol/kg, preferably of about 300 mOsmol/kg (e.g. 310 mOsmol/kg).
- the freezing point temperature of the buffer system and/or the pharmaceutical composition is between -10°C and -30°C, preferably at about -20°C.
- the glass transition temperature (Tg) of the buffer system and/or the pharmaceutical composition is between -30°C and -50°C, preferably at about -44°C.
- the Tg can be determined using Differential Scanning Calorimetry (DSC) as commonly used in the art.
- DSC Differential Scanning Calorimetry
- adjusting the pharmaceutical composition to a certain freezing point temperature / glass transition temperature may be advantageous in preserving lipid-based carriers at low temperatures below 0°C (e.g. -20°C or -40°C).
- the pharmaceutical composition comprising the herein defined components is in a gel-like semi-frozen state at -20°C or -40°C. Nonetheless, a semi-frozen or gel-like structure is considered to be a frozen storage in the context of the invention.
- the glass-liquid transition is the gradual and reversible transition in amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and relatively brittle "glassy" state into a viscous or rubbery state as the temperature is increased.
- An amorphous solid that exhibits a glass transition is called a glass.
- the reverse transition, achieved by supercooling a viscous liquid into the glass state, is called vitrification.
- the glass-transition temperature Tg of a material characterizes the range of temperatures over which this glass transition occurs.
- the buffer system / the pharmaceutical composition does not comprise or is essentially free of any of the excipients selected from ethanol, phenoxyethanol, phenylethyl alcohol, benzyl alcohol, PEG, DMSO or 3-(l- Pyridinio)-l-propanesulfonate (NDSB-201), ascorbic acid, citric acid, malic acid, monothioglycerol, phosphoric acid, potassium, metabisulfite, sodium metabisulfite, alpha-tocopherol, lithium acetate, lithium chloride, lithium formate, lithium nitrate, magnesium acetate, Triton, poloxamers, amino acids, cyclodextrin, Polysorbate, polysorbate 20, polysorbate 40, polysorbate, 60, or polysorbate 80, diethylenetriamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), iminodisuccinic acid, polyaspartic acid, ethylened
- the buffer system / the composition does not comprise phenoxyethanol or benzyl alcohol. In preferred embodiments, the buffer system / the pharmaceutical composition does not comprise ethanol. In preferred embodiments, the buffer system / the pharmaceutical composition comprises sodium acetate, e.g. sodium acetate at a concentration of about 1mM to 20mM, in particular 5mM, 10mM, or 15mM.
- Lipid based carriers contained in tfie buffer system Lipid based carriers contained in tfie buffer system
- lipid-based carriers of the invention are further specified.
- lipid-based carrier encompasses lipid-based delivery systems for RNA that comprise a lipid component.
- a lipid-based carrier may additionally comprise other components suitable for encapsulating/incorporating/complexing an RNA including a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
- a typical “lipid-based carried’ is selected from liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes.
- the RNA of the pharmaceutical composition may completely or partially be incorporated or encapsulated in a lipid-based carrier, wherein the RNA may be located in the interior space of the lipid- based carrier, within the lipid layer/membrane of the lipid-based carrier or associated with the exterior surface of the lipid- based carrier.
- the incorporation of RNA into lipid-based earners may be referred to as “encapsulation”.
- a “lipid-based carried’ is not restricted to any particular morphology, and include any morphology generated when e.g.
- an aggregation reducing lipid and at least one further lipid are combined, e.g. in an aqueous environment in the presence of RNA.
- RNA e.g., an LNP, a liposome, a lipid complex, a lipoplex and the like are within the scope of the term “lipid-based carrier' 1 .
- Lipid-based carriers can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50nm and 500nm in diameter.
- MLV multilamellar vesicle
- SUV small unicellular vesicle
- LUV large unilamellar vesicle
- Liposomes a specific type of lipid-based carrier, are characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
- the at least one RNA is typically located in the interior aqueous space enveloped by some or the entire lipid portion of the liposome.
- Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains.
- Lipid nanoparticles a specific type of lipid-based carrier, are characterized as microscopic lipid particles having a solid core or partially solid core.
- an LNP does not comprise an interior aqua space sequestered from an outer medium by a bilayer.
- the at least one RNA may be encapsulated or incorporated in the lipid portion of the LNP enveloped by some or the entire lipid portion of the LNP.
- An LNP may comprise any lipid capable of forming a particle to which the RNA may be attached, or in which the RNA may be encapsulated.
- the lipid-based carriers of the pharmaceutical composition are selected from lipid nanoparticles, liposomes, lipoplexes, nanoliposomes, or any combination thereof.
- the lipid-based carriers of the pharmaceutical composition encapsulate the RNA of the composition.
- encapsulated refers to the essentially stable combination of RNA with one or more lipids into lipid-based carriers (e.g. larger complexes or assemblies) preferably without covalent binding of the RNA.
- the lipid-based carriers - encapsulated RNA may be completely or partially located in the interior of the lipid-based carrier (e.g. the lipid portion and/or an interior space) and/or within the lipid layer/membrane of the lipid-based carriers.
- incorporation means the encapsulation of an RNA into lipid-based carriers.
- incorporation may be to protect the RNA from an environment which may contain enzymes, chemicals, or conditions that degrade the RNA.
- incorporating RNA into lipid-based carriers may promote the uptake of the RNA, and hence, may enhance the therapeutic effect of the RNA when administered to a cell or a subject.
- the lipid-based carriers are lipid nanoparticles (LNPs).
- the lipid-based carriers comprise one or more lipids selected from the list comprising an aggregationreducing lipid, a cationic lipid, a neutral lipid or phospholipid, a steroid or steroid analogue.
- the lipid-based carriers preferably the LNPs, comprise at least one aggregationreducing lipid, at least one cationic or ionizable lipid, at least one neutral lipid, and/or at least one steroid or steroid analogue.
- the lipid-based carriers preferably the LNPs, comprise an (i) aggregation-reducing lipid, (ii) a cationic lipid or ionizable lipid, (ill) a neutral lipid or phospholipid, (iv) and a steroid or steroid analogue.
- the lipid-based carriers preferably the LNPs, comprise an (i) aggregationreducing lipid, (II) a cationic lipid or ionizable lipid, (ill) two neutral lipid or phospholipids, (iv) and a steroid or steroid analogue.
- the lipid-based carriers preferably the LNPs, comprise an aggregation reducing lipid.
- aggregation reducing lipid refers to a molecule comprising both a lipid portion and a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
- the lipid- based carriers may undergo charge-induced aggregation, a condition which can be undesirable for the stability of the lipid-based carriers. Therefore, it can be desirable to include a lipid compound which can reduce aggregation, for example by sterically stabilizing the lipid-based carriers. Such a steric stabilization may occur when a compound having a sterically bulky but uncharged moiety that shields or screens the charged portions of a lipid-based carriers from close approach to other lipid-based carriers in the composition.
- stabilization of the lipid-based carriers is achieved by including lipids which may comprise a lipid bearing a sterically bulky group which, after formation of the lipid-based carrier, is preferably located on the exterior of the lipid-based carrier.
- Suitable aggregation reducing groups include hydrophilic groups, e.g. monosialoganglioside GM1 , polyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g. polyethylene glycol) or polypropylene glycol).
- the aggregation reducing lipid is a polymer conjugated lipid.
- polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion, wherein the polymer is suitable of reducing or preventing aggregation of lipid-based carriers comprising the RNA.
- a polymer has to be understood as a substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits.
- a suitable polymer of the invention may be a hydrophilic polymer.
- An example of a polymer conjugated lipid is a PEGylated or PEG-conjugated lipid.
- the aggregation reducing lipid preferably the polymer conjugated lipid is a PEG-conjugated lipid or a PEG-free lipid.
- the average molecular weight of the PEG moiety in the PEG- conjugated lipid preferably ranges from about 500 to about 8,000 Daltons (e.g., from about 1 ,000 to about 4,000 Daltons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Daltons.
- the PEG-conjugated lipid is selected from PEG-modified phosphatidylethanolamine, PEG- modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
- PEG-modified phosphatidylethanolamine PEG-modified phosphatidic acid
- PEG-modified ceramides e.g. PEG-CerC14 or PEG-CerC20
- PEG-modified dialkylamines e.g. PEG-CerC14 or PEG-CerC20
- Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
- the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid is DMG-PEG 2000. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).
- the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(uj- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), ora PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(t)
- the polymer conjugated lipid e.g. the PEG-conjugated lipid
- the polymer conjugated lipid is selected or derived from 1 ,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG 2000).
- PEG2000 DMG or DMG-PEG 2000 1 ,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000
- DMG-PEG 2000 is typically considered a mixture of 1 ,2-DMG PEG 2000 and 1 ,3-DMG PEG 2000 in ⁇ 97:3 ratio.
- the polymer conjugated lipid e.g. the PEG-conjugated lipid
- the polymer conjugated lipid is selected or derived from C10- PEG2K, or Cer8-PEG2K.
- the polymer conjugated lipid e.g. the PEG-conjugated lipid is selected or derived from formula (IV) of WO2018078053, preferably selected from formula (IVa) of WO2018078053.
- a PEG-conjugated lipid selected or derived from formula IVa may have the chemical term 2[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide, also referred to as ALC-0159.
- a PEG-free lipid in the context of the invention may be selected or derived from a POZ-lipid.
- the POZ lipids or respectively preferred polymer conjugated lipids are described in WO2023031394 (i.e. lipids derived from formula I, II, and III of WO2023031394, or lipid nanoparticles and/or lipids as specified in Claims 1 to 46 of WO2023031394), the full disclosure herewith incorporated by reference.
- the disclosure relating to polymer conjugated lipids as shown in any one of claims 1 to 8 of WO2023031394 are incorporated by reference.
- the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid. Accordingly, in embodiments, the polymer conjugated lipid is a POZ-lipid, which is defined as a compound according to formula (POZ): [H] - [linker] - [M], wherein
- [H] is a homopolymer moiety comprising at least one polyoxazoline (POZ) monomer unit wherein R is C1 -9 alkyl or C2-9 alkenyl and n has a mean value ranging from 2 to 200, preferably from 20 to 100, more preferably from 24 to 26 or 45 to 50; [linker] is an optional linker group, and [M] is a lipid moiety.
- POZ polyoxazoline
- [H] is a heteropolymer moiety or homopolymer moiety comprising multiple monomer units selected from the group consisting of poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly(2-propyl-2-oxazoline) (PPOZ), poly(2-butyl-2-oxazoline) (PBOZ), poly(2-isopropyl-2-oxazoline) (PIPOZ), poly(2- methoxymethyl-2-oxazoline) (PMeOMeOx), and poly(2-dimethylamino-2-oxazoline) (PDMAOx), preferably wherein [H] is a homopolymer moiety comprising multiple PMOZ or PEOZ monomer units, more preferably wherein [H] comprises or preferably consists of multiple PMOZ monomer units, wherein (i) n has a mean value ranging from 2 to 200, preferably from 20 to 100, more
- [H] is a heteropolymer moiety or homopolymer moiety comprising multiple monomer units selected from the group consisting of PmeOx, PETOx, PnPrOx, PcPrOx, PiPrOx, PsecBuOx, PiBuOx, PnBuOx, PPentOx, PheptOx, PNOx, PPheOx, PButEnOx, PPynOx, PDecEnOx, PiPrEnOx, and PIPOx.
- the [H] from the polymer conjugated lipid according to formula (POZ) is selected from the group consisting of poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) and poly(2-dimethylamino-2-oxazoline) (PDMAOx).
- the lipid moiety [M] as shown in formula (POZ) comprises at least one straight or branched, saturated or unsaturated alkyl chain containing from 6 to 30 carbon atoms, preferably wherein the lipid moiety [M] comprises at least one straight or branched saturated alkyl chain, wherein the alkyl chain is optionally interrupted by one or more biodegradable group(s) and/or optionally comprises one terminal biodegradable group, wherein the biodegradable group is selected from the group consisting of but not limited to a pH-sensitive moiety, a zwitterionic linker, non-ester containing linker moieties and ester-containing linker moieties ( — C(O)O — or — OC(O) — ), amido ( — C(O)NH — ), disulfide ( — S — S — ), carbonyl ( — C(O) — ), ether ( — O — ), thioether
- the lipid moiety [M] comprises at least one straight or branched, saturated or unsaturated alkyl chain comprising 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, preferably in the range of 10 to 20 carbon atoms, more preferably in the range of 12 to 18 carbon atoms, even more preferably 14, 16 or 18 carbon atoms, even more preferably 16 or 18 carbon atoms, most preferably 14 carbon atoms, wherein all selections are independent of one another.
- the linker group [linker] as shown in formula (POZ) is selected from the group consisting of but not limited to a pH-sensitive moiety, a zwitterionic linker, non-ester containing linker moieties and ester- containing linker moieties ( — C(O)O — or — OC(O) — ), amido ( — C(O)NH — ), disulfide ( — S — S — ), carbonyl ( — C(O) —
- ether — O —
- thioether — S —
- carbamate — NHC(O)O —
- urea — NHC(O)NH—
- succinyl — (O)CCH2CH2C(O)—
- succinamidyl — NHC(O)CH2CH2C(O)NH—
- the polymer conjugated lipid is selected or derived from PMOZ 1 , PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 of W02023031394.
- the polymer conjugated lipid is selected or derived from PMOZ4 of W02023031394.
- the at least one aggregation-reducing lipid is selected or derived from PMOZ4 according to
- the linker group [linker] comprises preferably an amide linker moiety. In a further very preferred embodiment in that context, the linker group [linker] comprises preferably an ester linker moiety. In a further very preferred embodiment in that context t, the linker group [linker] comprises preferably a succinate linker moiety. In another very preferred embodiment in that context, the linker group [linker] comprises both an ester linker and an amid linker moiety. In another preferred embodiment, the linker group [linker] comprises both an ester linker, an amine linker and an amid linker moiety.
- the at least one aggregation-reducing lipid preferably the PEG-conjugated lipid, is selected or derived from ALC-0159, DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K.
- the aggregation-reducing lipid is ALC-0159.
- the lipid-based carriers of the pharmaceutical composition comprise an aggregation reducing lipid, wherein the aggregation reducing lipid is not a PEG-conjugated lipid or a PEG-free lipid.
- the PEG-free lipid is a polymer-conjugated lipid that comprises a polymer different from PEG.
- the aggregation reducing lipid is selected or derived a POZ-lipid, which is defined as a compound according to formula (POZ) as defined herein.
- POZ formula
- the aggregation-reducing lipid is selected from a PMOZ-lipid as defined herein (e.g. a PMOZ 4 lipid as defined herein).
- lipid-based carriers include less than about 3mol%, 2mol%, or 1 mol% of aggregation reducing lipid, based on the total moles of lipid in the lipid-based carrier.
- lipid-based carriers comprise from about 0.1 % to about 10% of the aggregation reducing lipid on a molar basis, e.g. about 0.5% to about 10%, about 0.5% to about 5%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1 %, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).
- lipid-based carriers comprise from about 1 .0% to about 2.0% of the aggregation reducing lipid on a molar basis, e.g. about 1 .2% to about 1.9%, about 1.2% to about 1 .8%, about 1.3% to about 1.8%, about 1 .4% to about 1.8%, about 1 .5% to about 1.8%, about 1 .6% to about 1.8%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1 .8%, about 1.9%, most preferably 1 .7% (based on 100% total moles of lipids in the lipid-based carrier).
- lipid-based carriers comprise about 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, preferably 2.5% of the aggregation reducing lipid on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).
- the molar ratio of the cationic lipid to the aggregation reducing lipid ranges from about 100:1 to about 25:1.
- the lipid-based carriers preferably the LNPs, comprise a cationic or ionizable lipid.
- the cationic or ionizable lipid of the lipid-based carriers may be cationisable or ionizable, i.e. it becomes protonated as the pH is lowered below the pK ofthe ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
- the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
- the lipid-based carriers comprise a cationic or ionizable lipid that preferably carries a net positive charge at physiological pH, more preferably the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group.
- the lipid-based carriers comprise a cationic or ionizable lipid that is selected from or derived from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
- the lipid formulation comprises cationic or ionizable lipids as defined in Formula I of paragraph [00251] of WO2021222801 or a lipid selected from the disclosure of paragraphs [00260] or [00261] of WO2021222801.
- the lipid formulation comprises cationic or ionizable lipids selected from the group consisting of ATX-001 to ATX-132 as disclosed in claim 90 of WO2021183563, preferably ATX-0126.
- the disclosure of WO2021222801 and WO2021183563, especially aforementioned lipids, are incorporated herewith by reference.
- cationic or ionizable lipids may be selected from the lipids disclosed in WO2018078053 (i.e. lipids derived from formula I, II, and III of WO2018078053, or lipids as specified in claims 1 to 12 of WO2018078053), the disclosure of WO2018078053 hereby incorporated by reference in its entirety.
- lipids disclosed in Table 7 of WO2018078053 e.g. lipids derived from formula 1-1 to 1-41
- lipids disclosed in Table 8 of WO2018078053 e.g. lipids derived from formula 11-1 to il-36
- the lipid-based carriers comprise a cationic or ionizable lipid selected or derived from structures 111-1 to HI-36 of Table 9 of published PCT patent application W02018078053. Accordingly, formula ill-1 to HI-36 of WO2018078053, and the specific disclosure relating thereto, are herewith incorporated by reference. In preferred embodiments, the lipid-based carriers comprise a cationic lipid selected or derived from formula HI-3 of published PCT patent application WO2018078053.
- a preferred lipid of said formula ill-3 has the chemical term ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also referred to as ALC-0315, i.e. CAS Number 2036272-55-4.
- suitable cationic or ionizable lipids may be selected or derived from cationic lipids according to PCT claims 1 to 14 of published patent application WO2021123332, or table 1 of WO2021123332, the disclosure relating to claims 1 to 14 or table 1 of WO2021123332 herewith incorporated by reference. Accordingly, suitable cationic or ionizable lipids may be selected or derived from cationic lipids according Compound 1 to Compound 71 (C1-C27) of Table 1 of WO2021123332. In alternative embodiments, the lipid-based carriers (e.g.
- LNPs of the pharmaceutical composition comprise a cationic or ionizable lipid selected or derived from (COATSOME® SS-EC) SS-33/4PE-15 (see C23 in Table 1 of WO2021123332).
- the lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise a cationic or ionizable lipid selected or derived from HEXA-C5DE-PipSS (see C2 in Table 1 of WO2021123332).
- the lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise a cationic or ionizable lipid selected or derived from compound C26 as disclosed in Table 1 of WO2021123332:
- the lipid-based carriers of the pharmaceutical composition comprise a cationic lipid selected or derived from 9-Heptadecanyl 8- ⁇ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino)octanoate, also referred to as SM- 102.
- lipid-based carriers e.g. LNPs
- preferred lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise a squaramide ionizable amino lipid, more preferably a cationic lipid selected or derived from the group consisting of formulas (M1) and (M2): wherein the substituents (e.g. R1 , R2, R3, R5, R6, R7, R10, M, M1 , m, n, o, I) are defined in claims 1 to 13 of US10392341 B2; US10392341 B2 being incorporated herein in its entirety.
- substituents e.g. R1 , R2, R3, R5, R6, R7, R10, M, M1 , m, n, o, I
- the lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise a cationic or ionizable lipid selected or derived from ALC-0315 (lipid of formula ill-3), SM-102, SS-33/4PE-15, HEXA- C5DE-PipSS, or compound C26 (see C26 in Table 1 of WO2021123332).
- the cationic lipid is selected from ALC-0315 or C26.
- the lipid-based carriers comprise two or more (different) cationic or ionizable lipids as defined herein. In preferred embodiments, the lipid-based carriers comprise one cationic lipid as defined herein.
- the cationic or ionizable lipid as defined herein is present in the lipid-based carriers in an amount from about 30mol% to about 95mol%, relative to the total lipid content of the lipid-based carriers. If more than one cationic lipid is incorporated within the lipid-based carriers, such percentages apply to the combined cationic lipids.
- the cationic or ionizable lipid is present in the lipid-based carriers in an amount from about 30mol% to about 70mol%. In one embodiment, the cationic lipid is present in the lipid-based carriers in an amount from about 40mol% to about 60mol%, such as about 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60mol%, respectively.
- the cationic lipid is present in the lipid-based carriers in an amount from about 47mol% to about 48mol%, such as about 47.0, 47.1 , 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0mol%, respectively, wherein 47.4mol% are particularly preferred.
- the cationic lipid is present in the lipid-based carriers in an amount from about 55mol% to about 65mol%, such as about 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64 or65mol%, respectively, wherein 59mol% are particularly preferred.
- the cationic or ionizable lipid is present in a ratio of from about 20mol% to about 70mol% or 75mol% or from about 45mol% to about 65mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70mol% of the total lipid present in the lipid-based carriers.
- the LNPs comprise from about 25% to about 75% on a molar basis of cationic lipid, e.g., from about 20 to about 70%, from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 57.1 %, about 50% or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).
- the ratio of cationic or ionizable lipid to RNA is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11 .
