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WO2025104351A1 - Dosages par clhp pour détecter de multiples constructions d'arnm - Google Patents

Dosages par clhp pour détecter de multiples constructions d'arnm Download PDF

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Publication number
WO2025104351A1
WO2025104351A1 PCT/EP2024/082760 EP2024082760W WO2025104351A1 WO 2025104351 A1 WO2025104351 A1 WO 2025104351A1 EP 2024082760 W EP2024082760 W EP 2024082760W WO 2025104351 A1 WO2025104351 A1 WO 2025104351A1
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Prior art keywords
mrnas
mobile phase
mrna
composition
column
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Inventor
Kalavathi DASURI
Marc FRANCOIS-HEUDE
Daniel LIBERTA
Camille MALBURET
Laurine Thomas
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Sanofi Pasteur Inc
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Sanofi Pasteur Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids

Definitions

  • the present disclosure relates to the separation of nucleic acids, for example messenger RNAs (mRNAs), with similar lengths from a mixture.
  • mRNAs messenger RNAs
  • the disclosure relates to preparative and analytical RP-HPLC methods for the characterization and separation of at least a first mRNA and a second mRNA of similar lengths from a mixture.
  • the method can be utilized for characterizing and separating mRNAs from a mixture, before the mRNAs are packaged into lipid nanoparticles, or after the mRNAs have been extracted from lipid nanoparticles.
  • mRNA Messenger RNA
  • mRNA therapy is becoming an increasingly important approach for the treatment of a variety of diseases.
  • mRNA therapy requires effective delivery of the mRNA to the patient and efficient production of the protein encoded by the mRNA within the patient’s body.
  • mRNAs are typically encapsulated in lipid nanoparticles to protect mRNA from degradation before it can reach the target cells or tissue in a patient’s body.
  • Some mRNA therapies include multiple different mRNAs that are similar in length. These mRNAs may be encapsulated in the same lipid nanoparticle, or in different lipid nanoparticles.
  • mRNA-based multicomponent vaccines may be used to inoculate a patient against different strains or variants of a disease-causing agent (e.g., a multivalent influenza vaccine or a vaccine protection against multiple variants of SARS-CoV2), or against a combination of disease-causing agents (e.g., a combination vaccine protecting against infection with influenza and SARS-CoV2).
  • a disease-causing agent e.g., a multivalent influenza vaccine or a vaccine protection against multiple variants of SARS-CoV2
  • a combination of disease-causing agents e.g., a combination vaccine protecting against infection with influenza and SARS-CoV2
  • Development of such multicomponent mRNA vaccines necessitates the development of methods which can be used to assess the presence and/or quality/integrity of multiple mRNAs encoding different antigens in a composition, e.g., after formulation of the vaccine. For instance, it is desirable to confirm whether lipid nanoparticle encapsulation of each m
  • WO 2014/144039 describes methods for the characterization of mRNA molecules during the mRNA production process.
  • WO 2019/036683 describes liquid chromatography methods (e.g., HPLC-based methods) which enable separation of polynucleotides of various lengths, sequences, and/or base compositions.
  • WO 2019/036685 describes gradient-based reversed phase HPLC methods for selectively separating polynucleotides comprising one or more hydrophobic portions, including poly-A-tailed mRNAs.
  • WO 2021/254593 describes a method for determining the integrity of a mixture comprising at least two mRNAs with different sizes.
  • RP-HPLC reverse-phase high pressure liquid chromatography
  • the present disclosure relates to analytical RP-HPLC methods that are capable of separating two or more mRNAs in a multi-mRNA composition.
  • the methods are particularly suitable for analyzing multi-mRNA compositions comprising two or more mRNAs of similar or identical lengths.
  • the disclosed methods also find utility in assessing the quality and/or integrity of each of the two or more mRNAs in a multi-mRNA composition.
  • the disclosed methods can be used to calculate the proportion of each mRNA in a multi-mRNA composition.