- the lipid-based carriers preferably the LNPs, comprise at least one neutral lipid or phospholipid.
- neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Suitable neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
- the selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the bloodstream.
- the neutral lipid is a lipid having two acyl groups (e.g.
- the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20. In another embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
- the lipid-based carriers comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising DOPC, DPPC, DOPG, DPPG, DOPE, POPO, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, SOPE, transDOPE, or mixtures thereof.
- the neutral lipid of the lipid-based carriers of the pharmaceutical composition is selected or derived from 1 ,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).
- the neutral lipid of the lipid-based carriers of the pharmaceutical composition is selected or derived from 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE).
- the neutral lipid of the lipid-based carriers of the pharmaceutical composition is selected or derived from phosphatidylserine, preferably DPhyPS (1 ,2-diphytanoyl-sn-glycero-3-phospho-L-serine).
- the neutral lipid of the lipid-based carriers of the pharmaceutical composition is selected or derived from 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
- the lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise at least one neutral lipid selected or derived from DSPC, DHPC, DPhyPE, or DPhyPS.
- the neutral lipid is DSPC.
- the lipid-based carrier preferably the LNP, comprises DPhyPE and DPhyPS.
- the molar ratio of the cationic lipid to the neutral lipid in the lipid-based carriers ranges from about 2:1 to about 8:1.
- the neutral lipid is preferably from about 5mol% to about 90mol%, about 5mol% to about 10mol%, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90mol% of the total lipid present in the lipid-based carrier.
- the lipid-based carrier includes from about 0% to about 15% or 45% on a molar basis of neutral lipid, e.g. from about 3% to about 12% or from about 5% to about 10%.
- the lipid-based carrier may include about 15%, about 10%, about 7.5%, or about 7.1 % of neutral lipid on a molar basis (based upon 100% total moles of lipid in the lipid-based carrier).
- Steroid, steroid analogue or sterol Steroid, steroid analogue or sterol:
- the lipid-based carriers of the pharmaceutical composition comprise a steroid, steroid analogue or sterol.
- the steroid, steroid analogue or sterol may be derived or selected from cholesterol, cholesteryl hemisuccinate (CHEMS) and a derivate thereof.
- the lipid-based carriers of the pharmaceutical composition comprise a steroid, steroid analogue or sterol derived from a phytosterol (e.g., a sitosterol, such as beta-sitosterol), preferably from a compound having the structure of Formula I as disclosed in claim 1 of W02020061332; the disclosure ofW02020061332, especially the disclosure of Formula I and phytosterols being incorporated by reference herewith.
- a phytosterol e.g., a sitosterol, such as beta-sitosterol
- the steroid is an imidazole cholesterol ester or “ICE” as disclosed in paragraphs [0320] and [0339]-[0340] of WO2019226925; WO2019226925 being incorporated herein by reference in its entirety.
- the lipid-based carriers (e.g. LNPs) of the pharmaceutical composition comprise at least one steroid or steroid analogue selected or derived from cholesterol or cholesteryl hemisuccinate (CHEMS).
- the steroid or steroid analogue is cholesterol.
- the lipid-based carrier comprises about 10mol% to about 60mol% or about 25mol% to about 40mol% sterol (based on 100% total moles of lipids in the lipid-based carrier). In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60mol% of the total lipid present in the lipid-based carrier.
- the lipid-based carriers include from about 5% to about 50% on a molar basis of the sterol, e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31 .5% or about 30% on a molar basis (based upon 100% total moles of lipid in the lipid-based carrier).
- the lipid-based carrier comprises about 28%, about 29% or about 30% sterol (based on 100% total moles of lipids in the lipid-based carrier).
- the lipid-based carrier comprises about 40.9% sterol (based on 100% total moles of lipids in the lipid-based carrier).
- Lipid-based carrier compositions are Lipid-based carrier compositions:
- the lipid-based carriers of the pharmaceutical composition comprise RNA, a cationic or ionizable lipid as defined herein, an aggregation reducing lipid as defined herein, optionally, a neutral lipid as defined herein, and, optionally, a steroid or steroid analogue as defined herein.
- the lipid-based carriers are lipid-based carriers.
- the lipid-based carriers comprise
- At least one cationic lipid or ionizable lipid selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA- C5DE-PipSS or compound C26 (see C26 in Table 1 of WO2021123332);
- the lipid-based carriers preferably the LNPs comprise
- SM102-LNPs At least one aggregation reducing lipid selected from DMG-PEG 2000; and wherein the lipid-based carriers encapsulate the RNA.
- LNPs are herein referred to as SM102-LNPs.
- the lipid-based carriers preferably the LNPs comprise
- RNA-based carriers encapsulate the RNA.
- LNPs are herein referred to as GN-LNPs.
- the lipid-based carriers preferably the LNPs comprise
- LNPs At least one aggregation reducing lipid selected from ALC-0159; and wherein the lipid-based carriers encapsulate the RNA.
- Such LNPs are herein referred to as 315-LNPs.
- the cationic or ionizable lipid (as defined herein), neutral lipid (as defined herein), steroid or steroid analogue (as defined herein), and/or aggregation reducing lipid (as defined herein) may be combined at various relative ratios.
- the lipid-based carriers comprise (i) to (iv) in a molar ratio of about 20-60% cationic lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid.
- the lipid-based carriers preferably the LNPs, comprise (i) to (iv) in a molar ratio of about 45-55% cationic lipid or ionizable lipid, about 5-15% neutral lipid, about 35-45% steroid or steroid analogue, and about 0.5-2.5% aggregation reducing lipid e.g. polymer conjugated lipid, preferably wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
- the nucleic acid e.g. the RNA
- the lipid-based carriers preferably the LNPs, comprise (i) to (iv) in a molar ratio of about 47-51 % cationic lipid or ionizable lipid, about 8-12% neutral lipid, about 38-42% steroid or steroid analogue, and about 0.75-1.75% aggregation reducing lipid e.g. polymer conjugated lipid, preferably wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
- the nucleic acid e.g. the RNA
- the 315-LNPs comprise I) to (iv) in a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analogue, and about 1.7% aggregation reducing lipid, preferably wherein the lipid-based carriers encapsulate the RNA.
- the pharmaceutical composition comprises lipid nanoparticles (LNPs) which have a molar ratio of approximately 50:10:38.5:1 .5, preferably 47.5:10:40.8:1 .7 or more preferably 47.4:10:40.9:1 .7 (i.e.
- the wt/wt ratio of lipid to the RNA in the lipid-based carriers is from about 10: 1 to about 60: 1 , preferably from about 20:1 to about 30:1 , more preferably about 25:1 . In other preferred embodiments, the wt/wt ratio of lipid to RNA is in the range of 20 to 60, preferably from about 3 to about 15, about 5 to about 13, about 4 to about 8 or from about 7 to about 11.
- the amount of lipid comprised in the lipid-based carriers may be selected taking the amount of the RNA cargo into account. In one embodiment, these amounts are selected such as to result in an N/P ratio of the lipid-based carriers encapsulating the RNA in the range of about 0.1 to about 20.
- the N/P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid to the phosphate groups (“P”) of the RNA which is used as cargo.
- the N/P ratio may be calculated on the basis that, for example, 1 ug RNA typically contains about 3nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases.
- the “N”-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and - if present - cationisable groups.
- the N/P ratio can be in the range of about 1 to about 50. In other embodiments, the range is about 1 to about 20, and preferably about 1 to about 15. In preferred embodiments, the N/P ratio of the lipid-based earners encapsulating the RNA is in a range from about 1 to about 20, about 1 to about 10, preferably about 6.
- the pharmaceutical composition comprises lipid-based carriers (encapsulating RNA) that have a defined size (particle size, homogeneous size distribution).
- the size of the lipid-based carriers of the pharmaceutical composition is typically described herein as Z-average size.
- the terms “average diameter”, “mean diameter”, “diameter” or “size” for particles (e.g. lipid-based carrier) are used synonymously with the value of the Z-average.
- Z-average size refers to the mean diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321).
- DLS dynamic light scattering
- Suitable DLS protocols are known in the art.
- DLS instruments are commercially available (such as the Zetasizer Nano Series, Malvern Instruments, Worcestershire, UK). DLS instruments employ either a detector at 90° (e.g.
- DLS measurements are performed at a temperature of about 25°C.
- DLS is also used in the context of the present invention to determine the polydispersity index (PDI) and/or the main peak diameter of the lipid-based carriers incorporating RNA.
- the lipid-based carriers of the pharmaceutical composition have a Z-average size ranging from about 50nm to about 200nm, from about 50nm to about 190nm, from about 50nm to about 180nm, from about 50nm to about 170nm, from about 50nm to about 160nm, 50nm to about 150nm, 50nm to about 140nm, 50nm to about 130nm, 50nm to about 120nm, 50nm to about 110nm, 50nm to about 100nm, 50nm to about 90nm, 50nm to about 80nm, 50nm to about 70nm, 50nm to about 60nm, 60nm to about 200nm, from about 60nm to about 190nm, from about 60nm to about 180nm, from about 60nm to about 170nm, from about 60nm to about 160nm, 60nm to about 150nm, 60nm to about 140nm, 60nm to about 130nm, 60n
- the lipid-based carriers have a Z-average size in a range from about 50nm to about 200nm, about 50nm to about 150nm, about 50nm to about 120nm, preferably in a range of about 60nm to about 115nm, even more preferably in a range of about 65nm to about 90nm.
- the Z-average size is determined using DLS, in particular as described in the Example section.
- lipid-based carriers In embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the lipid-based carriers have a particle size exceeding about 500nm. In embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% lipid- based carriers that have a particle size smaller than about 20nm. Preferably, at least about 80%, 85%, 90%, 95% of lipid-based carriers of the composition have a spherical morphology.
- the polydispersity index (PDI) of the lipid-based carriers is in the range of 0.1 to 0.5.
- the lipid-based carriers have a polydispersity index (PDI) value of less than about 0.3, preferably of less than about 0.2, more preferably of less than about 0.1.
- the PDI is determined using DLS, in particular as described in the Example section.
- the composition of the invention has a certain level of free RNA and/or RNA encapsulation.
- free RNA or “non-complexed RNA” or “non-encapsulated RNA” comprise the RNA molecules that are not comprised (e.g. encapsulated) in the lipid-based carriers as defined herein.
- free RNA may represent a contamination or an impurity.
- a large proportion of non-encapsulated or free RNA may also be an indicator for destabilization of the lipid- based carriers of the composition (e.g. upon storage, thawing, dilution).
- free RNA detectable in the liquid composition may increase during storage, which may be used as a feature to determine the temperature stability of the composition.
- Free RNA in the liquid composition may be determined by chromatographic methods (e.g. AEX, SEC) or by using probes (e.g. dyes) that bind to free RNA in the composition.
- probes e.g. dyes
- the amount of free RNA or non-encapsulated RNA may be determined using a dye-based assay.
- Suitable dyes that may be used to determine the amount and/or the proportion of free RNA comprise RiboGreen®, PicoGreen® dye, OliGreen® dye, QuantiFluor® RNA dye, Qubit® RNA dye, Quant-iTTM RNA dye, TOTO®-1 dye, Y0Y0®-1 dye. Such dyes are suitable to discriminate between free RNA and encapsulated RNA. Reference standards consisting of defined amounts of free RNA or encapsulated RNA may be used and mixed with the respective reagent (e.g. RiboGreen® reagent (Excitation 485 nm/Emission 530 nm)) as recommended by the supplier's instructions. Typically, the free RNA of the liquid composition is quantitated using the Quant-iT RiboGreen RNA Reagent according to the manufacturer's instructions. The proportion of free RNA in the context of the invention is typically determined using a RiboGreen assay.
- encapsulated RNA comprises the RNA molecules that are encapsulated in the lipid-based carriers as defined herein.
- the proportion of encapsulated RNA in the context of the invention is typically determined using a RiboGreen assay or similar methods used for determining the amount of free RNA.
- the composition comprises less than about 30% free RNA, preferably less than about 20% free RNA, more preferably less than about 10% free RNA.
- RNA of the composition is encapsulated in lipid-based carriers.
- the percentage of encapsulation or the amount of free RNA may be determined by a RiboGreen assay, in particular as described in the Example section.
- the lipid-based carriers preferably encapsulating or comprising RNA are purified by at least one purification step, preferably by at least one step of TFF and/or at least one step of clarification and/or at least one step of filtration.
- the (main) phase transition temperature (Tm) of the lipid-based carriers encapsulating an RNA is at a temperature ranging from about 5CTC to about 100°C, from about 50°C to about 80°C, from about 60°C to about 70°C.
- the Tm of the lipid-based carriers encapsulating an RNA is at about 67 Q C.
- the phase transition temperature of the lipid-based carriers, preferably LNPs, is typically determined using Differential Scanning Calorimetry (DSC) as commonly used in the art.
- the lipid-based carriers as defined in the present section may be formulated in a buffer system as defined herein.
- the lipid-based carriers comprise (i) at least one cationic lipid as defined herein;
- the lipid-based carriers encapsulate the RNA, wherein the lipid based are preferably contained in a buffer system that comprises a) 50mM to 200mM, preferably 10OmM to 200mM, more preferably 10OmM to 150mM glycerol; b) 50mM to 200mM, preferably 10OmM to 200mM, more preferably 10OmM to 150mM sucrose; c) T ris as buffer agent at preferably at a concentration of at least 10mM or more than 20mM; and wherein the added total concentration for both glycerol and sucrose is preferably in a range of 200mM to 300mM.
- the lipid-based carriers are selected from 315-LNPs as defined herein, GN-LNPs as defined herein, or SM102- LNPs as defined herein.
- lipid-based carriers of the composition encapsulate an RNA and comprise (i) the cationic lipid ALC-0315 (lipid of formula III), (ii) the neutral lipid DSPC, (iii) cholesterol, and (iv) the aggregation reducing lipid ALC-0159 (lipid of formula IVa), preferably wherein (i) to (iv) are in a molar ratio of about 47.4% cationic lipid, 10% neutral lipid, 40.9% steroid or steroid analogue, and 1.7% aggregation reducing lipid, wherein the lipid-based carriers are preferably contained in a buffer system that comprises 100mM glycerol, 150mM sucrose (as sugar component), and at least 10mM or more than 20m M Tris as buffer agent, preferably 30mM.
- a buffer system that comprises 100mM glycerol, 150mM sucrose (as sugar component), and at least 10mM or more than 20m M Tris as buffer agent, preferably
- lipid-based carriers of the composition encapsulate an RNA and comprise (i) the cationic lipid ALC-0315 (lipid of formula III), (ii) the neutral lipid DSPC, (iii) cholesterol, and (iv) the aggregation reducing lipid ALC-0159 (lipid of formula IVa), preferably wherein (i) to (iv) are in a molar ratio of about 47.4% cationic lipid, 10% neutral lipid, 40.9% steroid or steroid analogue, and 1.7% aggregation reducing lipid, wherein the lipid-based carriers are preferably contained in a buffer system that comprises 150mM glycerol, 100mM sucrose (as sugar component), and at least 10mM or more than 20mM Tris as buffer agent, preferably 30mM.
- a buffer system that comprises 150mM glycerol, 100mM sucrose (as sugar component), and at least 10mM or more than 20mM Tris as buffer agent, preferably
- RNA comprised in the lipid-based carriers
- the RNA is selected from a single stranded RNA or double stranded RNA, and/or a coding RNA or a non-coding RNA, and/or a linear RNA or a circular RNA.
- the RNA may be a double-stranded non-coding RNA in circular form, or the RNA may be a single stranded non-coding RNA in linear form, or the RNA may be a double stranded RNA coding RNA in linear form, etc.
- the RNA is a single stranded coding RNA in linear or circular form.
- the RNA is selected from viral RNA, retroviral RNA, replicon RNA, small interfering RNA (siRNA), antisense RNA, saRNA (small activating RNA ), CRISPR RNA (small guide RNA, sgRNA), ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), self-replicating RNA, circular RNA, or mRNA.
- the RNA is a non-coding RNA, preferably a CRISPR/Cas9 guide RNA or a small interfering RNA (siRNA).
- the RNA is a coding RNA.
- a coding RNA can be any type of RNA construct (for example a double stranded RNA, a single stranded RNA, a circular double stranded RNA, or a circular single stranded RNA) characterized in that said coding RNA comprises at least one coding sequence (cds) that is translated into at least one amino-acid sequence (upon administration to e.g. a cell).
- the RNA comprises at least one coding sequence.
- the length the coding sequence may be at least or greater than about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 5000, or 6000 nucleotides. In embodiments, the length of the coding sequence may be in a range of from about 500 to about 2500 nucleotides.
- the RNA is a coding RNA.
- the RNA is selected from an mRNA, a (coding) circular RNA, a (coding) self-replicating RNA, a (coding) circular RNA, a (coding) viral RNA, ora (coding) replicon RNA.
- the RNA is a circular RNA.
- the terms “circular RNA” or “circRNAs” have to be understood as a circular polynucleotide construct that may encode at least one peptide or protein.
- a circRNA is a single stranded RNA molecule.
- said circRNA comprises at least one coding sequence encoding at least one peptide or protein as defined herein, or a fragment or variant thereof.
- the RNA of the liquid composition is a replicon RNA.
- the term “replicon RNA” is e.g. intended to be an optimized self-replicating RNA.
- Such constructs may include replicase elements derived from e.g. alphaviruses (e.g. SFV, SIN, VEE, or RRV) and the substitution of the structural virus proteins with the nucleic acid of interest (that is, the coding sequence encoding an antigenic peptide or protein as defined herein).
- the replicase may be provided on an independent RNA or DNA construct. Downstream of the replicase may be a sub-genomic promoter that controls replication of the replicon RNA.
- the RNA is an mRNA.
- the RNA encapsulated in lipid-based carriers is suitably an mRNA.
- RNA and mRNA are e.g. intended to be a ribonucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosinemonophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone.
- the backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer.
- the specific succession of the monomers is called the RNA-sequence.
- the mRNA messenger RNA
- the mRNA provides the nucleotide coding sequence that may be translated into an amino-acid sequence of a particular peptide or protein.
- the RNA has a length ranging from about 500 nucleotides to about 10000 nucleotides, ranging from about 1000 nucleotides to about 10000 nucleotides, ranging from about 1000 nucleotides to about 5000 nucleotides. In preferred embodiments, the RNA has a length of at least 3000 nucleotides, preferably ranging from about 3000 nucleotides to about 5000 nucleotides.
- the RNA may have a length ranging from about 1000 nucleotides to about 5000 nucleotides, preferably ranging from 2000 nucleotides to about 5000 nucleotides, ranging from 3000 nucleotides to about 5000 nucleotides, ranging from 3500 nucleotides to about 5000 nucleotides, ranging from about 3500 nucleotides to about 4500 nucleotides.
- the RNA has a length of not more than 3000 nucleotides, preferably ranging from about 500 nucleotides to about 3000 nucleotides.
- the RNA is a therapeutic RNA.
- RNA relates to an RNA providing a therapeutic modality.
- therapeutic in that context has to be understood as “providing a therapeutic function” or as “being suitable for therapy or administration”. However, “therapeutic” in that context should not at all to be understood as being limited to a certain therapeutic modality.
- therapeutic modalities may be the provision of a coding sequence (via said therapeutic RNA) that encodes for a peptide or protein (wherein said peptide or protein has a certain therapeutic function, e.g. an antigen for a vaccine, or an enzyme for protein replacement therapies).
- a further therapeutic modality may be genetic engineering, wherein the RNA provides or orchestrates factors to e.g.
- RNA does not include natural RNA extracts or RNA preparations (e.g. obtained from bacteria or obtained from plants) that are not suitable for administration to a subject (e.g. animal, human).
- RNA of the invention may be an artificial RNA.
- the RNA is an artificial RNA.
- an artificial RNA as used herein is intended to refer to an RNA that does not occur naturally.
- an artificial RNA may be understood as a non-natural RNA molecule.
- Such RNA molecules may be non-natural due to its individual sequence (e.g. G/C content modified coding sequence, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides.
- artificial RNA may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides.
- an artificial RNA is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type or reference sequence/the naturally occurring sequence by at least one nucleotide (via e.g. codon modification as further specified below).
- the term “artificial RNA” is not restricted to mean “one single molecule” but is understood to comprise an ensemble or plurality of essentially identical RNA molecules.
- the RNA is a modified and/or stabilized RNA.
- the RNA may thus be provided as a “stabilized RNA” that is to say an RNA showing improved resistance to in vivo degradation and/or an RNA showing improved stability in vivo, and/or an RNA showing improved translatability in vivo.
- the RNA of the present invention may be provided as a “stabilized RNA”.
- the RNA comprises at least one codon modified coding sequence.
- the amino acid sequence encoded by the at least one codon modified coding sequence is not being modified compared to the amino acid sequence encoded by the corresponding wild type or reference coding sequence.
- codon modified coding sequence relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference coding sequence.
- a codon modified coding sequence in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably to optimize/modify the coding sequence for in vivo applications.
- the at least one coding sequence is a codon modified coding sequence, wherein codon modified coding sequence is selected from a C maximized coding sequence, a CAI maximized coding sequence, human codon usage adapted coding sequence, a G/C content modified coding sequence, and a G/C optimized coding sequence, or any combination thereof.
- the at least one codon modified coding sequence is a G/C optimized coding sequence
- the at least one coding sequence of the RNA has a G/C content of at least about 50%, 55%, or 60%. In particular embodiments, the at least one coding sequence of the RNA has a G/C content of at least about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
- the coding sequence has a GC content of at least 54%, 57%, 60%, 63%.
- the RNA has a G/C content of at least about 50%, 55%, or 60%. In particular embodiments, the RNA has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
- the RNA sequence has a GC content of at least 50%, preferably at least 55%, more preferably at least 60%.
- the RNA comprising the codon modified coding sequence When transfected into mammalian host cells, the RNA comprising the codon modified coding sequence has a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and is capable of being expressed by the mammalian host cell (e.g. a muscle cell).
- the mammalian host cell e.g. a muscle cell.
- the RNA comprising the codon modified coding sequence is translated into protein, wherein the amount of protein is at least comparable to, or preferably at least 10% more than, or at least 20% more than, or at least 30% more than, or at least 40% more than, or at least 50% more than, or at least 100% more than, or at least 200% or more than the amount of protein obtained by a naturally occurring or wild type or reference coding sequence transfected into mammalian host cells.
- the RNA may be modified, wherein the C content of the at least one coding sequence may be increased, preferably maximized, compared to the C content of the corresponding wild type or reference coding sequence (herein referred to as “C maximized coding sequence”).
- C maximized coding sequence The generation of a C maximized nucleic acid sequences may suitably be carried out using a modification method according to WO2015062738. In this context, the disclosure of WO2015062738 is included herewith by reference.
- the RNA may be modified, wherein the G/C content of the at least one coding sequence may be optimized compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as “G/C optimized coding sequence”).
- G/C optimized coding sequence refers to a coding sequence wherein the G/C content is preferably increased to the essentially highest possible G/C content.
- the generation of a G/C content optimized nucleic acid sequence may be carried out using a method according to W02002098443. In this context, the disclosure of W02002098443 is included in its full scope in the present invention.
- G/C optimized coding sequences are indicated by the abbreviations “gc”.
- the RNA may be modified, wherein the codons in the at least one coding sequence may be adapted to human codon usage (herein referred to as “human codon usage adapted coding sequence”). Codons encoding the same amino acid occur at different frequencies in humans. Accordingly, the coding sequence of the RNA is preferably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage.
- the wild type or reference coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (see e.g. Table 2 of published PCT patent application WO2021156267). Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the RNA to obtain sequences adapted to human codon usage.
- the RNA may be modified, wherein the G/C content of the at least one coding sequence may be modified compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as “G/C modified coding sequence”).
- G/C optimization or “G/C content modification” relate to a nucleic acid that comprises a modified, preferably an increased number of guanosine and/or cytosine nucleotides as compared to the corresponding wild type or reference coding sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides.
- RNA sequences having an increased G/C content are more stable or show a better expression than sequences having an increased A/U.
- the G/C content of the coding sequence of the RNA is increased by at least 10%, 20%, 30%, preferably by at least 40% compared to the G/C content of the coding sequence of the corresponding wild type or reference nucleic acid sequence (herein referred to “gc mod”).
- the RNA may be modified, wherein the codon adaptation index (CAI) may be increased or preferably maximised in the at least one coding sequence (herein referred to as “CAI maximized coding sequence”).
- CAI maximized coding sequence it is preferred that all codons of the wild type or reference nucleic acid sequence that are relatively rare in e.g. a human are exchanged for a respective codon that is frequent in the e.g. a human, wherein the frequent codon encodes the same amino acid as the relatively rare codon.
- the most frequent codons are used for each amino acid of the encoded protein (see Table 2 of published PCT patent application WO2021156267, most frequent human codons are marked with asterisks).
- the RNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of the at least one coding sequence is 1 (CAM).
- CAI codon adaptation index
- the wild type or reference coding sequence may be adapted in a way that the most frequent human codon “GCC” is always used for said amino acid. Accordingly, such a procedure (as exemplified for Ala) may be applied for each amino acid encoded by the coding sequence of the nucleic acid to obtain CAI maximized coding sequences.
- the at least one coding sequence of the RNA of the invention is G/C optimized coding sequence.
- the length of an RNA and the codon usage of an RNA may have an impact on RNA stability. Accordingly, in the context of the invention, the amount of glycerol, the sugar component, and the buffer agent may be adjusted based on the length of an RNA and the codon usage of an RNA.
- the RNA has a length of at least 3000 nucleotides, preferably a length ranging from about 3000 nucleotides to about 5000 nucleotides, and optionally the RNA has a GC content of at least about 55%. In preferred embodiments in that context, the RNA has a length ranging from about 3500 nucleotides to about 4500 nucleotides, optionally wherein the RNA has a GC content of at least about 60%.
- the buffer system comprises more sugar component (e.g. sucrose) than glycerol.
- sugar component e.g. sucrose
- the molar ratio of glycerol to the sugar component is ranging from about 1 to 1.1 to about 1 to 2, preferably it is 1 to 1.5.
- the concentration the sugar component, preferably sucrose, in the buffer system is at least 10mM, 20mM, 30mM, 40mM, or 50mM higher than the concentration of glycerol.
- the buffer system comprises 50mM to 200mM glycerol and 50mM to 200mM sucrose (as sugar component), and the added total concentration for both glycerol and sucrose is preferably in a range of 200mM to 300mM, and the concentration of sucrose is higher than the concentration of glycerol.
- the term “higher than the concentration of glycerol” may mean that the concentration of sucrose is at least 10mM, 20mM, 30mM, 40mM, or 50mM higher than the concentration of sucrose.
- the buffer system comprises 100mM glycerol, 150mM sucrose, and Tris at preferably more than 20mM Tris (e.g. 30mM).
- the RNA has a length of not more than 3000 nucleotides, preferably a length ranging from about 500 nucleotides to about 3000 nucleotides, and optionally the RNA has a GC content of at least about 55%. In preferred embodiments in that context, the RNA has a length ranging from about 1500 nucleotides to about 2500 nucleotides, and optionally the RNA has a GC content of at feast about 57%.
- the buffer system comprises more glycerol than sugar component (e.g. sucrose).
- sugar component e.g. sucrose
- the molar ratio of glycerol to the sugar component is ranging from about 1.1 to 1 to about 2 to 1 , preferably it is 1.5 to 1.
- the concentration glycerol in the buffer system is at least 10mM, 20mM, 30mM, 40mM, or 50mM higher than the concentration the sugar component, preferably sucrose.
- the buffer system comprises 50mM to 200mM glycerol, and 50mM to 200mM sucrose (as sugar component), and the added total concentration for both glycerol and sucrose is preferably in a range of 200mM to 300mM, and the concentration of glycerol is higher than the concentration of sucrose.
- the term “higher than the concentration of sucrose” may mean that the concentration of glycerol is at least 10mM, 20mM, 30mM, 40mM, or 50mM higher than the concentration of sucrose.
- the buffer system comprises 150mM Glycerol, 100mM Sucrose, and Tris at preferably more than 20mM (e.g. 30mM).
- the at least one coding sequence comprises more than one stop codon to allow sufficient termination of translation.
- the at least one coding sequence comprises two or three stop codons to allow sufficient termination of translation. This more than one stop codon may optionally be positioned in alternative reading frames.
- the RNA comprises at least one untranslated region (UTR).
- UTR untranslated region
- UTR untranslated region
- UTR element The term “untranslated region” or “UTR” or “UTR element” will be recognized and understood by the person of ordinary skill in the art and are e.g. intended to refer to a part of a nucleic acid molecule typically located 5’ or 3' of a coding sequence.
- An UTR is not translated into protein.
- An UTR may be part of the RNA.
- An UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements etc.
- the RNA comprises a protein-coding region (“coding sequence” or “cds”), and a 5-UTR and/or 3-UTR.
- UTRs may harbour regulatory sequence elements that determine RNA turnover, stability, and localization.
- UTRs may harbour sequence elements that enhance translation.
- translation of the RNA into at least one peptide or protein is of paramount importance to therapeutic efficacy.
- Certain combinations of 3’-UTRs and/or 5’-UTRs may enhance the expression of operably linked coding sequences encoding peptides or proteins as defined herein.
- RNA molecules harbouring said UTR combinations advantageously enable rapid and transient expression of antigenic peptides or proteins after administration to a subject, preferably after intramuscular administration.
- RNA of the invention comprising certain combinations of 3’-UTRs and/or 5’-UTRs is particularly suitable for administration as a vaccine, in particular, suitable for administration into the muscle, the dermis, or the epidermis of a subject.
- the RNA comprises at least one heterologous 5-UTR and/or at least one heterologous 3-UTR.
- Said heterologous 5-UTRs or 3'-UTRs may be derived from naturally occurring genes or may be synthetically engineered.
- the RNA comprises at least one coding sequence as defined herein operably linked to at least one (heterologous) 3-UTR and/or at least one (heterologous) 5-UTR.
- the RNA comprises least one 5'-UTR and/or at least one 3-UTR.
- the RNA of the invention comprises at least one 3’-UTR.
- 3'-untranslated region or “3’-UTR” will be recognized and understood by the person of ordinary skill in the art and are e.g. intended to refer to a part of an RNA molecule located 3' (i.e. downstream) of a coding sequence and which is not translated into protein.
- a 3 -UTR may be part of a nucleic acid located between a coding sequence and an (optional) terminal poly(A) sequence.
- a 3-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.
- the RNA comprises at least one 3 -UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
- the 3’-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.
- the RNA comprises at least one 3’-UTR, wherein the at least one 3’-UTR comprises a nucleic acid sequence derived or selected from a 3-UTR of a gene selected from PSMB3, alpha-globin, ALB7, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment, or variant of any one of these genes.
- the at least one 3-UTR derived or selected from PSMB3, alpha-globin, ALB7, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 or RPS9 comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 253-268 of WO2021156267, or a fragment or a variant of any of these.
- the RNA comprises a 3-UTR derived or selected from a PSMB3 gene.
- the 3-UTR derived or selected from PSMB3 comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3 or 4, or a fragment or a variant thereof.
- the RNA of the invention comprises at least one 5-UTR.
- 5'-untranslated region or “5’-UTR” will be recognized and understood by the person of ordinary skill in the art and are e.g. intended to refer to a part of an RNA located 5’ (i.e. “upstream”) of a coding sequence and which is not translated into protein.
- a 5-UTR may be part of a nucleic acid located 5' of the coding sequence. Typically, a 5-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence.
- a 5-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.
- the 5-UTR may be modified, e.g. by enzymatic or co-transcriptional addition of a 5’-cap structure (e.g. for mRNA as defined below).
- the RNA comprises at least one 5-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
- the 5’-UTR comprises one or more of a binding site for proteins that affect an RNA stability or RNA location in a cell, or one or more miRNA or binding sites for miRNAs (as defined above).
- miRNA or binding sites for miRNAs as defined above may be removed from the 5’-UTR or introduced into the 5-UTR in order to tailor the expression of the nucleic acid to desired cell types or tissues (e.g. muscle cells).
- the RNA comprises at least one 5’-UTR, wherein the at least one 5’-UTR comprises a nucleic acid sequence derived or selected from a 5-UTR of gene selected from HSD17B4, RPL32, ASAH1 , ATP5A1 , MP68, NDUFA4, NOSIP, RPL31 , SLC7A3, TUBB4B, and UBQLN2, or from a homolog, a fragment or variant of any one of these genes,
- the at least one 5-UTR derived or selected from HSD17B4, RPL32, ASAH1 , ATP5A1 , MP68, NDUFA4, NOSIP, RPL31 , SLC7A3, TUBB4B, and UBQLN2 comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 231 -252 of WO2021156267, or a fragment or a variant of any of these.
- the RNA comprises a 5-UTR derived or selected from a HSD17B4 gene.
- the 5-UTR derived or selected from HSD17B4 comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1 or 2, or a fragment or a variant thereof.
- the RNA of the invention comprises at least one coding sequence as specified herein operably linked to a 3 -UTR and/or a 5-UTR selected from the following 5'-UTR/3’-UTR combinations (also referred to “UTR designs”): a-1 (HSD17B4/PSMB3), a-2 (NDUFA4/PSMB3), a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1 ), b-5 (NOSIP/COX6B1 ), 0-1 (NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4 (NDUFA4 /NDUFA1),
- the at least one 5-UTR is selected from HSD17B4 and the at least one 3’ UTR is selected from PSMB3
- the RNA comprises at least one coding sequence as defined herein, wherein said coding sequence is operably linked to a HSD17B45-UTR and a PSMB33-UTR (HSD17B4/PSMB3 (a-1)).
- the RNA comprises at least one poly(N) sequence, e.g. at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or combinations thereof.
- the RNA ofthe invention comprises at least one poly(A) sequence.
- poly(A) sequence “poly(A) tail” or ”3'-poly(A) tail” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to be a sequence of adenosine nucleotides, typically located at the 3’-end of a linear RNA of up to about 1000 adenosine nucleotides.
- said poly(A) sequence is essentially homopolymeric, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides has essentially the length of 100 nucleotides.
- the poly(A) sequence is interrupted by at least one nucleotide different from an adenosine nucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide).
- a poly(A) sequence of e.g. 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide).
- the RNA comprises at least one poly(A) sequence, preferably wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosine nucleotides, preferably about 60 to about 250 adenosine nucleotides, more preferably about 60 to about 150 adenosine nucleotides.
- the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides, preferably consecutive adenosine nucleotides.
- the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.
- the RNA comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, preferably by about 10 nonadenosine (N) nucleotides (e.g. A30-N10-A70 or A70-N10-A30)).
- N nonadenosine
- the poly(A) sequence as defined herein may be located directly at the 3’ terminus of the RNA.
- the 3’-terminal nucleotide (that is the last 3’- terminal nucleotide in the polynucleotide chain) is the 3’-terminal A nucleotide of the at least one poly(A) sequence.
- the term “directly located at the 3’ terminus” has to be understood as being located exactly at the 3’ terminus - in other words, the 3’ terminus of the RNA consists of a poly(A) sequence terminating with an A.
- Ending on an adenosine nucleotide decreases the induction of interferons, e.g. IFNalpha, by the RNA ofthe invention if for example administered as a vaccine. This is particularly important as the induction of interferons, e.g. IFNalpha, is thought to be one main factor for induction of fever in vaccinated subjects.
- the poly(A) sequence comprises 100 consecutive adenosine nucleotides and is located directly at the 3’ terminus of the (linear) RNA, preferably the mRNA.
- the poly(A) sequence of the RNA is obtained from a DNA template during RNA in vitro transcription.
- the RNA comprises at least one poly(A) sequence obtained by enzymatic polyadenylation, wherein the majority of RNA molecules comprise about 100 (+/-20) to about 500 (+/-100) adenosine nucleotides, preferably 100 (+/-20) to 200 (+/-40) adenosine nucleotides. In some embodiments, the RNA comprises at least one polyadenylation signal.
- the RNA comprises at least one poly(C) sequence.
- a poly(C) sequence in the context of the invention may be located in an UTR region, preferably in the 3 -UTR.
- poly(C) sequence as used herein is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides.
- the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides.
- the poly(C) sequence comprises about 30 cytosine nucleotides.
- the RNA comprises at least one histone stem-loop (hSL) or histone stem loop structure.
- hSL histone stem-loop
- a hSL in the context of the invention may be located in an UTR region, preferably in the 3-UTR.
- histone stem-loop is intended to refer to nucleic acid sequences that forms a stem-loop secondary structure predominantly found in histone mRNAs.
- Histone stem-loop sequences/structures may suitably be selected from hSL sequences as disclosed in W02012019780, the disclosure relating to histone stem-loop sequences/histone stem-loop structures incorporated herewith by reference.
- a hSL sequence that may be used within the present invention may be derived from formulae (I) or (II) of W02012019780.
- the RNA comprises at least one hSL sequence derived from at least one of the specific formulae (la) or (Ila) of W02012019780.
- the RNA comprises at least one hSL, wherein said hSL comprises or consists of an RNA sequence identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 178 or 179 of WO2021156267, or fragments or variants of those.
- the RNA does not comprise a histone stem-loop as defined herein.
- the RNA of the invention is a modified RNA, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.
- a modified RNA may comprise nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications.
- a backbone modification in the context of the invention is a modification in which phosphates of the backbone of the nucleotides of the RNA are chemically modified.
- a sugar modification in the context of the invention is a chemical modification of the sugar of the nucleotides of the RNA.
- a base modification in the context of the invention is a chemical modification of the base moiety of the nucleotides of the RNA.
- nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
- the RNA of the invention comprises at least one modified nucleotide.
- the RNA ofthe invention comprises at least one modified Uracil nucleotide.
- the at least one modified nucleotide is selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydrupseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2’-O-
- the RNA of the invention comprises at least one modified nucleotide selected from pseudouridine (up) and/or N1 -methylpseudouridine (m1qj). Particularly preferred in the context of the invention is N1 -methylpseudouridine (m1qj).
- essentially all, e.g. essentially 100% of the uracil in the coding sequence (or the full RNA sequence) have a chemical modification, preferably a chemical modification in the 5-position of the uracil.
- all Uracils in the RNA sequence are replaced by modified nucleotides, preferably modified Uracil nucleotides, more preferably by pseudouridine (qj) and/or N1- methylpseudouridine (m1qj), most preferably by N1 -methylpseudouridine (m1qj).
- modified nucleotides preferably modified Uracil nucleotides, more preferably by pseudouridine (qj) and/or N1- methylpseudouridine (m1qj), most preferably by N1 -methylpseudouridine (m1qj).
- the RNA of the invention does not comprise N1 -methylpseudouridine (m1'4 J ) substituted positions or pseudouridine (qj) substituted positions.
- the RNA comprises a coding sequence that consists only of G, C, A and U nucleotides and therefore does not comprise modified nucleotides.
- the coding sequence / the RNA sequence does not comprise modified nucleotides (except of an optional 5’ cap structure as further defined below).
- the RNA comprises a 5’-cap structure, which suitably stabilizes the RNA and/or enhances expression of the encoded antigen and/or reduces the stimulation of the innate immune system (after administration to a subject, e.g. a human subject).
- the RNA comprises a 5’-cap structure, preferably m7G, capO, cap1 , cap2, a modified capO ora modified cap1 structure.
- 5’-cap structure as used herein will be recognized and understood by the person of ordinary skill in the art and is e.g. intended to refer to a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5’-end of an RNA, e.g. an mRNA.
- the 5’-cap structure is connected via a 5’-5’-triphosphate linkage to the RNA.
- 5’-cap structures which may be suitable in the context of the present invention are capO (methylation of the first nucleobase, e.g. m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.
- a 5’-cap (capO or cap1 ) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co- transcriptional capping) using cap analogues.
- cap analogue as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of an RNA molecule when incorporated at the 5’-end of the nucleic acid molecule.
- Non-polymerizable means that the cap analogue will be incorporated only at the 5'-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3'-direction by a templatedependent polymerase, particularly, by template-dependent RNA polymerase.
- a cap1 structure is generated using tri-nucleotide cap analogue as disclosed in WO2017053297, WO2017066793, WO2017066781 , WO2017066791 , WO2017066789, WO2017066782, WO2018075827 and WO2017066797.
- cap structures derivable from the structure disclosed in claim 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate a cap1 structure.
- any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to generate a cap1 structure.
- the 5’-cap structure may suitably be added co-transcriptionally using tri-nucleotide cap analogue as defined herein, preferably in an RNA in vitro transcription reaction as defined herein.
- the RNA of the invention in particular the mRNA, comprises a cap1 structure.
- the cap1 structure of the RNA is formed via co-transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG.
- a particularly preferred cap1 analogue in that context is m7G(5’)ppp(5’)(2’OMeA)pG.
- the cap1 structure of the RNA is formed using co-transcriptional capping using trinucleotide cap analogue 3’0Me-m7G(5’)ppp(5’)(2’0MeA)pG.
- the 5’-cap structure is formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases) to generate capO or cap1 or cap2 structures.
- capping enzymes e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases
- RNA (species) comprises a cap structure, preferably a cap1 structure as determined by a capping assay.
- about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises a cap structure, preferably a cap1 structure, upon storage of the composition as determined by a capping assay.
- a capping assay as described in W02015101416, in particular, as described in claims 71 to 46 of W02015101416 can be used.
- the A/U (A/T) content in the environment of the ribosome binding site of the RNA may be increased compared to the A/U (A/T) content in the environment of the ribosome binding site of its respective wild type nucleic acid.
- This modification an increased A/U (A/T) content around the ribosome binding site
- An effective binding of the ribosomes to the ribosome binding site in turn has the effect of an efficient translation the RNA.
- the RNA of the composition comprises a ribosome binding site, also referred to as “Kozak sequence” identical to or at least 80%, 85%, 90%, 95% identical to any one of the sequences SEQ ID NOs: 180 or 181 of WO2021156267, or fragments or variants thereof.