  • the disclosure relates to a method for detecting at least a first mRNA and a second mRNA in a composition using reverse-phase high pressure liquid chromatography (RP-HPLC), wherein said first mRNA and said second mRNA are of similar length, comprising: (i) providing an RP-HPLC column comprising a stationary phase, wherein the RP-HPLC column has a length of 150 mm or greater and the stationary phase comprises porous particles with a pore size of at least about 500 A; (ii) contacting the stationary phase at a flow rate with a mobile phase A comprising a first ion pairing reagent; (iii) adding the composition comprising the at least first and second mRNAs to the stationary phase; (iv) applying a gradient of the mobile phase A with a mobile phase B to the stationary phase, thereby separating the at least first and second mRNAs, wherein the mobile phase B comprises a first organic modifier; and (v) detecting the at least first and second mRNA
  • the disclosure also relates to a method for detecting two or more mRNAs in a composition using reverse-phase high pressure liquid chromatography (RP-HPLC), comprising: (i) providing an RP-HPLC column comprising a stationary phase, wherein the RP-HPLC column is held at a temperature of less than 55°C ; (ii) contacting the stationary phase at a flow rate of less than 0.6 ml/min with a mobile phase A comprising a first ion pairing reagent; (iii) adding the composition comprising the two or more mRNAs to the stationary phase; (iv) applying a mobile phase B comprising a first organic modifier to the stationary phase, thereby separating the two or more mRNAs; and (v) detecting the two or more mRNAs using ultra violet (UV) detection.
  • RP-HPLC reverse-phase high pressure liquid chromatography
  • step (v) yields a chromatogram providing information about characteristics of the at least first and second mRNAs or the two or more mRNAs.
  • the characteristics include integrity and purity of said at least first and second mRNAs or the two or more mRNAs.
  • the chromatogram is used to calculate the ratios of said at least first and second mRNAs within the composition.
  • the at least first and second mRNAs or two or more mRNAs differ in length by no more than 50 nucleotides.
  • the porous particles have a size equal to or greater than about 4 pm. In some embodiments, the porous particles have a size of 8 pm or less. In some embodiments, the porous particles have a size from about 4 pm to about 8 pm. In a specific embodiment, the porous particles have a size of about 5 pm.
  • the porous particles have a pore size from about 500 A to about 5000 A. In some embodiments, the porous particles have a pore size of at least about 1000 A. In some embodiments, the porous particles have a pore size greater than 2000 A. In some embodiments, the porous particles have a pore size greater than 3000 A. In some embodiments, the porous particles have a pore size from about 2000 A to about 5000 A. In some embodiments, the porous particles have a pore size from about 3000 A to about 5000 A. In some embodiments, the porous particles have a pore size of about 4000 A.
  • the RP-HPLC column has a length of greater than 150 mm. In some embodiments, the RP-HPLC column has a length of greater than 200 mm. In some embodiments, the RP-HPLC column has a length from about 200 mm to about 300 mm. In a specific embodiment, the RP-HPLC column has a length of about 250 mm.
  • the RP-HPLC column has an inner diameter smaller than 5 mm. In some embodiments, the RP-HPLC column has an inner diameter from about 2 mm to about 5 mm, optionally wherein the column has an inner diameter of about 2 mm.
  • the first ion pairing reagent is hexylammonium acetate (HAA).
  • the first ion pairing reagent is at a concentration from about 75 mM to about 500 mM. In some embodiments, the first ion pairing reagent has a concentration of about 100 mM to about 400 mM. In some embodiments, the first ion pairing reagent has a concentration of about 150 mM to about 400 mM. In some embodiments, the first ion pairing reagent has a concentration of about 150 mM. In some embodiments, the first ion pairing reagent has a concentration of about 200 mM to about 400 mM. In some embodiments, the first ion pairing reagent has a concentration of about 200 mM. In some embodiments, the first ion pairing reagent is at a concentration of about 250 mM.
  • the mobile phase B further comprises the first ion pairing reagent. In some embodiments, the mobile phase B comprises the first ion pairing reagent at the same concentration as the mobile phase A.
  • the mobile phase A further comprises an organic modifier.
  • the organic modifier is at a concentration of 20% (v/v) to 60% (v/v). In some embodiments, the organic modifier is at a concentration of about 40% (v/v). In some embodiments, the organic modifier is the same as the organic modifier in mobile phase B.
  • the organic modifier is acetonitrile (ACN).
  • the organic modifier is about 40% (v/v) to about 100% (v/v) of the mobile phase B, e.g., about 50% (v/v) or 75% (v/v). In some embodiments, the organic modifier is about 50% (v/v) of the mobile phase B. In some embodiments, the organic modifier is about 70% (v/v) to about 80% (v/v) of the mobile phase B. In some embodiments, the organic modifier is about 75% (v/v) of the mobile phase B.
  • the flow rate is less than 0.6 ml/min. In some embodiments, the flow rate is about 0.2 ml/min to about 0.5 ml/min. In some embodiments, the flow rate is about 0.3 ml/min or about 0.4 ml/min.
  • the RP-HPLC column is held at a temperature from about 45°C to about 60°C. In some embodiments, the RP-HPLC column is held at a temperature of less than 55°C. In some embodiments, the RP-HPLC column is held at a temperature from about 45°C to about 55°C. In a specific embodiment, the RP-HPLC column is held at a temperature of about 50°C.