- Kozak sequence identical to or at least 80%, 85%, 90%, 95% identical to any one of the sequences SEQ ID NOs: 180 or 181 of WO2021156267, or fragments or variants thereof.
- the RNA is an in vitro transcribed RNA.
- the RNA of the invention has been produced by RNA in vitro transcription as defined herein.
- the DNA template for RNA in vitro transcription is linearized with a suitable restriction enzyme before it is subjected to RNA in vitro transcription.
- Reagents typically used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5, preferably T7); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein preferably Cap1 analog); optionally, modified nucleotides as defined herein (preferably ml qj); a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g.
- RNA polymerase preferably T7; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, pyrophosphatase; MgCI2; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine.
- RNase ribonuclease
- the nucleotide mixture i.e. the fraction of each nucleotide in the mixture
- the nucleotide mixture used for RNA in vitro transcription reactions is optimized for the given RNA sequence, preferably as described WO2015188933.
- RNA-grade RNA is produced using a manufacturing process approved by regulatory authorities.
- RNA production is performed under current good manufacturing practice (GMP), implementing various quality control steps on DNA and RNA level, preferably quality control steps selected from methods described in WO2016180430.
- GMP-grade RNA is a GMP-grade RNA, particularly a GMP-grade mRNA.
- the RNA of the invention is a purified RNA, preferably a purified mRNA.
- purified RNA or “purified mRNA” as used herein has to be understood as RNA which has a higher purity after certain purification steps (e.g. HPLC, TFF, Oligo d(T) purification, precipitation steps) than the starting material (e.g. in vitro transcribed RNA).
- Typical impurities that are essentially not present in purified RNA comprise peptides or proteins (e.g. enzymes derived from DNA dependent RNA in vitro transcription, e.g.
- RNA polymerases RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double stranded RNA (dsRNA)), free nucleotides (modified nucleotides, conventional NTPs, cap analogue), template DNA fragments, buffer components (HEPES, TRIS, MgCI2) etc.
- Other potential impurities that may be derived from e.g. fermentation procedures comprise bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents etc.). Accordingly, it is desirable in this regard for the “degree of RNA purity” to be as close as possible to 100%.
- purified RNA as used herein has a degree of purity of more than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favourably 99% or more.
- the degree of purity is e.g. determined by an analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of the peak for the target RNA and the total area of all peaks including the peaks representing the byproducts.
- the degree of purity is e.g. determined by an analytical agarose gel electrophoresis or capillary gel electrophoresis.
- purification of the RNA of the invention may be performed by means of (RP)-HPLC, AEX, size exclusion chromatography, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, and/or cellulose-based purification.
- the RNA has been purified using RP-HPLC (preferably as described in W02008077592) and/or tangential flow filtration (preferably as described in WO2016193206) and/or oligo d(T) purification (preferably as described in WO2016180430) to e.g. to remove dsRNA, non-capped RNA and/or RNA fragments.
- RP-HPLC preferably as described in W02008077592
- tangential flow filtration preferably as described in WO2016193206
- oligo d(T) purification preferably as described in WO2016180430
- the RNA is a purified RNA, preferably wherein the RNA has been purified by RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof, more preferably wherein the RNA has been purified by RP-HPLC and/or TFF.
- the RNA of the invention has a certain RNA integrity.
- RNA integrity generally describes whether the complete RNA sequence is present in the pharmaceutical composition. Low RNA integrity could be due to, amongst others, RNA degradation, RNA cleavage, incorrect or incomplete chemical synthesis of the RNA, incorrect base pairing, integration of modified nucleotides or the modification of already integrated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete RNA in vitro transcription.
- RNA is a fragile molecule that can easily degrade, which may be caused e.g. by temperature, ribonucleases, pH or other factors (e.g. nucleophilic attacks, hydrolysis etc.), which may reduce the RNA integrity and, consequently, the functionality of the RNA.
- a large proportion of RNA with low integrity may also be an indicator for destabilization of the lipid-based carriers of the composition (e.g. upon storage, thawing, dilution).
- RNA integrity may be determined using analytical (RP)HPLC, in particular IP-RP-HPLC.
- a test sample of the liquid composition comprising lipid-based carrier encapsulating or comprising RNA may be treated with a detergent (e.g.
- RNA samples may be diluted to a concentration of 0.1 g/l using e.g. water for injection (WFI). About 10 l of the diluted RNA sample may be injected into an HPLC column (e.g.
- RNA integrity in the context of the invention is determined using analytical RP-HPLC, preferably IP-RP-HPLC, more preferably using CuPe integration.
- the RNA of the invention has an RNA integrity ranging from about 40% to about 100%. In embodiments, the RNA has an RNA integrity ranging from about 50% to about 100%. In embodiments, the RNA has an RNA integrity ranging from about 60% to about 100%. In embodiments, the RNA integrity is for example about 50%, about 60%, about 70%, about 80.
- the RNA has an RNA integrity of at least about 50%, preferably of at least about 60%, more preferably of at least about 70%, most preferably of at least about 80% or 90%.
- RNA integrity is suitably determined using analytical RP-HPLC, preferably IP-RP-HPLC, more preferably using CuPe integration.
- the RNA of the invention has a low content of late eluting peaks (LNPs) species.
- late eluting peaks or the corresponding abbreviation “LEPs” generally describes a certain RNA proportion of RNA that has been formulated in lipid-based carriers as defined herein, wherein said proportion of RNA has a longer retention time in an RP-HPLC method than the actual expected RNA (as further outlined below). Accordingly, that certain proportion of RNA having a longer retention time in an RP-HPLC method elutes later in an RP-HPLC (in comparison to the expected RNA main peak), giving rise to a certain typically heterogeneous region in the chromatogram, and is therefore generally described as late eluting peaks (LEPs).
- LEPs may be an RNA population that is associated with (covalently or non-covalently) or that is modified with a lipid or a fraction of a lipid or a chemical group or moiety of a lipid or a precursor or adduct thereof.
- LEPs may represent the fraction of a lipid-RNA impurity.
- These LEPs species have also been described in the literature, e.g. in Packer, Meredith, et al. "A novel mechanism for the loss ofmRNA activity in lipid nanoparticle delivery systems.” Nature communications 12.1 (2021): 1-11. As these LEPs species may be present in the formulated pharmaceutical composition, and as the effect of such LEPs when e.g. applied to a human as medicament are not well understood, it is desired to minimize the level of LEPs.
- LEPs levels are expressed as content in %.
- the RNA extracted from the lipid-based carriers is analysed using analytical (RP)HPLC, preferably IP-RP-HPLC, used in a similar fashion as described for the determination of RNA integrity.
- the obtained chromatograms may be evaluated using a software and the relative peak area of the non-late eluting peak (NEP) and the LEPs are determined in percent (%) of the total chromatographic peak area. Accordingly, the main peak that comprises the expected RNA would represent the NEP fraction, and the RNA that is potentially associated with a lipid would be present in the LEPs fraction.
- LEPs species may also be analysed using an analytical IP-RP-HPLC method as outlined in Packer, Meredith, et al. "A novel mechanism for the loss ofmRNA activity in lipid nanoparticle delivery systems.” Nature communications 12.1 (2021 ): 1-11.
- the RNA comprises less than about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% late eluting peaks as determined using an HPLC method on RNA isolated from lipid-based carriers as defined herein.
- the HPLC method is IP-RP-HPLC as defined herein.
- an RNA integrity analysis as defined herein may be performed, preferably
- the RNA is suitable for intranasal, intramuscular, intradermal, transdermal, or subcutaneous administration. In particularly preferred embodiments, the RNA is suitable for intramuscular administration.
- the RNA of the composition may provide at least one coding sequence encoding a peptide or protein that is translated into a (functional) peptide or protein after administration (e.g. after administration to a subject, e.g. a human subject).
- the RNA of the invention comprises at least one coding sequence, wherein the coding sequence encodes at least one peptide or protein suitable for use in treatment or prevention of a disease, disorder, or condition.
- the at least one coding sequence encodes at least one therapeutic peptide or protein.
- the length of the encoded peptide or protein may be at least or greater than about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1500 amino acids.
- the at least one therapeutic peptide or protein is selected or derived from an antibody, an intrabody, a receptor, a receptor agonist, a receptor antagonist, a binding protein, a CRISPR-associated endonuclease, a chaperone, a transporter protein, an ion channel, a membrane protein, a secreted protein, a transcription factor, an enzyme, a peptide or protein hormone, a growth factor, a structural protein, a cytoplasmic protein, a cytoskeletal protein, a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope, an allergen, a tumor antigen or epitope, or fragments, variants, or combinations of any of these.
- the at least one therapeutic peptide or protein is selected or derived from an antigen or epitope of a pathogen (e.g. a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope).
- a pathogen e.g. a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope.
- the peptide or protein is selected from an antigen or epitope of a pathogen selected or derived from List 1 provided below.
- Acinetobacter baumannii Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans
- the at least one therapeutic peptide or protein is selected or derived from an antigen or epitope of a pathogen preferably selected from a Coronavirus or Influenza virus.
- the RNA comprises a coding sequence encoding at least one antigen or epitope selected or derived from a Coronavirus, preferably SARS-CoV-2.
- a Coronavirus preferably SARS-CoV-2 antigen or epitope
- the Coronavirus preferably SARS-CoV-2 antigen or epitope is selected or derived from a spike protein.
- the spike antigen is preferably a prefusion stabilized SARS-CoV-2 spike protein preferably comprising a K986P and/or a V987P amino acid substitution.
- the least one peptide or protein is selected or derived from a SARS-CoV-2 spike protein and comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the SARS-CoV2 spike proteins or fragments thereof disclosed in the Sequence listings of WO2021156267, in particular being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-26, 341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22947-22
- the least one peptide or protein is selected or derived from a SARS-CoV-2 spike protein and comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12-14, ora fragment or variant of any of these sequences.
- the RNA comprises a coding sequence (encoding a SARS-CoV-2 spike protein or fragment) which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequences encoding a SARS-CoV-2 spike protein disclosed in the Sequence listings of WO2021156267, in particular being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 136-138, 140, 141, 148, 149, 152, 155, 156, 159, 162, 163, 166, 169, 170, 173, 11731-11813, 11815, 11817-11966, 12271
- the RNA encoding the SARS-CoV-2 antigen or epitope comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 148- 175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069- 24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829- 26044, 26049-26264, 26269-26484, 26489-26704, 26709-26937 of WO2021156267
- the RNA encoding the SARS-CoV-2 antigen or epitope comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5-11 , preferably SEQ ID NOs: 7 or 8, or a fragment or variant of that sequence.
- the RNA comprises a 5’ Cap1 structure.
- the RNA sequence comprises ml qj instead of Uracils.
- the least one peptide or protein is selected or derived from an antigen or epitope of an Influenza virus (HA or NA) and comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 14178-14229 of WO2021239880 or a fragment or variant of any of these sequences (see Table 8 of WO2021239880).
- the RNA comprises a coding sequence encoding an antigen or epitope of an Influenza virus (HA or NA) which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14230- 14333 of WO2021239880 or a fragment or variant of any of these sequences (see Table 8 of WO2021239880).
- HA or NA Influenza virus
- the RNA encoding an antigen or epitope of an Influenza virus comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14334-14541 , 26947-26955 ofWO2021239880 or a fragment or variant of any of these sequences (see Table 9 of WO2021239880).
- the RNA comprises a 5’ Cap1 structure.
- the RNA sequence comprises m1qj instead of Uracils.
- the RNA comprises at least the following elements:
- the RNA preferably the mRNA, comprises the following elements:
- histone stem-loop optionally, histone stem-loop preferably as specified herein;
- the mRNA comprises the following elements preferably in 5’- to 3'-direction:
- a 5-UTR preferably derived from a 5-UTR of a HSD17B4 gene as defined herein;
- a 3' -UTR preferably derived from a 3-UTR of a PSMB3 gene as defined herein;
- G optionally, chemically modified nucleotides, e.g. pseudo uridine (i ) or N1 -methylpseudouridine (ml i ).
- pseudo uridine i
- N1 -methylpseudouridine ml i
- the pharmaceutical composition comprises a plurality of RNA species, preferably at least 2, 3, 4, or more RNA species, each encoding a different therapeutic peptide or protein as defined herein.
- the pharmaceutical composition comprises at least one or more RNA species encoding a different antigen or epitope of a SARS-CoV2 and/or at least one or more RNA species encoding a different antigen or epitope of an Influenza virus.
- the pharmaceutical composition comprises at least 2, 3, 4 RNA species encoding a different antigen or epitope of a SARS-CoV2 (for example, Spike proteins or fragments thereof derived from a different SARS-CoV2 variant).
- the pharmaceutical composition comprises at least 2, 3, 4 RNA species encoding a different antigen or epitope of an Influenza virus (for example, derived from HA or NA of different Influenza virus strains).
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises d) 50mM to 200mM, preferably 10OmM to 200mM, more preferably 10OmM to 150mM glycerol; e) 50mM to 200mM, preferably 10OmM to 200mM, more preferably 100mM to 150mM sucrose; f) Tris as buffer agent at preferably at a concentration of at least 10mM or more than 20mM; and wherein the added total concentration of glycerol and sucrose is preferably in a range of 200mM to 300mM, in particular at about 250mM, and the lipid-based carriers, preferably LNPs, comprise
- RNA that has a length preferably ranging from about 500 nucleotides to about 5000 nucleotides.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1.
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) 100mM glycerol; b) 150mM sucrose (as sugar component); c) T ris as buffer agent at preferably at least 10mM or more than 20mM, suitably at 30mM; and wherein the lipid-based carriers, preferably LNPs, comprise
- RNA preferably has a length of at least 3000 nucleotides, preferably a length ranging from about 3000 nucleotides to about 5000 nucleotides.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1 , in particular SARS-CoV2 (e.g. Spike protein).
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) 150mM glycerol; b) 10OmM sucrose (as sugar component); c) Tris as buffer agent at preferably at least 10mM more than 20mM, suitably at 30mM; and wherein the lipid-based carriers, preferably LNPs, comprise
- RNA preferably has a length of not more than 3000 nucleotides, preferably a length ranging from about 1000 nucleotides to about 3000 nucleotides.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1 , in particular Influenza virus.
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid- based carriers comprise RNA and are contained in a buffer system that comprises a) 50mM to 200mM glycerol, preferably 10OmM to 150mM glycerol; b) 50mM to 200mM sucrose (as sugar component), preferably 10OmM to 150mM sucrose; c) T ris as buffer agent at preferably at least 10mM or more than 20mM, suitably at 30mM, and wherein the added total concentration for both glycerol and sucrose is preferably in a range of 200mM to 300mM, in particular at about 250mM, and the lipid-based carriers are 315-LNPs or GN-LNPs as defined herein, and the lipid- based carriers encapsulate an RNA.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1.
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid- based carriers comprise RNA and are contained in a buffer system that comprises a) 100mM glycerol; b) 150mM sucrose (as sugar component); c) T ris as buffer agent at preferably at least 10mM or more than 20mM, suitably at 30mM, and wherein the lipid-based carriers are 315-LNPs or GN-LNPs as defined herein, and the lipid-based carriers encapsulate an RNA that preferably has a length of at least 3000 nucleotides, preferably a length ranging from about 3000 nucleotides to about 5000 nucleotides.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1 , in particular SARS-CoV2 (e.g. Spike protein).
- the pharmaceutical composition comprises lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) 150mM glycerol; b) 10OmM sucrose (as sugar component); c) T ris as buffer agent at preferably at least 10mM or more than 20mM, suitably at 30mM, and wherein the lipid-based carriers are 315-LNPs or GN-LNPs as defined herein, and the lipid-based carriers encapsulate an RNA that preferably has a length of not more than 3000 nucleotides, preferably a length ranging from about 1000 nucleotides to about 3000 nucleotides.
- the RNA encodes a therapeutic peptide or protein, preferably an antigen of a pathogen of List 1 , in particular Influenza virus.
- the lipid-based carriers are LNPs encapsulating an RNA and are contained in a buffer system that comprises a) 100mM glycerol; b) 150mM sucrose (as sugar component); c) T ris as buffer agent at preferably at least 10mM or more than 20mM, suitably at 30mM; wherein the lipid-based carriers comprise (i) the cationic lipid ALC-0315 (lipid of formula III), (ii) the neutral lipid DSPC, (iii) cholesterol, and (iv) the aggregation reducing lipid ALC-0159 (lipid of formula IVa), preferably wherein (i) to (iv) are in a molar ratio of about 47.4% cationic lipid, 10% neutral lipid, 40.9% steroid or steroid analogue, and 1.7% aggregation reducing lipid, wherein the RNA encodes a SARS-CoV-2 antigen or epitope, optionally wherein
- the lipid-based carriers comprising an RNA as defined herein have been formulated in a sucrose PBS buffer followed by a buffer exchange to the advantageous buffer system as defined herein (e.g. comprising glycerol).
- the lipid-based carriers may be prepared by combining an ethanolic lipid solution (comprising lipids of the lipid-based carriers as defined herein) and an RNA solution (comprising RNA as defined herein) to allow the formation of lipid-based carriers encapsulating the RNA.
- the RNA solution comprises citrate or acetate.
- the combining step involves a T-piece or a Y-piece based mixing under specific flow rates.
- the obtained lipid-based carriers may be transferred to a sucrose PBS buffer (e.g. by filtration or TFF) or a T ris buffer. This step is important to remove ethanol, citrate, unwanted lipid species, or other by-products.
- the concentration of sucrose in the sucrose PBS buffer (used in filtration or TFF) is in a range from about 50mM to about 300mM, preferably about 150mM.
- the sucrose PBS buffer comprises a salt, preferably NaCI.
- the concentration of the salt comprised in the composition is in a range from about 10mM to about 200mM, preferably about 75mM.
- the salt comprised in the sucrose PBS buffer is NaCI, preferably in a concentration of about 75mM.
- the sucrose PBS buffer comprises a buffer agent, preferably NaPO4.
- the buffer agent is in a concentration ranging from about 1mM to about 100mM.
- the buffer agent is NaPO4, preferably in a concentration of about 10mM.
- the lipid-based earners comprising an RNA as defined herein have been formulated in a sucrose PBS buffer that comprises sucrose in a concentration of about 150mM, NaCI in a concentration of about 75mM and Na3PO4 in a concentration of about 10mM, followed by a buffer exchange to the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component).
- the concentration of Tris in the Tris buffer (used in filtration orTFF) is in a range from about 10mM to about 50mM.
- the lipid-based carriers comprising an RNA as defined herein have been formulated in a Tris buffer as defined herein, followed by a buffer exchange to the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component).
- Said buffer exchange to the advantageous buffer system as defined herein may be performed by filtration, dialysis, TFF, or dilution.
- the buffer exchange is performed by dilution (dilution as further specified below).
- the pharmaceutical composition has been prepared by diluting a sucrose PBS buffer or a Tris buffer with the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component).
- the resulting pharmaceutical composition of the invention may comprise NaCI or Phosphate.
- the obtained lipid-based carriers may be (directly) transferred (e.g. by filtration or TFF) to the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component).
- the resulting pharmaceutical composition of the invention does not comprise NaCI or Phosphate or is essentially free of NaCI or Phosphate.
- the lipid-based carriers are transferred to a sucrose PBS buffer (e.g. by filtration orTFF) or a Tris buffer followed by a buffer exchange to the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component), preferably by dilution.
- a sucrose PBS buffer e.g. by filtration orTFF
- a Tris buffer followed by a buffer exchange to the advantageous buffer system as defined herein (e.g. comprising glycerol and a sugar component), preferably by dilution.
- the buffer system advantageously allows a stable storage of lipid-based carriers comprising RNA at low concentrations of said lipid-based carriers comprising RNA. This is particularly important for a storage of a pharmaceutical composition that is ready to use, e.g., that comprises one dose in an appropriate volume (e.g. 500pl volume).
- an appropriate volume e.g. 500pl volume
- the total concentration of lipid in the composition is in a range from about 25 pg/ml to about 5000 pg/ml, in a range from about 250 pg/ml to about 2500 pg/ml, in a range from about 500 pg/ml to about 2500 pg/ml, in a range from about 500 pg/ml to about 1250 pg/ml.
- the total concentration of lipid in that context may relate to the sum of cationic lipid, cholesterol, neutral lipid, and aggregation reducing lipid comprised in lipid-based carriers, e.g. LNPs.
- the total concentration of lipid in the composition is in a range from about 500 pg/ml to about 2500 pg/ml.
- the concentration of RNA in the composition is in a range from about 1 pg/ml to about 500 pg/ml, in a range from about 1 pg/ml to about 100 pg/ml, in a range from about 10 pg/ml to about 100 pg/ml, in a range from about 20 pg/ml to about 100 pg/ml, in a range from about 20 pg/ml to about 50 pg/ml.
- the concentration of RNA in the composition is in a range from about 20 pg/ml to about 100 pg/ml.
- the total concentration of lipid in the composition is in a range from about 500 pg/ml to about 2500 pg/ml and the total concentration of RNA in the composition is in a range from about 20 pg/ml to about 100 pg/ml.
- said dose would comprise 5 to 50pg RNA formulated in 125 to 1250pg total lipid (e.g. comprising cationic lipid, cholesterol, neutral lipid, and aggregation reducing lipid).
- the pharmaceutical composition as defined herein is in liquid form. Accordingly, in embodiments, the pharmaceutical composition is a liquid pharmaceutical composition.
- the liquid pharmaceutical composition has been subjected to at least one freeze step and to at least one thaw step.
- a freeze-thaw step does not impact quality attributes of the lipid-based carriers as defined herein comprising an RNA as defined herein.
- This has advantages in the supply of the pharmaceutical composition as for example a centralized storage of the pharmaceutical composition at -80°C, -60°C, or -40°C is possible, followed by shipping of the pharmaceutical composition at -20°C, followed by a further storage, after thawing, at fridge temperature (about 5°C).
- the pharmaceutical composition as defined herein is in frozen form or at freezing temperature. Accordingly, in embodiments, the pharmaceutical composition is a frozen pharmaceutical composition. In preferred embodiments in that context, the pharmaceutical composition is stored or frozen at -80°C, preferably at -60°C, more preferably at -40°C, even more preferably at -20 Q C.
- the pharmaceutical composition as defined herein is in dried form. Accordingly, in embodiments, the pharmaceutical composition is a dried pharmaceutical composition (e.g. powder, granules).
- the pharmaceutical composition has been dried using lyophilization (e.g. essentially according to protocols disclosed in WO2016165831 or WO2011069586) to yield a dried composition.
- the pharmaceutical composition has been dried using spray-drying or spray-freeze drying (e.g. essentially according to protocols disclosed in WO2016184575 or WO2016184576) to yield a dried composition.
- the pharmaceutical composition as defined herein comprises lipid-based carriers comprising RNA as defined herein and are contained in a buffer system as defined herein is characterized by advantageous stability characteristics as shown in the Example section.
- the pharmaceutical composition is stable upon dilution (as further defined herein). In preferred embodiments, the pharmaceutical composition is stable upon thawing (as further defined herein).
- the pharmaceutical composition is stable upon storage (as further defined herein).
- ''stable refers to a composition comprising lipid-based carriers comprising RNA where the measured values for various physiochemical parameters are within a defined range after storage, dilution, or thawing.
- the composition comprising lipid-based carriers comprising RNA is analyzed to assess stability according to various parameters.
- Suitable stability parameters include, without limitation, RNA integrity, Z-average particle size, polydispersity index (PDI), the amount of free RNA in the composition, RNA encapsulation (proportion of the RNA in percent associated with lipid-based carriers), the amount of late-eluting peaks (LEPs), shape and morphology of the lipid-based carriers encapsulating an RNA, pH, osmolality, or turbidity.
- RNA integrity Z-average particle size
- PDI polydispersity index
- LEPs late-eluting peaks
- shape and morphology of the lipid-based carriers encapsulating an RNA pH, osmolality, or turbidity.
- stable refers to a composition comprising lipid-based carriers comprising RNA where the measured values for various functional parameters are within a defined range after storage, dilution, or thawing.
- the liquid composition comprising lipid-based carriers comprising an RNA is analyzed to assess the potency of the liquid composition including for example the expression of the encoded peptide or protein, the induction of specific antibody titers, the induction of neutralizing antibody titers, the induction of T-cell, the reactogenicity of the liquid composition including for example the induction of innate immune responses etc.
- the pharmaceutical composition is characterized by stable or preserved quality attributes (or physiochemical parameters) of the lipid-based carriers comprising RNA, preferably wherein the quality attributes are selected from RNA integrity, RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs).
- the quality attributes are selected from RNA integrity, RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs).
- the RNA of the pharmaceutical composition has a stable or preserved RNA integrity, preferably upon dilution, thawing, and/or storage of the composition.
- the RNA integrity value decreases less than about 30%, less than about 20%, less than about 10%.
- the delta change of the RNA integrity in % is less than about -30%, less than about -20%, less than about -10%.
- Delta change of the RNA integrity is calculated as defined herein.
- the integrity of the RNA decreases less than about 0.4% per day of storage, less than about 0.3% per day of storage, less than about 0.2% per day of storage, less than 0.1 % per day of storage.
- the delta change of the RNA integrity in % is less than about -0.4% per day of storage, less than about -0.3% per day of storage, less than about -0.2% per day of storage, less than about -0.1 % per day of storage.
- the RNA upon dilution, thawing, or storage of the composition, the RNA has an integrity value of at least about 50%, of at least about 60%, of at least about 70%.
- RNA integrity is determined using analytical IP RP HPLC as described herein.
- the integrity of the RNA decreases less than about 10%, 5%, 4%, 3%, 2%, 1 % over a storage period of about 80 days.
- the delta change of the RNA integrity in % is less than about -10%, -5%, -4%, -3%, -2%, -1 % upon storage of the composition at -20°C or -40°C.
- RNA integrity may be considered to be an essentially linear process over time of storage, when stored at -20°C or -40°C, the integrity of the RNA decreases less than about 0.15% per day of storage, less than about 0.1% per day of storage, less than about 0.05% per day of storage, less than about 0.01% per day of storage.
- the integrity of the RNA decreases less than about 30%, 20%, 10% over a storage period of about 80 days.
- the delta change of the RNA integrity in % is less than about -30%, less than about -20%, less than about -10% upon storage of the composition at 5°C.
- RNA integrity may be considered to be an essentially linear process, when stored at 5°C, the integrity of the RNA decreases less than about less than about 0.5% per day of storage, less than about 0.4% per day of storage, less than about 0.3% per day of storage, less than about 0.2% per day of storage, less than about 0.1% per day of storage.
- the RNA of the pharmaceutical composition has a stable or preserved amount of free RNA or a stable or preserved RNA encapsulation, preferably upon dilution, thawing, and/or storage of the composition.
- the RNA encapsulation decreases less than about 15%, 10%, 5%.
- the delta change of the RNA encapsulation in % is less than about -15%, -10%, -5%.
- Delta change of RNA encapsulation is calculated as defined herein.
- the RNA encapsulation decreases less than about 0.25% per day of storage, less than about 0.20% per day of storage, less than about 0.15% per day of storage, less than about 0.10% per day of storage, less than about 0.05% per day of storage.
- the delta change of the RNA encapsulation is less than about -0.25% per day of storage, less than about -0.2% per day of storage, less than about -0.15% per day of storage, less than about -0.10% per day of storage.
- the RNA encapsulation in % is at least about 70%, at least about 80%, at least about 90%.
- RNA encapsulation is determined by a RiboGreen assay as described herein.
- the RNA encapsulation decreases less than about 10%, 5%, 4%, 3%, 2% over a storage period of about 80 days.
- the delta change of the RNA encapsulation in % is less than about -10%, -5%, -4%, -3%, -2%, upon storage of the composition at 5°C, -20°C, or -40°C.
- RNA encapsulation may be considered to be an essentially linear process, when stored at 5°C, -20°C, or -40°C, the RNA encapsulation in % decreases less than about 0.2% per day, 0.1% per day, 0.05% per day.
- the RNA of the pharmaceutical composition has a stable or preserved PDI value, preferably upon dilution, thawing, and/or storage of the composition.
- the PDI value of the lipid-based carriers encapsulating the RNA does not increase by more than a value of about 0.2, by not more than a value of about 0.1 , by not more than a value of about 0.05.
- the delta change of the PDI is not more than a value of about 0.2, not more than a value of about 0.1 , not more than a value of about 0.05.
- Delta change of PDI is calculated as defined herein.
- the PDI value does not increase by more than a value of about 0.003 per day of storage, by not more than a value of about 0.002 per day of storage, by not more than a value of about 0.001 per day of storage, by not more than a value of about 0.0005 per day of storage.
- the delta change of the PDI value is less than about 0.003 per day of storage, less than about 0.002 per day of storage, less than about 0.001 per day of storage, less than about 0.0005 per day of storage.
- the PDI value upon dilution, thawing, or storage of the composition, is less than about 0.3, less than about 0.2, less than about 0.1.
- the PDI is determined by dynamic light scattering as described herein.
- the PDI value increases by not more than a value of about 0.2, by not more than a value of about 0.1 , by not more than a value of about 0.05 over a storage period of about 80 days.
- the delta change of the PDI value is not more than about 0.2, not more than about 0.1 , not more than about 0.05 upon storage of the composition at 5 Q C, -20°C, or -40°C.
- the increase in PDI value may be considered to be an essentially linear process, when stored at 5°C, -20°C, or -40°C, the PDI value increases by not more than a value of about 0.003 per day of storage, by not more than a value of about 0.002 per day of storage, by not more than a value of about 0.001 per day of storage, by not more than a value of about 0.0005 per day of storage.
- the pharmaceutical composition has a stable or preserved Z-average size of the lipid-based carriers, preferably upon dilution, thawing, and/or storage of the composition.
- the Z-average size of the lipid-based carriers does not increase by more than about 30nm, by not more than about 20nm, by not more than about 10nm, by not more than about 5nm.
- the delta change of the Z-average size of the lipid-based carriers is not more than about 30nm, not more than about 20nm, not more than about 10nm, not more than about 5nm.
- Delta change of Z-average size is calculated as defined herein.
- the Z-average size does not increase by more than about 0.5nm per day of storage, by not more than about 0.4nm per day of storage, by not more than about 0.3nm per day of storage, by not more than about 0.2nm per day of storage, by not more than about 0.1 nm per day of storage, by not more than about 0.05nm per day of storage.
- the delta change the Z-average size is less than about 0.5nm per day of storage, less than about 0.4nm per day of storage, less than about 0.3nm per day of storage, less than about 0.2nm per day of storage, less than about 0.1 nm per day of storage, less than about 0.05nm per day of storage.
- the Z-average size of the lipid- based carriers are in a range from about 50nm to about 200nm, about 50nm to about 150nm, about 50nm to about 120nm, about 60nm to about 115nm.
- the Z-average size is determined by dynamic light scattering as described herein.
- the Z-average size of the lipid-based carriers increases by not more than a value of about 30nm, by not more than about 20nm, by not more than about 10nm, by not more than about 5nm over a storage period of about 80 days.
- the delta change of the Z- average size is not more than about 30nm, by not more than 20nm, by not more than about 10nm, by not more than about 5nm upon storage of the composition at 5°C, -20°C, or -40°C.
- the Z-average size may be considered to be an essentially linear process, when stored at 5°C, -20°C, or -40°C, the Z-average size increases by not more than 0.5nm per day of storage, by not more than about 0.4nm per day of storage, by not more than about 0.3nm per day of storage, by not more than about 0.2nm per day of storage, by not more than about 0.1 nm per day of storage, by not more than about 0.05nm per day of storage.
- the pharmaceutical composition upon dilution, thawing, or storage of the composition, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the lipid-based carriers have a particle size exceeding about 500nm and/or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% lipid-based carriers that have a particle size smaller than about 20nm.
- the pharmaceutical composition has a stable or preserved amount of late-eluting peaks, preferably upon dilution, thawing, and/or storage of the composition.
- the amount of late- eluting peaks does not increase by more than 20%, by not more than 10%, by not more than 5%, by not more than 1%.
- the delta change of the LEPs is less than about 20%, less than about 10%, less than about 5%, less than about 1%.
- Delta change of the LEPs is calculated as defined herein.
- the LEPs increase less than about 0.3% per day of storage, less than about 0.2% per day of storage, less than about 0.1% per day of storage, less than about 0.05% per day of storage.
- the delta change of the LEPs in % is less than about 0.3% per day, less than about 0.2% per day, less than about 0.1% per day, less than about 0.05% per day of storage.
- the RNA upon dilution, thawing, or storage of the composition, the RNA comprises less than about 15%, less than about 10%, less than about 5% late eluting peaks.
- LEPs are determined using analytical IP RP HPLC as described herein.
- the LEPs in % upon storage of the composition at -20°C or-40°C, increase less than about 10%, 5%, 4%, 3%, 2%, 1% overa storage period of about 80 days.
- the delta change of the LEPs in % is less than about 10%, 5%, 4%, 3%, 2%, 1 % upon storage of the composition at -20°C or -40°C.
- the increase in LEPs may be considered to be an essentially linear process, when stored at -20°C or -40°C, the LEPs in % increase less than about less than about 0.2% per day, less than about 0.1 % per day, less than about 0.05% per day, less than about 0.01% per day.
- the LEPs in % upon storage of the composition at 5°C or 25°C, increase less than about 20%, 10%, 5% overa storage period of about 80 days.
- the delta change of the LEPs in % is less than about 20%, 20%, 5% upon storage of the composition at 5°C or 25°C.
- the increase of the LEPs in % may be considered to be an essentially linear process, when stored at 5°C or 25°C, the LEPs in % increase less than about 0.3% per day, less than about 0.2% per day, less than about 0.1 % per day.
- the pharmaceutical composition has a stable or preserved potency, preferably upon dilution, thawing, and/or storage of the composition.
- potency can be considered as the expression of the encoded peptide or protein upon administration of the composition to a cell. If the composition is a vaccine, potency can be considered as the induction of specific antibody titers upon administration of the composition/vaccine to a cell, and/or the induction of neutralizing antibody titers upon administration of composition/vaccine to a cell, and/or the induction of antigen-specific T-cell responses upon administration of the composition/vaccine to a cell.
- the potency of the composition decreases less than about 30%, preferably less than about 20%, more preferably less than about 10%. Potency is typically determined using a cell-based protein expression assay.
- the pharmaceutical composition is more stable or more preserved upon dilution, thawing, and/or storage of the pharmaceutical composition compared to a reference composition that is stored, diluted, or thawed in an essentially identical fashion.
- the reference composition in the context of the invention comprises the same lipid-based carriers comprising RNA and are formulated in a phosphate buffer that contains about 150mM Sucrose, about 75mM NaCI and about 10mM Na3PO4.
- the quality attributes of the lipid-based carriers encapsulating the RNA are more stable or more preserved upon dilution, thawing, and/or storage of the pharmaceutical composition compared to the reference composition (that is stored, diluted, or thawed in an identical fashion).
- the quality attributes are selected from RNA integrity as defined herein, RNA encapsulation as defined herein, the PDI value as defined herein, the Z-average size of the lipid- based carriers as defined herein, the amount of late-eluting peaks (LEPs) as defined herein, the potency as defined herein.
- the dilution, thawing, and/or storage may take place in a container, a vial, or an article for administration.
- the dilution, thawing, and/or storage may take place in a syringe.
- the thawing and/or storage may take place in a syringe or a pre-filled syringe as defined in the context of the third aspect.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step. In preferred embodiments, the storage comprises at least two storage steps. In other preferred embodiments, the storage comprises at least three storage steps.
- the term “storage step” as used herein defines a certain storage step that is maintained for at least 2 hours at a certain defined temperature. During such a storage step, the temperature is essentially kept at a specified value (deviating from that value by not more than 20%). Accordingly, the temperature may include fluctuations that are typical for commercially available freezing devices or fridges. Accordingly, another storage step starts if the temperature is changed for at least 2h and then essentially maintained at that specified temperature. For example, if the pharmaceutical composition would be stored at about -60°C for 2 days, at about -20°C for 2 days, and at about 5°C for one day, that storage would comprise three storage steps.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step in frozen form (or at freezing temperature).
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least two storage steps in frozen form, e.g. 2, 3, 4 or more steps.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step in liquid form.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least two storage steps in liquid form, in particular 2 steps.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step in frozen form (or at freezing temperature) and at least one storage step in liquid form.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least two storage steps in frozen form (or at freezing temperature) and at least one storage step in liquid form.
- the pharmaceutical composition is stable as defined herein, when subjected to at least one freeze-thaw step or to at least one step of thawing.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one freeze-thaw step. In embodiments, the storage comprises at least 2, 3, or even more freeze-thaw steps.
- the at least one storage step in frozen form has been performed at or below -10°C, at or below - 20°C, at or below -40°C, at or below -60°C, or at -80°C.
- the at least one storage step in frozen form has been performed at about -20°C or at about -4CTC.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step in frozen form (or at freezing temperature), wherein the storage step in frozen form (or at freezing temperature) has been performed at about -20°C.
- the at least one storage step in frozen form has been performed at or below -60°C as a first storage step and at about -20 Q C as a second storage step.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least two storage step in frozen form (or at freezing temperature), wherein one storage step in frozen form (or at freezing temperature) has been performed at or below -60°C or -40°C and one storage step in frozen form (or at freezing temperature) has been performed at about -20°C.
- the at least one storage step in frozen form has been performed for at least 1 months, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, preferably longer than 6 months. In preferred embodiments, the at least one storage step in frozen form (or at freezing temperature) has been performed for at least 1 year, 2 years or even longer.
- the at least one storage step in liquid form has been performed at fridge temperature, for example at about 5°C or more (e.g. between 5°C and 20°C).
- the storage comprises at least one storage step in liquid form
- the at least one storage step in liquid form has been performed at about 5°C or more, for example at 5°C, 10°C, or even 20°C.
- the at least one storage step in liquid form has been performed at fridge temperature (about 5°C) as a first storage step in liquid form and at room temperature (about 20 Q C) as a second storage step in liquid form.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least two storage step in liquid form, wherein one storage step in liquid form has been performed at fridge temperature (about 5°C) and one storage step in liquid form has been performed at about 20°C (room temperature).
- the at least one storage step in liquid form has been performed for at least 2 weeks, 1 months, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, preferably longer than 6 months. In preferred embodiments, the at least one storage step in liquid form has been performed for at least 1 year.
- the pharmaceutical composition as defined herein is stable upon storage as defined herein, wherein the storage comprises at least one storage step in frozen form (or at freezing temperature) as defined herein, preferably at about -20°C for at least 2 months followed by at least one thaw step and followed by at least one storage step in liquid form as defined herein, preferably at about 5°C for at least 2 weeks.
- the pharmaceutical composition as defined herein is stable upon dilution as defined herein, wherein the dilution comprises at least one dilution step.
- the dilution comprises at least one step of dilution from a higher concentration to a lower concentration with a dilution factor.
- the higher concentration is more than 500mg/l RNA and/or more than 12.5 mg/l lipid and the lower concentration is less than 50mg/l RNA and/or less than 1 .25 mg/l lipid.
- the dilution factor is between 1 :5 and 1 :200, preferably between 1 :10 and 1 :100.
- the dilution is performed using the buffer system (comprising glycerol) as defined herein.
- a higher concentrated composition in sucrose-PBS buffer is diluted to a lower concentration using the buffer system (comprising glycerol) as defined herein with a dilution factor between 1 :5 and 1 :200, preferably between 1 : 10 and 1 : 100.
- the pharmaceutical composition comprise one therapeutically effective dose of lipid-based carriers comprising RNA.
- an “effective dose” means an amount of the lipid-based carriers comprising RNA that significantly induces a positive modification of a disease or disorder e.g. a disease related to an infection with a pathogen (e.g. a virus, a bacterium, a protozoan) as specified herein.
- a pathogen e.g. a virus, a bacterium, a protozoan
- an “effective dose” is small enough to avoid serious sideeffects.
- An “effective dose” may preferably mean an amount of the pharmaceutical composition that is suitable for stimulating the adaptive immune system against a pathogen as specified herein in such a manner that no excessive or damaging immune reactions are achieved.
- a “effective dose” of the composition or as defined herein will vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the skilled person.
- the “safe and effective amount” of the composition may depend on application/delivery route (intradermal, intramuscular, intranasal), application device (jet injection, needle injection, microneedle patch, electroporation device) and/or complexation/formulation.
- the “effective dose” of the composition may depend on the physical condition of the treated subject (infant, pregnant women, immunocompromised human subject etc.).
- the pharmaceutical composition contains one therapeutically effective dose. Accordingly, the pharmaceutical composition is a mono-dose composition.
- the pharmaceutical composition is a ready-to-use composition.
- a ready-to-use composition according to the invention is configured for direct administration into a subject and does not require any step handling / preparation / dilution (as for e.g. a multidose composition) or tonicity adjustment (e.g. by dilution with NaCI solution etc.).
- the amount of RNA in the pharmaceutical composition is at about 10Ong to about 500pg, 1 pg to about 200pg, 5pg to about 10Opg, 5pg to about 50pg.
- the pharmaceutical composition may contain one therapeutically dose of RNA that is at about 10Ong to about 500pg, 1 pg to about 200pg, 5pg to about 10Opg, 5pg to about 50pg.
- the amount of lipid in the composition is at about 2500ng to about 12.5mg, 25pg to about 5mg, 125pg to about 2,5mg, 250pg to about 1 ,25mg.
- the pharmaceutical composition has a total volume of about 50pl to 2ml, about 10Opi to 1ml, about 250pl to 1 ml, preferably about 500pl.
- the pharmaceutical composition has a total volume of about 50pl to about 2ml, 10Opi to 1 ml, 250pl to 1 ml, preferably about 500pl and comprises one therapeutically effective dose of RNA that is at about 10Ong to about 500pg, 1 pg to about 200pg, 5pg to about 10Opg, 5pg to about 50pg.
- the pharmaceutical composition is a mono-dose composition, has a total volume of about 500pl, and comprises one therapeutically effective dose comprising RNA in an amount ranging from about 1 pg to about 200pg, 5pg to about 10Opg, or 5pg to about 50pg.
- a pharmaceutical composition as described in the context of the second first aspect may be obtained by the method of manufacturing as described herein.
- the present invention provides a vaccine comprising or consisting of the pharmaceutical composition as defined in the context of the first aspect.
- features and embodiments that are described in the context of the pharmaceutical composition of the first aspect may also be applicable to the vaccine of the second aspect.
- features and embodiments that are described in the context of the vaccine of the second aspect may also be applicable to the pharmaceutical composition of the first aspect.
- the “buffer system” e.g. comprising glycerol, sucrose, and Tris
- the “the lipid-based carriers” e.g. LNPs comprising cationic lipid or ionizable lipid, neutral lipid or phospholipid, steroid or steroid analogue, aggregation reducing lipid
- the RNA e.g.
- mRNA encoding an antigen may also be understood as suitable features for the vaccine of the second aspect.
- advantageous stability properties of the pharmaceutical composition of the first aspect that e.g. relate to stability upon storage, dilution and/or thawing (e.g. integrity of the RNA, the amount of free RNA, RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs), the potency) may also be understood as suitable features for the vaccine of the second aspect.
- the vaccine comprises the pharmaceutical composition comprising lipid-based carriers comprising RNA contained in a buffer system as defined in the first aspect, wherein the RNA encodes at least one antigenic peptide or protein selected from or derived from a pathogen, preferably as defined in the context of the first aspect.
- the RNA of the vaccine comprises at least one coding sequence as defined herein that encodes at least one antigenic peptide or protein selected from or derived from a pathogen as defined herein.
- pathogens may be bacterial, viral, or protozoological (multicellular) pathogenic organisms.
- the pathogen evokes an immunological reaction or an infection in a subject, in particular a mammalian subject, preferably a human subject.
- the vaccine is against at least one pathogen selected from any one of List 1.
- the vaccine is against a pathogen, for example against a virus, against a bacterium, or against a protozoan, preferably a virus.
- the vaccine is against a virus.
- the vaccine is against at least one Coronavirus (e.g. SARS-CoV-2 coronavirus).
- the vaccine is against at least one Influenza virus.
- the vaccine is a polyvalent vaccine preferably against at least one Coronavirus (e.g. SARS-CoV-2 coronavirus) and against at least one Influenza virus.
- the vaccine comprises lipid-based earner, preferably LNPs, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) at least 50mM glycerol, preferably 50mM to 200mM; b) at least 50mM sucrose as sugar component, preferably 50mM to 200mM sucrose; c) at least 10mM or more than 20mM T ris, preferably 30mM T ris; and the added total concentration for both glycerol and sucrose is in a range of 10OmM to 400mM, preferably in a range of 200mM to 300mM, and the lipid-based carriers are LNPs as defined herein, preferably GN-LNPs as defined herein, more preferably 315-LPNs as defined herein, and the lipid-based carriers encapsulate an RNA that comprises a coding sequence encoding an antigen or epitope selected or derived from a pathogen.
- a buffer system that comprises
- the RNA encodes an antigen or epitope selected or derived from a pathogen of List 1 , preferably a virus, more preferably a SARS-CoV-2 virus and/or an Influenza virus.
- the vaccine elicits an adaptive immune response against the encoded antigen or epitope when administered to a cell or a subject.
- the vaccine comprises lipid-based carrier, preferably LNPs, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) 100mM glycerol; b) 150mM sucrose (as sugar component); c) at least 10mM or more than 20mM T ris, preferably 30mM T ris, and the lipid-based carriers are LNPs as defined herein, preferably 315-LPNs as defined herein, and the lipid-based carriers encapsulate an RNA that encodes an antigen or epitope selected or derived from a pathogen of List 1 , preferably a virus, more preferably a SARS-CoV-2 virus.
- a buffer system that comprises a) 100mM glycerol; b) 150mM sucrose (as sugar component); c) at least 10mM or more than 20mM T ris, preferably 30mM T ris, and the lipid-based carriers are LNPs as defined here
- the RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID NOs: 5-11 , preferably SEQ ID NOs: 7 or 8, wherein the RNA preferably comprises a Cap1 structure and wherein the RNA optionally comprises ml ip instead of Uracils.
- the vaccine elicits an adaptive immune response against the encoded antigen or epitope, preferably a Spike antigen, when administered to a cell or a subject.
- an adaptive immune response against the encoded antigen or epitope preferably a Spike antigen
- the vaccine comprises lipid-based carrier, preferably LNPs, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) 150mM glycerol; b) 10OmM sucrose (as sugar component); c) at least 10mM or more than 20mM T ris, preferably 30mM T ris, and the lipid-based carriers are LNPs as defined herein, preferably 315-LPNs as defined herein, and the lipid-based carriers encapsulate an RNA that encodes an antigen or epitope selected or derived from a pathogen of List 1 , preferably a virus, more preferably an Influenza virus.
- a buffer system that comprises a) 150mM glycerol; b) 10OmM sucrose (as sugar component); c) at least 10mM or more than 20mM T ris, preferably 30mM T ris, and the lipid-based carriers are LNPs as defined herein,
- the RNA comprises a Cap1 structure, and the RNA optionally comprises ml i instead of Uracils.
- the vaccine elicits an adaptive immune response against the encoded antigen or epitope, preferably a HA or NA antigen, when administered to a cell or a subject.
- the vaccine of the second aspect is preferably administered locally.
- Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, intraarticular and sublingual injections.
- the vaccine may be administered by an intradermal, subcutaneous, or intramuscular route, preferably by injection, which may be needle-free and/or needle injection.
- Preferred in the context of the invention is intramuscular injection.
- the vaccine is adjusted for intramuscular injection, preferably by adjusting the tonicity of the vaccine to a well tolerable range as defined in the context of the first aspect.
- the vaccine may be used according to the invention for human medical purposes and also for veterinary medical purposes (mammals, vertebrates, or avian species).
- the vaccine elicits an adaptive immune response against at least one pathogen when administered to a cell or a subject, wherein the at least one pathogen may be selected from a bacterium, a protozoan, or a virus, for example from a pathogen provided in List 1.
- the vaccine elicits an adaptive immune response against at least one SARS-CoV-2 virus and/or at least one Influenza virus when administered to a cell or a subject.
- the vaccine elicits antigen-specific immune responses in a subject.
- the vaccine e.g. SARS-CoV-2 virus and/or the Influenza virus vaccine
- the vaccine elicits antigen-specific immune responses in a subject that has an age of about 5 years old or younger. Accordingly, the vaccine is suitable for infants.
- the temperature stable vaccine elicits antigen-specific immune responses in a subject that has an age of about 60 years old or older. Accordingly, the vaccines is suitable for the elderly.
- the vaccine upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, the vaccine elicits antigen-specific immune responses in a subject upon storage as defined herein.
- the vaccine elicits an adaptive immune response against a Coronavirus (e.g. SARS-CoV-2) or an Influenza virus when administered to a cell or a subject.
- a Coronavirus e.g. SARS-CoV-2
- the adaptive immune response upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, the adaptive immune response does not decrease by more than 20%, preferably by not more than 10%.
- administration of a therapeutically effective amount of the vaccine elicits neutralizing antibody titers against at least one pathogen, wherein the at least one pathogen may be selected from a bacterium, a protozoan, or a virus, for example from a pathogen provided in List 1.
- the vaccine elicits neutralizing antibody titers against a Coronavirus (e.g. SARS-CoV-2) or an Influenza virus when administered to a cell or a subject.
- the neutralizing antibody titers upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, do not decrease by more than 20%, preferably by not more than 10%.
- detectable levels of the respective antigen are produced after about 1 day in the serum of the subject post administration of the vaccine.
- detectable levels of the respective antigen are produced in the serum of the subject after about 1 day.
- the neutralizing antibody titer that is induced upon administration of the vaccine to a subject is at least 100 neutralizing units per milliliter (NU/mL), at least 500NU/mL, or at least 10OONU/mL.
- the neutralizing antibody titer induced upon administration of the vaccine to a subject is at least 100 neutralizing units per milliliter (NU/mL), at least 500NU/mL, or at least 10OONU/mL.
- a neutralizing antibody titer of at least 100NU/ml, at least 500NU/ml, or at least WOONU/ml is produced in the serum of the subject post administration of the vaccine.
- a neutralizing antibody titer of at least 100NU/ml, at least 500NU/ml, or at least 10OONU/ml is produced in the serum of the subject post administration of the vaccine.
- the neutralizing antibody titer is sufficient to reduce infection with a pathogen by at least 50% relative to a neutralizing antibody titer of an unvaccinated control subject or relative to a neutralizing antibody titer of a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine.
- the neutralizing antibody titer upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, the neutralizing antibody titer is sufficient to reduce infection with a pathogen by at least 50% relative to a neutralizing antibody titer of an unvaccinated control subject or relative to a neutralizing antibody titer of a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein sub unit viral vaccine.
- administration of a therapeutically effective amount of the vaccine to a subject induces a T cell immune response against a pathogen (e.g. a Coronavirus or an Influenza virus) in the subject, preferably wherein the T cell immune response comprises a CD4+ T cell immune response and/or a CD8+ T cell immune response.
- a pathogen e.g. a Coronavirus or an Influenza virus
- administration of a therapeutically effective amount of the vaccine to a subject upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, administration of a therapeutically effective amount of the vaccine to a subject induces a T cell immune response against a pathogen (e.g. a Coronavirus or an Influenza virus) in the subject, preferably wherein the T cell immune response comprises a CD4+ T cell immune response and/or a CD8+ T cell immune response
- the neutralizing antibody titer is sufficient to block fusion of a pathogen (e.g. a Coronavirus or an Influenza virus) with epithelial cells of the subject.
- a pathogen e.g. a Coronavirus or an Influenza virus
- the neutralizing antibody titer upon storage as defined herein, upon dilution as defined herein, or upon thawing as defined herein, the neutralizing antibody titer is sufficient to block fusion of a pathogen (e.g. a Coronavirus or an Influenza virus) with epithelial cells of the subject
- the neutralizing antibody titer is induced within 20 days following a single 1 ug-200ug dose of the vaccine, or within 40 days following a second 1 ug-200 g dose of the vaccine, wherein the dose relates to the amount of the RNA.
- a dose comprises about 1ug-200ug RNA, about 5ug-100ug RNA, about 10ug- 50ug RNA.
- the vaccine comprises one therapeutically effective dose.
- the vaccine is a mono-dose vaccine, preferably a ready-to-use mono-dose vaccine.
- a mono-dose vaccine, preferably a ready- to-use mono-dose vaccine comprises one therapeutically effective dose as defined herein, preferably ranging from about 1 pg to about 200pg, 5pg to about 10Opg, or 5pg to about 50pg, formulated in a total volume of about 100 to 10OOpi, preferably 500pl.
- a vaccine as described in the context of the second aspect may be obtained by the method of manufacturing as described herein.
- a syringe containing the pharmaceutical composition or vaccine is a syringe containing the pharmaceutical composition or vaccine:
- the present invention provides a syringe containing the pharmaceutical composition as defined in the context of the first aspect or containing the vaccine as defined in the context of the second aspect.
- the contained pharmaceutical composition as defined herein or vaccine as defined herein may be in liquid form, in frozen form, or in dried form (e.g. lyophilized orspray(fieeze)dried).
- features and embodiments that characterize the “buffer system” e.g. comprising glycerol
- the “the lipid- based carriers” e.g. LNPs comprising cationic lipid or ionizable lipid, neutral lipid or phospholipid, steroid or steroid analogue, aggregation reducing lipid
- the RNA e.g. mRNA encoding an antigen
- advantageous stability properties of the pharmaceutical composition of the first or second aspect that e.g. relate to stability upon storage, dilution and/or thawing (e.g.
- RNA integrity of the RNA may also be understood as suitable features for the third aspect.
- syringes can cause RNA agglomeration in lipid-based carrier compositions, which can be reduced or prevented by selecting syringes with certain characteristics, e.g. silicone-oil free syringes.
- syringe containing the pharmaceutical composition or the vaccine are selected from syringes as described in published PCT patent application WO2022207862, in particular characterized by any one of the features of claims 70 to 83.
- the whole disclosure of WO2022207862, in particular the disclosure relating to claims 1 to 83, preferably the disclosure relating to claims 70 to 83 are herewith incorporated by reference.
- the syringe is a syringe for injection and contains the pharmaceutical composition of the first aspect or the vaccine of the second aspect, wherein the syringe is characterized in that the inner surface of the syringe barrel is essentially free or free of silicone oils and/or the syringe plunger stopper is essentially free or free of silicone oils.
- the syringe barrel comprises a polymer preferably selected from olefin polymer, cyclic olefin copolymer (COP), polypropylene, polystyrene, polyethylene, polycarbonate, or a combination of any of these, more preferably COP or polypropylene.
- the syringe barrel comprises glass.
- the syringe barrel comprises a glass coating of the inner surface ora silicon dioxide coating of the inner surface.
- the syringe plunger stopper comprises a thermoplastic elastomer, a silicone polymer, or a rubber and, optionally, comprises a coating to reduce the gliding force needed for an injection.
- the syringe e.g. the pre-filled syringe
- the syringe plunger stopper e.g. a crystallization of the rubber or the elastomer.
- Such a crystallization typically occurs at freezing temperatures below -50°C.
- a stable storage of a pre-filled syringe containing a pharmaceutical composition of lipid-based carriers comprising RNA
- very low storage temperatures e.g. -60°C or -80°C
- the syringe is configured for intramuscular injection, intradermal injection, intertumoral injection, intravenous injection, or intraocular injection, preferably intramuscular injection.
- the syringe is a pre-filled syringe that contains one effective dose of the pharmaceutical composition or the vaccine.
- the pre-filled syringe contains a ready-to use pharmaceutical composition or vaccine.
- one effective dose comprises about 10Ong to about 500pg RNA, 1 pg to about 200pg RNA, 5pg to about 10Opg RNA, 5pg to about 50pg RNA. In preferred embodiments in that context, one effective dose has a total volume of about 50pl to about 2ml, 10Opi to 1 ml, 250pl to 1 ml, preferably about 500pl.
- the syringe is a pre-filled mono-dose syringe containing the pharmaceutical composition as defined herein or vaccine as defined herein in a total volume of about 500pl that comprises one therapeutically effective dose of RNA in an amount ranging from about 1 pg to about 200pg, 5pg to about 10Opg, or 5pg to about 50pg.
- the syringe suitably contains one dose of a vaccine or a pharmaceutical composition that comprises lipid-based carrier, preferably LNPs, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) at least 50mM glycerol, preferably 50mM to 200mM; b) at least 50mM sucrose as sugar component, preferably 50mM to 200mM sucrose; c) at least 10mM or more than 20mM T ris, preferably 30mM T ris; and the added total concentration of glycerol and sucrose is in a range of 100mM to 400mM, preferably in a range of 200mM to 300mM, and the lipid-based carriers are LNPs as defined herein, preferably GN-LNPs as defined herein, more preferably 315-LPNs as defined herein, and the lipid-based carriers encapsulate an RNA that comprises a coding sequence encoding an antigen or
- compositions or vaccines that may be contained in the syringe are described in the context of the first aspect and in the context of the second aspect.
- the pharmaceutical composition as defined in the first aspect, or the vaccine of the second aspect is stable upon dilution, thawing, and/or storage in the syringe as defined herein.
- the pharmaceutical composition as defined herein or vaccine as defined herein is stable upon storage in the syringe as defined herein.
- stability is characterized by stable or preserved quality attributes of the lipid- based carriers comprising RNA, wherein the quality attributes are selected from RNA integrity, RNA encapsulation, the PDI, the Z-average size of the lipid-based carriers, the amount of LEPs.
- the present invention provides a kit or kit of parts comprising the pharmaceutical composition of the first aspect, and/or vaccine of the second aspect and/or the syringe of the third aspect.
- the kit or kit of parts comprises pharmaceutical composition of the first aspect, and/or the vaccine of the second aspect and/or the syringe of the third aspect and optionally additionally technical instructions providing information on administration of the components and dosage of the components.
- the pharmaceutical composition as defined herein and/or the vaccine as defined herein are provided in a separate part of the kit, for example in a storage container or vial.
- the kit or kit of parts comprises at least one empty syringe (e.g. single use empty syringe) for administering the pharmaceutical compositions and/or the vaccine.
- the at least one empty syringe is selected from syringes (e.g. silicone oil free syringes) as described in published PCT patent application WO2022207862.
- the kit or kit of parts comprises at least one buffer system as defined herein for diluting the pharmaceutical composition as defined herein and/or the vaccine as defined herein.
- the buffer is provided in a separate storage container or vial.
- the buffer for diluting the pharmaceutical composition and/or the vaccine is a buffer system as defined in the context of the first aspect.
- the present invention relates to the medical use of the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe of the third aspect, or the kit or kit of parts of the fourth aspect.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect may likewise be read on and be understood as suitable embodiments of medical uses of the invention.
- the pharmaceutical composition as defined in the first aspect for use as a medicament the vaccine as defined in the second aspect for use as a medicament, the syringe containing the pharmaceutical composition or the vaccine as defined in the third aspect for use as a medicament, or the kit or kit of parts as defined in the fourth aspect for use as a medicament.
- the pharmaceutical composition as defined herein, vaccine as defined herein, the syringe as defined herein, or the kit or kit of parts as defined herein may be used for human medical purposes and also for veterinary medical purposes, preferably for human medical purposes.
- the pharmaceutical composition as defined herein, vaccine as defined herein, the syringe as defined herein, or the kit or kit of parts as defined herein may be in particular used and suitable for human medical purposes, in particular for young infants, newborns, immunocompromised recipients, pregnant and breast-feeding women, and elderly people.
- the invention relates to the medical use of the pharmaceutical composition as defined herein, vaccine as defined herein, the syringe as defined herein, or the kit or kit of parts as defined herein for use as a medicament in treating or preventing an infectious, a tumour, or a genetic disease, disorder or condition.
- the invention relates to the medical use of the pharmaceutical composition as defined herein, vaccine as defined herein, the syringe as defined herein, or the kit or kit of parts as defined herein for use as a medicament in treating or preventing an infectious disease, disorder or condition.
- an infection may be caused a pathogen selected from a bacterium, a protozoan, or a virus, for example from a pathogen provided in List 1.
- the pathogen is a virus, e.g. a Coronavirus (e.g. SARS- CoV-2) or an Influenza virus
- the invention relates to the medical use of the pharmaceutical composition as defined herein, the vaccine as defined herein, the syringe as defined herein, or the kit or kit of parts as defined herein, for use as a medicament in treating or preventing an infection with a Coronavirus, preferably a SARS-CoV-2 coronavirus, or of a disorder related to such an infection.
- the pharmaceutical composition, the vaccine, the syringe, or the kit or kit of may be administered locally or systemically.
- administration may be by an intradermal, subcutaneous, intranasal, intraocular (intravitreal), or intramuscular route.
- administration may be by conventional needle injection.
- infectious diseases is intramuscular injection.
- the pharmaceutical composition, the vaccine may be transferred to a salt solution, in particular an NaCI solution before administration to a subject.
- concentration of the salt comprised in the solution is in a range from about 10mM to about 300mM, preferably about 150mM.
- the salt comprised in solution is NaCI, preferably in a concentration of about 150mM.
- a transfer to a salt solution (e.g. 150mM NaCI) with a subsequent administration, e.g. intramuscular administration, may be advantageous for inducing immune responses.
- the present invention relates to a method of treating or preventing a disorder.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe of the third aspect, or the kit or kit of parts of the fourth aspect, or embodiments relating to medical uses of the fifth aspect may also be read and understood as suitable embodiments relating to method of treatments as provided herein.
- specific features and embodiments relating to method of treatments as provided herein may also apply for medical uses of the invention.
- Preventing (Inhibiting) or treating a disease relates to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a virus infection.
- a disease e.g. an infection, cancer, a genetic disorder
- Treatment relates to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a virus infection.
- Tumenf refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
- the term “ameliorating”, with reference to a disease or pathological condition refers to any observable beneficial effect of the treatment.
- Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection.
- the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease.
- a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
- the present invention relates to a method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof pharmaceutical composition of the first aspect, the vaccine of the first aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect.
- the disease, disorder or condition is an infectious, a tumour, or a genetic disease, disorder or condition.
- the disorder is an infection with a pathogen selected from a bacterium, a protozoan, or a virus, for example from a pathogen provided in List 1.
- the pathogen is a virus.
- the disorder an infection with a Coronavirus, or a disorder related to such infections, in particular an infection with SARS-CoV-2, or a disorder related to such infections (e.g. COVID-19).
- the disorder an infection with an Influenza virus, or a disorder related to such infections.
- applying or administering is performed via intramuscular injection, intradermal injection, transdermal injection, intradermal injection, intralesional injection, intracranial injection, subcutaneous injection, intracardial injection, intratumoral injection, intravenous injection, or intraocular injection, intrapulmonal inhalation, intraarticular injection, sublingual injection.
- administering is performed via intramuscular injection.
- the subject in need is a mammalian subject, preferably a human subject, e.g. new-born human subject, pregnant human subject, immunocompromised human subject, and/or elderly human subject.
- a human subject e.g. new-born human subject, pregnant human subject, immunocompromised human subject, and/or elderly human subject.
- the present invention provides a buffer system for stabilizing, storing, preparing, freezing, and/or diluting lipid-based carriers comprising RNA.
- the buffer is characterized by any of the features of the first aspect relating to the optimized buffer system.
- the buffer system as defined herein stabilizes lipid-based carriers comprising RNA.
- Lipid- based carriers comprising RNA are defined in the context of the first aspect.
- “Stabilizing” in that context has to be understood as essentially preserving the physiochemical properties or quality attributes of lipid-based carriers comprising RNA in producing the same, upon storage as defined herein (see first aspect), upon dilution as defined herein (see first aspect), or upon freezing as defined herein (see first aspect).
- Physiochemical properties or quality attributes that are suitably stabilized are selected from the integrity of the RNA, the amount of free RNA, encapsulation efficiency, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs), the potency.
- Advantageous features of the buffer that suitably stabilizes lipid-based carriers comprising RNA are outlined in the foregoing aspects, in particular the first aspect (see e.g. Table A).
- the buffer system comprises a) at least 50mM glycerol; b) at least 50mM of at least one sugar component, preferably sucrose; c) at least one buffer agent, preferably T ris; and wherein the added total concentration of glycerol and sucrose is preferably in a range of 100mM to 400mM.
- the buffer system comprises a) 50mM to 250mM glycerol; b) 50mM to 250mM sucrose (as sugar component); c) T ris (as buffer agent) preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration for glycerol and sucrose is preferably in a range of 200mM to 300mM.
- the buffer system comprises a) 50mM to 200mM glycerol; b) 50mM to 200mM sucrose (as sugar component); c) T ris (as buffer agent) preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration for glycerol and sucrose is preferably in a range of 200mM to 300mM, in particular at about 250mM.
- the buffer system comprises a) 10OmM to 200mM glycerol, preferably 10OmM to 150mM glycerol; b) 10OmM to 200mM sucrose, preferably 10OmM to 150mM sucrose; c) at least 10mM or more than 20mM Tris, preferably 30mM T ris, and wherein the added total concentration for both glycerol and sucrose is in a range of 200mM to 300mM, preferably at about 250mM.
- the buffer system comprises 100mM glycerol, 150mM sucrose, and at least 10mM or more than 20mM T ris, preferably 30mM T ris.
- the buffer system comprises 150mM glycerol, 100mM sucrose, and at least 10mM or more than 20mM T ris, preferably 30mM T ris.
- the buffer system is particularly suitable for preparing, storing, diluting, and/or freezing compositions or vaccines comprising lipid-based carriers comprising or encapsulating RNA.
- the present invention provides uses of the buffer system as defined herein.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect, or the buffer of the seventh aspect may likewise be read on and be understood as suitable embodiments of the use of the present aspect.
- the buffer system is characterized by any of the features of the first aspect, the second aspect, or the seventh aspect.
- the aspect relates to the use of a buffer characterized by any of the features of the first aspect, the second aspect, or the foregoing aspect for storing, diluting, stabilizing, producing, or freezing lipid-based carriers comprising or encapsulating RNA, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized as defined herein (for details, see first aspect).
- one use of the buffer system may be the use in the preparation of compositions (e.g. pharmaceutical compositions or vaccines or syringes comprising the same) comprising lipid-based carriers as defined herein comprising RNA as defined herein.
- compositions e.g. pharmaceutical compositions or vaccines or syringes comprising the same
- lipid-based carriers as defined herein comprising RNA as defined herein.
- one use of the buffer system may be the use in storing of lipid-based carriers as defined herein comprising RNA as defined herein. Further embodiments and features of that use are provided in conjunction with the first and second aspect.
- one use of the buffer system may be the use in diluting of lipid-based carriers as defined herein comprising RNA as defined herein. Further embodiments and features of that use are provided in conjunction with the first and second aspect.
- one use of the buffer system may be the use in freezing (and/or thawing) of lipid-based carriers as defined herein comprising RNA as defined herein. Further embodiments and features of that use are provided in conjunction with the first and second aspect.
- one use of the buffer system may be the use in stabilizing of lipid-based carriers as defined herein comprising RNA as defined herein. Further embodiments and features of that use are provided in conjunction with the first and second aspect.
- the use in stabilizing relates to a stabilization at -20°C or -40°C.
- the use of the buffer system suitably preserves or stabilizes quality attributes of the lipid- based carriers comprising RNA, preferably wherein the quality attributes are selected from the integrity of the RNA, the amount of free RNA, RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs), the potency.
- the quality attributes are selected from the integrity of the RNA, the amount of free RNA, RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, the amount of late-eluting peaks (LEPs), the potency.
- the present invention provides the use of glycerol (or other polyol compounds) for preserving and/or stabilizing quality attributes of lipid-based carriers comprising or encapsulating RNA in frozen form or stored at freezing temperatures, preferably at -20°C or -40°C.
- the preferred polyol component glycerol may be substituted by a functionally equivalent polyol component.
- Suitable functionally equivalent polyols may be selected from a diol, a triol, or a sugar alcohol.
- glycerol may be substituted by any one of the compounds selected from 1 ,2-propanediol, 1 ,3- propanediol, (+/-)-2-methyl-2,4-pentanediol, 1 ,6-hexanediol, 1 ,2-butanediol, 2,3-butanediol, 1,4-butanediol, ethylene glycol, or diethylene glycol, or any combination thereof.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect, or the buffer of the seventh aspect may likewise be read on and be understood as suitable embodiments of the use of the present aspect.
- glycerol has advantageous effects on the storage stability of lipid-based carriers as defined herein comprising or encapsulating RNA as defined herein. These advantageous effects have been observed for a storage at - 20°C and -40°C in a glycerol comprising buffer system, and typically relate to quality attributes selected from RNA encapsulation, Z-average particle size, RNA integrity and PDI.
- the present aspect relates to the use of glycerol for preserving quality attributes of lipid-based carriers comprising or encapsulating RNA when stored at -20°C or -40 Q C, wherein the quality attributes are selected from RNA encapsulation, Z-average particle size, PDI and/or RNA integrity, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of glycerol relates to preserving RNA encapsulation of lipid-based carriers encapsulating RNA when stored at -2CTC and/or -40°C, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of glycerol relates to preserving Z-average particle size of lipid-based carriers encapsulating RNA when stored at -20°C and/or -40°C, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of glycerol relates to preserving RNA integrity of lipid-based carriers encapsulating RNA when stored at -20°C, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the present invention provides the use of T ris as a buffer agent for preserving and/or stabilizing quality attributes of lipid-based carriers comprising or encapsulating RNA.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect, or the buffer of the seventh aspect may likewise be read on and be understood as suitable embodiments of the use of the present aspect.
- T ris has advantageous effects on the storage stability of lipid-based carriers as defined herein comprising or encapsulating RNA as defined herein. These advantageous effects have been observed for a storage at +25°C, 5°C, -20°C and -40°C, and typically relate to quality attributes selected from Z-average particle size, the prevention/reduction of LEPs and the PDI.
- the present aspect relates to the use of T ris for preserving quality attributes of lipid-based carriers comprising or encapsulating RNA, wherein the quality attributes are selected from Z-average particle size, the prevention/reduction of LEPs and the PDI, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of Tris relates to preserving the Z-average particle size of lipid-based carriers encapsulating RNA when stored at temperatures ranging from -20°C to 25°C, for example +25°C, 5°C, -20°C, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- Tris relates to preventing or reducing LEPs in compositions comprising lipid-based carriers, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of Tris relates to preserving the PDI of lipid-based carriers encapsulating RNA when stored at temperatures ranging from -20°C to 25°C, for example +25°C, 5°C, -20°C, preferably wherein the lipid-based carriers comprising or encapsulating RNA are characterized by any one of the features described in the context of the first aspect.
- the use of Tris relates to preserving the potency and/or the encapsulation efficiency of lipid- based carriers encapsulating RNA when used in TFF or dialysis of lipid-based carriers, e.g. LNPs.
- the present invention provides a method of manufacturing a pharmaceutical composition or a vaccine comprising lipid-based carriers comprising or encapsulating RNA.
- embodiments relating to the pharmaceutical composition of the first aspect, the vaccine of the second aspect, the syringe containing the pharmaceutical composition or the vaccine of the third aspect, or the kit or kit of parts of the fourth aspect, or the buffer of the seventh aspect may likewise be read on and be understood as suitable embodiments of method of manufacturing of the present aspect.
- the present aspect relates to a method of manufacturing a pharmaceutical composition or vaccine comprising lipid-based carriers, wherein the lipid-based carriers comprise RNA, the method comprises the steps
- composition comprising lipid-based carriers comprising RNA
- step B) Concentrating and/or purifying and/or clarifying and/or conditioning the composition of step A) using a first buffer;
- the first buffer or the second buffer is the optimized buffer system as defined in the context of the invention (e.g. first aspect or seventh aspect), in particular, a buffer system comprising glycerol and sucrose.
- the first buffer or the second buffer comprises at least 50mM glycerol; at least 50mM sucrose (as a sugar component) and T ris (as buffer agent) preferably at a concentration of more than at least 10mM or more than 20mM; and wherein the added total concentration for both glycerol and sucrose is preferably in a range of 100mM to 400mM.
- the first buffer or the second buffer comprises 50mM to 250mM glycerol; 50mM to 250mM sucrose; T ris preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration of glycerol and sucrose is preferably in a range of 10OmM to 400mM.
- the first buffer or the second buffer comprises 50mM to 200mM glycerol; 50mM to 200mM sucrose; T ris preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration of glycerol and sucrose is preferably in a range of200mM to 300mM.
- the first buffer or the second buffer comprises 150mM glycerol, 10OmM sucrose, and at least 10mM or more than 20mM Tris, preferably 30mM Tris.
- the first buffer or the second buffer comprises 10OmM glycerol, 150mM sucrose, and at least 10mM or more than 20mM Tris, preferably 30mM Tris.
- step A comprises a step A1 ) RNA synthesis and a step A2) lipid-based carrier formation.
- Step A1 is suitable selected from chemical RNA synthesis or RNA in vitro transcription.
- step A1 is selected from RNA in vitro transcription as defined herein (see first aspect), suitably using a Cap analog and, optionally, modified nucleotides such as e.g. ml i .
- the obtained RNA of step A1 is purified using RNA purification methods as defined herein (see first aspect).
- Step A2) may comprise a step of A2a) providing an RNA solution comprising the purified RNA.
- the RNA solution comprising the purified RNA is subjected to a citrate or acetate buffer (e.g. 50mM) at low pH (e.g. pH 4.0) with a desired RNA concentration (e.g. 100 pg/ml to about 1 mg/ml RNA).
- a citrate or acetate buffer e.g. 50mM
- pH e.g. pH 4.0
- a desired RNA concentration e.g. 100 pg/ml to about 1 mg/ml RNA
- Step A2) may comprise a step of A2b) providing an ethanolic lipid solution comprising lipids of the lipid-based carrier.
- the lipids are selected from preferred lipid-based carrier formulations as defined in the context of aspect 1 .
- at least one cationic or ionizable lipid at least one or two neutral lipids, at least one steroid or steroid analogue, at least one aggregation reducing lipid.
- the lipids are selected from lipids of 315-LNPs as defined herein, GN- LNPs as defined herein, or SM102-LNPs as defined herein.
- all lipids are dissolved in ethanol. The obtained ethanolic lipid solution is used for the lipid-based carrier complexation.
- Step A2) may suitably comprise a step A2c) of combining the ethanolic lipid solution (e.g. obtained in step A2b) and the RNA solution (e.g. obtained in step A2a) to allow the formation of lipid-based carriers comprising the RNA.
- the ethanolic lipid solution and the RNA solution are combined at a ratio of about 1 :5 to 1 :3 (vol/vol).
- the ethanolic lipid solution with a flow rate F1 and the RNA solution with a flow rate F2 are combined at a ratio of about 1 :5 to 1 :3 (vol/vol), wherein total flow rates are above 15ml/min.
- F1 and/or F2 are in a range of about 10ml/min to about 500ml/min.
- F1 :F2 is in a range of about 0.2 to 0.5, preferably 0.3 to 0.4, e.g. 0.33.
- the combining of the ethanolic lipid solution occurs in a mixing means, preferably a static mixing means or mixing reactor, e.g. a T-piece or a Y-piece to allow the instant formation lipid-based carriers encapsulating the RNA, preferably of lipid nanoparticles encapsulating the RNA.
- the mixing means is microfluidic mixing device.
- the ethanol is exchanged to an aqueous matrix.
- the obtained lipid-based carriers are suitably subjected to a concentrating and/or purifying and/or clarifying and/or conditioning step B).
- Step B) suitably comprises a step of concentrating the composition comprising lipid-based carries comprising RNA by tangential flow filtration (TFF; ultrafiltration) or dialysis.
- the concentrating step is performed until a desired concentration is achieved.
- Step B is carried out using tangential flow filtration using a hollow fiber system or a membrane cassette.
- Step B) suitably comprises a step of tangential flow filtration using a first buffer.
- the first buffer is a buffer as defined in the context of the optimized buffer system of the invention (in particular, a buffer comprising glycerol and sucrose).
- the method does optionally comprise step C) (e.g. for further diluting).
- Components of the optimized buffer system of the invention may have a negative impact on the quality attributes of lipid- based carriers comprising RNA (e.g. potency) when used in dialysis or TFF, therefore it is preferred to perform step B) with a different buffer system.
- RNA e.g. potency
- the first buffer is a PBS buffer.
- the PBS buffer comprises sucrose, preferably in a concentration of about 150mM, NaCI, preferably in a concentration of about 75mM, NaPO4, preferably in a concentration of about 10mM, and has a pH of about pH 7.4.
- the method preferably comprises step C).
- a PBS buffer in step C) in particular in combination with TFF or dialysis, has the advantage of improved qualify attributes of lipid-based carriers comprising RNA (e.g. potency).
- the first buffer is a Tris buffer lacking sucrose or glycerol.
- the Tris buffer comprises at least 10mMor more than 20mM Tris.
- the method preferably comprises step C). Using a Tris buffer in step C), in particular in combination with TFF or dialysis, has the advantage of improved qualify attributes of lipid-based carriers comprising RNA (e.g. potency, encapsulation efficiency).
- Step C) suitably comprises a step of exchanging the first buffer for a second buffer in embodiments where the first buffer is not selected from an optimized buffer system of the invention (in particular, a buffer comprising glycerol) or as defined above.
- an optimized buffer system of the invention in particular, a buffer comprising glycerol
- the first buffer (in step B) is a PBS buffer as defined herein (e.g. PBS-Sucrose) and the second buffer (in step C) is an optimized buffer system as defined in the context of the first aspect (in particular, a buffer comprising glycerol) or as defined above.
- PBS buffer as defined herein (e.g. PBS-Sucrose)
- the second buffer (in step C) is an optimized buffer system as defined in the context of the first aspect (in particular, a buffer comprising glycerol) or as defined above.
- the buffer exchange step C) may be performed by TFF or by dilution (which means the addition of certain components e.g. the optimized buffer as such or the addition of sucrose and/or glycerol and/or Tris).
- the dilution comprises at least one step of dilution from a higher concentration to a lower concentration with a dilution factor.
- the higher concentration is more than 500mg/l RNA and/or more than 12.5 mg/l lipid and the lower concentration is less than 50mg/l RNA and/or less than 1.25 mg/l lipid.
- the dilution factor is between 1 :5 and 1 :200, preferably between 1 :10 and 1 :100.
- the dilution is performed using the optimized buffer system of the first aspect.
- a higher concentrated composition in a first buffer (Sucrose-PBS buffer or T ris buffer) is diluted to a lower concentration using a second buffer (optimized buffer system as defined herein, e.g. comprising glycerol) with a dilution factor between 1 :5 and 1 :200, preferably between 1 :10 and 1 : 100.
- the dilution is performed using T ris, glycerol and sucrose to achieve the final desired buffer composition.
- that step increases encapsulation efficiency.
- the obtained pharmaceutical composition or vaccine of step D) is suitably characterized by any one of the features of the first aspect or second aspect.
- the obtained pharmaceutical composition or vaccine is subjected to a freezing step (either before aseptic filling or after aseptic filling), preferably frozen below the Tg of the pharmaceutical composition, which may activate or improve the potency of the pharmaceutical composition.
- the frozen state of the is pharmaceutical composition or vaccine is preferably maintained for at least 1 h or at least 1 day.
- the method comprises a step E) drying and F) aseptic filling.
- Step E) suitably, drying of the obtained pharmaceutical composition or vaccine is performed using lyophilization or spray(freeze) drying.
- the dried powder may be used for the aseptic filling step F).
- Step F) suitably comprises an aseptic filling of the pharmaceutical composition or vaccine obtained in step D) into a syringe to obtain a pre-filled syringe.
- syringes may be used as described in published PCT patent application PCT/EP2022/058690, in particular characterized by any one of the features of claims 70 to 83.
- PCT/EP2022/058690 in particular the disclosure relating to claims 1 to 83, preferably the disclosure relating to claims 70 to 83 are herewith incorporated by reference.
- the obtained pre-filled syringe is a syringe containing the pharmaceutical composition or the vaccine as defined in the third aspect.
- the method of manufacturing comprises the steps:
- composition comprising lipid-based carriers comprising RNA
- step A) Concentrating and/or purifying and/or clarifying and/or conditioning the composition of step A) using a first buffer system comprising PBS and sucrose, preferably using TFF;
- step E Optionally, drying of the obtained pharmaceutical composition or vaccine (before or after step F);
- the method of manufacturing comprises the steps:
- composition comprising lipid-based carriers comprising RNA
- step A) Concentrating and/or purifying and/or clarifying and/or conditioning the composition of step A) using a first buffer system comprising Tris, preferably comprising at least 10mM Tris, preferably using TFF;
- step E Optionally, drying of the obtained pharmaceutical composition or vaccine (before or after step F);
- the method of manufacturing comprises the steps:
- composition comprising lipid-based carriers comprising RNA
- step A) Concentrating and/or purifying and/or clarifying and/or conditioning the composition of step A) using a buffer system as defined in the context of the first aspect, preferably using a buffer comprising 150mM Glycerol, 100mM Sucrose, and at least 10mM or more than 20mM Tris, preferably 30mM Tris or using a buffer comprising 10OmM Glycerol, 150mM Sucrose, and at least 10mM or more than 20mM T ris, preferably 30mM Tris;
- step E Optionally, drying of the obtained pharmaceutical composition or vaccine (before or after step F);
- the method of manufacturing is a method of manufacturing a pharmaceutical composition of the first aspect or a method of manufacturing a vaccine of the second aspect.
- the method of manufacturing is a method of manufacturing a syringe of the third aspect.
- the invention also relates to a pharmaceutical composition, a vaccine, or a syringe obtainable by the method of manufacturing as defined herein.
- the present invention provides a method of stabilizing lipid-based carriers comprising RNA, in particular lipid-based carriers contained in a pharmaceutical composition or vaccine.
- RNA in particular lipid-based carriers contained in a pharmaceutical composition or vaccine.
- embodiments relating any of the foregoing aspects may likewise be read on and be understood as suitable embodiments of method of stabilizing of the present aspect and vice versa.
- the method for stabilization comprises the steps of
- composition comprising lipid-based carriers comprising or encapsulating RNA to a buffer system as defined in the context of the first aspect or as defined in the seventh aspect “A buffer system for stabilizing lipid-based carriers!’, thereby stabilizing the lipid-based carriers comprising RNA.
- the initial composition or vaccine is contained in a buffer that comprises less than 50mM glycerol or is essentially free of T ris or comprises more than 1 mM, preferably more than 10mM phosphate.
- the initial composition or vaccine is contained in a sucrose-PBS buffer or in a citrate or acetate buffer.
- the method further comprises a step selected from storing the lipid-based carriers comprising or encapsulating RNA, diluting the lipid-based carriers comprising or encapsulating RNA, freezing the lipid-based carriers comprising or encapsulating RNA, and/or thawing the lipid-based carriers comprising or encapsulating RNA.
- the lipid-based carriers comprising or encapsulating RNA are characterized as defined in the context of the first aspect.
- the storing, the diluting, the freezing, or the thawing of lipid-based carriers comprising or encapsulating RNA is stabilized as defined herein, for example as defined in the context of the first aspect.
- stabilizing is characterized by stable or preserved quality attributes of the lipid-based carriers comprising RNA as defined herein, preferably wherein the quality attributes are selected from the integrity of the RNA, the RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, and/or the amount of late-eluting peaks (LEPs), for example as defined in the context of the first aspect.
- the quality attributes are selected from the integrity of the RNA, the RNA encapsulation, the PDI value, the Z-average size of the lipid-based carriers, and/or the amount of late-eluting peaks (LEPs), for example as defined in the context of the first aspect.
- the transferring is performed during producing the lipid-based carriers comprising or encapsulating RNA or after producing the lipid-based carriers comprising or encapsulating RNA.
- the transferring may be a concentrating and/or purifying and/or clarifying and/or conditioning and/or a diluting.
- the transferring may comprise a step of adding or adjusting the glycerol concentration of the initial composition to obtain a final glycerol concentration of at least 50mM, preferably a concentration ranging from 50mM to 200mM.
- the transferring may comprise a step of adding or adjusting the sucrose concentration of the initial composition to obtain a final sucrose concentration of at least 50mM, preferably a concentration ranging from 50mM to 200mM.
- the transferring may comprise a step of adding or adjusting the Tris concentration of the initial composition to obtain a final Tris concentration of at least 10mM, preferably Tris concentration of more than 20mM, e.g. 30mM.
- the lipid-based carriers are contained in a buffer system as defined in the context of the first aspect or as defined in aspect “A buffer system for stabilizing lipid-based carrier ⁇ ’.
- a pharmaceutical composition comprising lipid-based carriers, wherein the lipid-based carriers comprise RNA and are contained in a buffer system that comprises a) at least 50mM glycerol; b) at least 50mM of at least one sugar component; c) at least one buffer agent; wherein the added total concentration of glycerol and the at least one sugar component is in a range of 10OmM to 400mM.
- composition of item 1 wherein the concentration of glycerol is at least 75mM, 10OmM, 125mM, 150mM.
- composition of items 1 to 2 wherein the concentration of glycerol ranges from 50mM to 300mM, 50mM to 250mM, 50mM to 200mM, 75mM to 200mM, 100mM to 200mM, preferably wherein the concentration of glycerol ranges from 50mM to 200mM.
- composition of items 1 to 3, wherein the concentration of glycerol is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM.
- composition of items 1 to 7, wherein the concentration of the at least one sugar component is at about 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM.
- composition of any one of the preceding items, wherein the at least one sugar component is selected from a disaccharide.
- the pharmaceutical composition of any one of the preceding items wherein the added total concentration of glycerol and the at least one sugar component is at about 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, preferably at about 250mM. 16.
- the buffer system comprises 50mM to 200mM glycerol and 50mM to 200mM sugar component, preferably wherein the added total concentration of glycerol and sugar component is in a range of 200mM to 300mM.
- composition of item 26, wherein the concentration of the at least one buffer agent is at about 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31 mM, 32mM, 33mM, 34mM, 35mM.
- the at least one buffer agent is selected from Tris, Bis-tris-methane, triethanolamine (TEA), imidazole, histidine (e.g. histidine-HCI), citrate (e.g. sodium citrate), MES, MOPS, HEPES, sodium succinate, sodium malate, sodium carbonate.
- composition of any one of the preceding items wherein the sugar component is selected from sucrose and the at least one buffer agent is selected from Tris, and wherein the buffer system comprises a) 50mM to 200mM glycerol; b) 50mM to 200mM sucrose; c) T ris preferably at a concentration of at least 10mM or more than 20mM, and wherein the added total concentration of glycerol and sucrose is preferably in a range of 200mM to 300mM.
- composition of items 32 or 33, wherein the buffer system comprises 10OmM glycerol, 150mM Sucrose, and more than 20mM Tris, preferably 30mM Tris.
- composition of items 32 or 33, wherein the buffer system comprises 150mM glycerol, 100mM sucrose, and more than 20mM Tris, preferably 30mM Tris.
- the buffer system comprises less than 20mM phosphate, preferably less than 10mM phosphate, more preferably less than 1mM phosphate.
- the buffer system has an osmolality of 100 mOsmol/kg to 400 mOsmol/kg, 150 mOsmol/kg to 400 mOsmol/kg, 200mOsmol/kg to 400mOsmol/kg, 300mOsmol/kg to 400mOsmol/kg, preferably of about 310 mOsmol/kg.
- lipid-based carriers are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes, preferably LNPs.
- LNPs lipid nanoparticles
- RNA is encapsulated in the lipid-based carriers.
- the lipid-based carriers, preferably the lipid nanoparticles comprise at least one aggregation-reducing lipid, at least one cationic or ionizable lipid, at least one neutral lipid, and/or at least one steroid or steroid analogue.
- the pharmaceutical composition of item 45 wherein the aggregation reducing lipid is a polymer conjugated lipid.
- n has a mean value ranging from 30 to 60, preferably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, more preferably wherein n has a mean value of 45 or 49; or wherein the aggregation reducing lipid is selected or derived a POZ-lipid, which is defined as a compound according to formula (POZ): [H] - [linker] - [M]formula (POZ), wherein
- [H] is a homopolymer moiety comprising at least one polyoxazoline (POZ) monomer unit wherein R is C1 -9 alkyl or C2-9 alkenyl and n has a mean value ranging from 2 to 200, preferably from 20 to
- [linker] is an optional linker group
- [M] is a lipid moiety.
- the pharmaceutical composition of items 45 to 49, wherein the at least one cationic lipid or ionizable lipid is selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
- composition of items 45 to 54 wherein the at least one steroid or steroid analogue is selected or derived from cholesterol or cholesteryl hemisuccinate (CHEMS).
- CHEMS cholesterol or cholesteryl hemisuccinate
- the pharmaceutical composition of item 56 wherein (i) to (iv) are in a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid.
- the pharmaceutical composition of any one of the preceding items, wherein the N/P ratio of the lipid-based carriers encapsulating the RNA is in a range from about 1 to about 20, about 1 to about 10, preferably about 6.
- the pharmaceutical composition of any one of the preceding items, wherein the lipid-based carriers have a Z- average size in a range from about 50nm to about 200nm, about 50nm to about 150nm, about 50nm to about 120nm, about 60nm to about 115nm.
- the pharmaceutical composition of any one of the preceding items, wherein the lipid-based carriers have a polydispersity index (PDI) value of less than about 0.3, preferably of less than about 0.2.
- PDI polydispersity index
- composition of items 1 to 62 wherein the lipid-based carriers encapsulate an RNA and comprise (I) the cationic lipid SM-102, (ii) the neutral lipid DSPC, (iii) cholesterol, and (iv) DMG-PEG 2000, preferably wherein (I) to (iv) are in a molar ratio of about 48.5% cationic lipid, 11.1% neutral lipid, 38.9% steroid or steroid analogue, and 1 .5% aggregation reducing lipid.
- the lipid-based carriers encapsulate an RNA and comprise (I) the cationic lipid SM-102, (ii) the neutral lipid DSPC, (iii) cholesterol, and (iv) DMG-PEG 2000, preferably wherein (I) to (iv) are in a molar ratio of about 48.5% cationic lipid, 11.1% neutral lipid, 38.9% steroid or steroid analogue, and 1 .5% aggregat
- RNA is selected from a single stranded RNA or double stranded RNA; and/or a coding RNA or a non-coding RNA; and/or a linear RNA or a circular RNA.
- RNA comprises at least one coding sequence.
- RNA is selected from an mRNA, a circular RNA, replicon RNA, or viral RNA.
- RNA is an mRNA
- RNA has a length ranging from about 500 nucleotides to about 10000 nucleotides, ranging from about 1000 nucleotides to about 10000 nucleotides, ranging from about 1000 nucleotides to about 5000 nucleotides.
- RNA has a length of at least 3000 nucleotides, preferably a length ranging from about 3000 nucleotides to about 5000 nucleotides.
- composition of item 74 wherein the concentration the sugar component in the buffer system is at least 10mM, 20mM, 30mM, 40mM, or 50mM higher than the concentration of glycerol.
- RNA has a length of not more than 3000 nucleotides, preferably a length ranging from about 500 nucleotides to about 3000 nucleotides.
- RNA comprises least one 5'- UTR and/or at least one 3’-UTR.
- the pharmaceutical composition of item 78, wherein the at least one 3’-UTR comprises or consists of a nucleic acid sequence derived from a 3-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1 , COX6B1 , GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes.
- the at least one 5’-UTR comprises or consists of a nucleic acid sequence derived from a 5-UTR of a gene selected from HSD17B4, RPL32, ASAH1 , ATP5A1 , MP68, NDUFA4, NOSIP, RPL31 , SLC7A3, TUBB4B and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.
- RNA comprises at least one poly(A) sequence, preferably wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosine nucleotides, about 60 to about 250 adenosine nucleotides, about 60 to about 150 adenosine nucleotides.
- composition of item 82, wherein the at least one poly(A) sequence comprises about 100 adenosine nucleotides.
- RNA comprises at least one poly(C) sequence and/or at least one miRNA binding site and/or at least one histone-stem loop sequence.
- RNA comprises at least one modified nucleotide, preferably selected from pseudouridine (i ) or N1 -methylpseudouridine (m 1 i ), more preferably selected from N1 -methylpseudouridine (m1qj).
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Abstract
La présente invention concerne, entre autres, des compositions pharmaceutiques ou des vaccins comprenant des transporteurs à base de lipides, les transporteurs à base de lipides comprenant de l'ARN et étant contenus dans un système de tampon qui comprend au moins un composant polyol (par exemple le glycérol), au moins un composant sucre (par exemple le saccharose), et au moins un agent tampon (par exemple Tris). Les formulations améliorées stabilisent avantageusement les transporteurs à base de lipides comprenant de l'ARN pendant le stockage (par exemple à 5 °C et/ou à -20 °C). D'autres aspects, entre autres, concernent des procédés de fabrication, des procédés de stabilisation et diverses utilisations. L'invention concerne également des méthodes de traitement ou de prévention de troubles ou de maladies, ainsi que des première, deuxième et autres utilisations médicales.
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Citations (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (fr) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Composition pharmaceutique contenant un arnm stabilise et optimise pour la traduction dans ses regions codantes |
WO2008077592A1 (fr) | 2006-12-22 | 2008-07-03 | Curevac Gmbh | Procédé de purification d'arn à l'échelle préparative par hplc |
WO2011069586A1 (fr) | 2009-12-09 | 2011-06-16 | Curevac Gmbh | Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques |
WO2012019780A1 (fr) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Acide nucléique comprenant ou codant pour une tige-boucle d'histone et une séquence poly(a) ou un signal de polyadénylation pour augmenter l'expression d'une protéine codée |
WO2015062738A1 (fr) | 2013-11-01 | 2015-05-07 | Curevac Gmbh | Arn modifié à propriétés immunostimulantes réduites |
WO2015101416A1 (fr) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Procédés d'analyse d'arn |
WO2015188933A1 (fr) | 2014-06-10 | 2015-12-17 | Curevac Ag | Procédés et moyen d'amélioration de la production d'arn |
WO2015199952A1 (fr) | 2014-06-25 | 2015-12-30 | Acuitas Therapeutics Inc. | Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques |
WO2016165831A1 (fr) | 2015-04-17 | 2016-10-20 | Curevac Ag | Lyophilisation de l'arn |
WO2016180430A1 (fr) | 2015-05-08 | 2016-11-17 | Curevac Ag | Procédé de production d'arn |
WO2016184575A1 (fr) | 2015-05-20 | 2016-11-24 | Curevac Ag | Composition de poudre sèche comprenant de l'arn à chaîne longue |
WO2016184576A2 (fr) | 2015-05-20 | 2016-11-24 | Curevac Ag | Composition de poudre sèche comprenant de l'arn à chaîne longue |
WO2016193206A1 (fr) | 2015-05-29 | 2016-12-08 | Curevac Ag | Procédé de production et de purification d'arn, comprenant au moins une étape de filtration à flux tangentiel |
WO2017004143A1 (fr) | 2015-06-29 | 2017-01-05 | Acuitas Therapeutics Inc. | Formulations de lipides et de nanoparticules de lipides pour l'administration d'acides nucléiques |
WO2017053297A1 (fr) | 2015-09-21 | 2017-03-30 | Trilink Biotechnologies, Inc. | Compositions et procédés de synthèse d'arn coiffés en 5' |
WO2017066791A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm à substitution sucre |
WO2017066789A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm avec sucre modifié |
WO2017066793A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes arnm et procédés de coiffage d'arnm |
WO2017066782A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes d'arnm hydrophobes |
WO2017066797A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes d'arnm trinucléotidiques |
WO2017066781A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm à liaison phosphate modifié |
WO2017075531A1 (fr) | 2015-10-28 | 2017-05-04 | Acuitas Therapeutics, Inc. | Nouveaux lipides et nouvelles formulations de nanoparticules de lipides pour l'administration d'acides nucléiques |
WO2018075827A1 (fr) | 2016-10-19 | 2018-04-26 | Arcturus Therapeutics, Inc. | Analogues de coiffes d'arnm de type trinucléotidique |
WO2018078053A1 (fr) | 2016-10-26 | 2018-05-03 | Curevac Ag | Vaccins à arnm à nanoparticules lipidiques |
WO2018089540A1 (fr) * | 2016-11-08 | 2018-05-17 | Modernatx, Inc. | Formulations stabilisées de nanoparticules lipidiques |
US10392341B2 (en) | 2015-09-17 | 2019-08-27 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2019226925A1 (fr) | 2018-05-24 | 2019-11-28 | Translate Bio, Inc. | Lipides cationiques de thioester |
WO2020061332A1 (fr) | 2018-09-19 | 2020-03-26 | Modernatx, Inc. | Analogues de stérol et leurs utilisations |
WO2020127959A1 (fr) | 2018-12-21 | 2020-06-25 | Curevac Ag | Procédés d'analyse d'arn |
WO2021123332A1 (fr) | 2019-12-20 | 2021-06-24 | Curevac Ag | Nanoparticules lipidiques pour l'administration d'acides nucléiques |
WO2021156267A1 (fr) | 2020-02-04 | 2021-08-12 | Curevac Ag | Vaccin contre un coronavirus |
WO2021183564A1 (fr) * | 2020-03-09 | 2021-09-16 | Arcturus Therapeutics, Inc. | Compositions et méthodes pour l'induction de réponses immunitaires |
WO2021222801A2 (fr) | 2020-05-01 | 2021-11-04 | Arcturus Therapeutics, Inc. | Acides nucléiques et procédés de traitement de la fibrose kystique |
WO2021239880A1 (fr) | 2020-05-29 | 2021-12-02 | Curevac Ag | Vaccins combinés à base d'acide nucléique |
WO2022135993A2 (fr) * | 2020-12-22 | 2022-06-30 | Curevac Ag | Composition pharmaceutique comprenant des vecteurs lipidique encapsulant de l'arn pour une administration multidose |
WO2022207862A2 (fr) | 2021-03-31 | 2022-10-06 | Curevac Ag | Seringues contenant des compositions pharmaceutiques comprenant de l'arn |
WO2022218891A2 (fr) * | 2021-04-12 | 2022-10-20 | BioNTech SE | Compositions d'arn comprenant une substance tampon et procédés de préparation, de stockage et d'utilisation de celles-ci |
WO2023031394A1 (fr) | 2021-09-03 | 2023-03-09 | CureVac SE | Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques |
-
2023
- 2023-10-27 WO PCT/EP2023/080036 patent/WO2024089229A1/fr unknown
Patent Citations (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (fr) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Composition pharmaceutique contenant un arnm stabilise et optimise pour la traduction dans ses regions codantes |
WO2008077592A1 (fr) | 2006-12-22 | 2008-07-03 | Curevac Gmbh | Procédé de purification d'arn à l'échelle préparative par hplc |
WO2011069586A1 (fr) | 2009-12-09 | 2011-06-16 | Curevac Gmbh | Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques |
WO2012019780A1 (fr) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Acide nucléique comprenant ou codant pour une tige-boucle d'histone et une séquence poly(a) ou un signal de polyadénylation pour augmenter l'expression d'une protéine codée |
WO2015062738A1 (fr) | 2013-11-01 | 2015-05-07 | Curevac Gmbh | Arn modifié à propriétés immunostimulantes réduites |
WO2015101416A1 (fr) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Procédés d'analyse d'arn |
WO2015188933A1 (fr) | 2014-06-10 | 2015-12-17 | Curevac Ag | Procédés et moyen d'amélioration de la production d'arn |
WO2015199952A1 (fr) | 2014-06-25 | 2015-12-30 | Acuitas Therapeutics Inc. | Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques |
WO2016165831A1 (fr) | 2015-04-17 | 2016-10-20 | Curevac Ag | Lyophilisation de l'arn |
WO2016180430A1 (fr) | 2015-05-08 | 2016-11-17 | Curevac Ag | Procédé de production d'arn |
WO2016184575A1 (fr) | 2015-05-20 | 2016-11-24 | Curevac Ag | Composition de poudre sèche comprenant de l'arn à chaîne longue |
WO2016184576A2 (fr) | 2015-05-20 | 2016-11-24 | Curevac Ag | Composition de poudre sèche comprenant de l'arn à chaîne longue |
WO2016193206A1 (fr) | 2015-05-29 | 2016-12-08 | Curevac Ag | Procédé de production et de purification d'arn, comprenant au moins une étape de filtration à flux tangentiel |
WO2017004143A1 (fr) | 2015-06-29 | 2017-01-05 | Acuitas Therapeutics Inc. | Formulations de lipides et de nanoparticules de lipides pour l'administration d'acides nucléiques |
US10392341B2 (en) | 2015-09-17 | 2019-08-27 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2017053297A1 (fr) | 2015-09-21 | 2017-03-30 | Trilink Biotechnologies, Inc. | Compositions et procédés de synthèse d'arn coiffés en 5' |
WO2017066782A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes d'arnm hydrophobes |
WO2017066793A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes arnm et procédés de coiffage d'arnm |
WO2017066789A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm avec sucre modifié |
WO2017066797A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffes d'arnm trinucléotidiques |
WO2017066781A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm à liaison phosphate modifié |
WO2017066791A1 (fr) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Analogues de coiffe d'arnm à substitution sucre |
WO2017075531A1 (fr) | 2015-10-28 | 2017-05-04 | Acuitas Therapeutics, Inc. | Nouveaux lipides et nouvelles formulations de nanoparticules de lipides pour l'administration d'acides nucléiques |
WO2018075827A1 (fr) | 2016-10-19 | 2018-04-26 | Arcturus Therapeutics, Inc. | Analogues de coiffes d'arnm de type trinucléotidique |
WO2018078053A1 (fr) | 2016-10-26 | 2018-05-03 | Curevac Ag | Vaccins à arnm à nanoparticules lipidiques |
WO2018089540A1 (fr) * | 2016-11-08 | 2018-05-17 | Modernatx, Inc. | Formulations stabilisées de nanoparticules lipidiques |
WO2019226925A1 (fr) | 2018-05-24 | 2019-11-28 | Translate Bio, Inc. | Lipides cationiques de thioester |
WO2020061332A1 (fr) | 2018-09-19 | 2020-03-26 | Modernatx, Inc. | Analogues de stérol et leurs utilisations |
WO2020127959A1 (fr) | 2018-12-21 | 2020-06-25 | Curevac Ag | Procédés d'analyse d'arn |
WO2021123332A1 (fr) | 2019-12-20 | 2021-06-24 | Curevac Ag | Nanoparticules lipidiques pour l'administration d'acides nucléiques |
WO2021156267A1 (fr) | 2020-02-04 | 2021-08-12 | Curevac Ag | Vaccin contre un coronavirus |
WO2021183564A1 (fr) * | 2020-03-09 | 2021-09-16 | Arcturus Therapeutics, Inc. | Compositions et méthodes pour l'induction de réponses immunitaires |
WO2021183563A1 (fr) | 2020-03-09 | 2021-09-16 | Arcturus Therapeutics, Inc. | Méthodes et compositions de vaccin contre le coronavirus |
WO2021222801A2 (fr) | 2020-05-01 | 2021-11-04 | Arcturus Therapeutics, Inc. | Acides nucléiques et procédés de traitement de la fibrose kystique |
WO2021239880A1 (fr) | 2020-05-29 | 2021-12-02 | Curevac Ag | Vaccins combinés à base d'acide nucléique |
WO2022135993A2 (fr) * | 2020-12-22 | 2022-06-30 | Curevac Ag | Composition pharmaceutique comprenant des vecteurs lipidique encapsulant de l'arn pour une administration multidose |
WO2022207862A2 (fr) | 2021-03-31 | 2022-10-06 | Curevac Ag | Seringues contenant des compositions pharmaceutiques comprenant de l'arn |
WO2022218891A2 (fr) * | 2021-04-12 | 2022-10-20 | BioNTech SE | Compositions d'arn comprenant une substance tampon et procédés de préparation, de stockage et d'utilisation de celles-ci |
WO2023031394A1 (fr) | 2021-09-03 | 2023-03-09 | CureVac SE | Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques |
Non-Patent Citations (3)
Title |
---|
CAS , no. 2036272-55-4 |
KOPPEL, D., J. CHEM. PHYS., vol. 57, 1972, pages 4814 - 4820 |
PACKERMEREDITH ET AL.: "A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems", NATURE COMMUNICATIONS, vol. 12, no. 1, 2021, pages 1 - 11 |
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