  • the mobile phase A and/or mobile phase B has/have a pH from about 6 to about 8. In some embodiments, the pH is about 7, about 7.5 or about 8.
  • the composition comprising the at least first and second mRNAs or the two or more mRNAs, the mobile phase A and/or the mobile phase B comprise(s) an agent for chelating divalent cations.
  • the composition comprising the at least first and second mRNAs or the two or more mRNAs comprises an agent for chelating divalent cations.
  • the agent for chelating divalent cations is ethylenediaminetetraacetic acid (EDTA).
  • the agent for chelating divalent cations in the mobile phase has a concentration of about 0.1 mM to about 0.9 mM.
  • the agent for chelating divalent cations has a concentration of about 0.2 mM to about 0.3 mM, optionally where the concentration of the agent for chelating divalent cations is about 0.25 mM.
  • the mobile phase does not comprise an agent for chelating divalent cations.
  • the composition comprises the at least first and second mRNAs or the two or more mRNAs encapsulated in one or more lipid nanoparticles. In some embodiments, the composition comprises the at least first and second mRNAs or the two or more mRNAs encapsulated in the same lipid nanoparticle. In some embodiments, the composition comprises the at least first and second mRNAs or the two or more mRNAs encapsulated in different lipid nanoparticles. In some embodiments, the mRNAs are extracted from the lipid nanoparticles. In some embodiments, the extraction is performed using one or more organic solvent(s) or a detergent. In some embodiments, the detergent comprises Triton X-100. In some embodiments, the extraction is performed using a mixture of phenol and chloroform. In some embodiments, the extraction is performed using ammonium acetate in isopropanol.
  • the composition comprises the at least first and second mRNAs or the two or more mRNAs in purified form.
  • the at least first and second mRNAs or the two or more mRNAs encode homologous proteins.
  • the homologous proteins are antigens.
  • the at least first and second mRNAs or the two or more mRNAs encode antigens from at least a first virus and a second virus.
  • the first and second viruses are phylogenetically related to each other.
  • the at least first and second viruses are influenza viruses.
  • the antigens are hemagglutinin (HA) or neuraminidase (NA) proteins.
  • the porous particles are made of styrene and/or divinylbenzene.
  • the gradient ranges from about 40% of mobile phase B to about 70% of mobile phase B. In some embodiments, the at least first and second mRNAs or the two or more mRNAs separate at a gradient ranging from about 50% to about 68% of mobile phase B.
  • the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 45 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 40 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 35 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 30 nucleotides.
  • the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 25 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 20 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 15 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 10 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs differ in length by no more than 5 nucleotides. In some embodiments, the at least first and second mRNAs or the two or more mRNAs are of the same length.
  • the at least first and second mRNAs or the two or more mRNAs differ in G/C content.
  • Figure 1 illustrates an initial attempt of separating four similarly sized mRNAs in a mixture. Chromatograms of each of the four individual mRNAs that form part of the mixture are shown as an overlay with a representative chromatogram of the mixture. Only incomplete separation of the four mRNAs was achieved when non-optimized conditions were used. Two major peaks were observed.
  • Figure 2 illustrates the effect of preheating the sample prior to chromatographic separation. An overlay of two representative chromatograms obtained with the same conditions as for Figure 1 are shown. Heating the sample prior to loading did not improve separation of the four mRNAs when non-optimized conditions were used. Two peaks were observed.
  • Figure 3 illustrates the effect of alternative ion pairing reagents and organic modifiers on the separation of four similarly sized mRNAs in a mixture. Changing the reagents improved separation of the four mRNAs only slightly. Two peaks, each with a small shoulder indicating the emergence of an additional peak, were observed.
  • Figure 4 illustrates the effects of using a longer column with a smaller inner diameter.
  • a polymer-based stationary phase composed of smaller particles was used. Chromatograms of each of the four individual mRNAs that form part of the mixture are shown as an overlay with a representative chromatogram of the mixture. Using the column and stationary phase resulted in effective separation of the four mRNAs in the mixture.
  • Figure 5 illustrates the effect of increasing the concentration of the first ion pairing reagent in mobile phase A or adding the first ion pairing reagent also to mobile phase B. Representative chromatograms are shown demonstrating the separation of the four mRNAs in the mixture.
  • Figure 6 illustrates the effect of the column temperature on the separation of four similarly sized mRNAs. Representative chromatograms are shown.
  • Figure 7 illustrates the effect of including an organic modifier in both mobile phases on the separation and detection of four mRNAs. Representative chromatograms are shown.
  • Figure 8 illustrates the effect of including a second organic modifier in mobile phase B before and after adjustments of the gradient. Representative chromatograms are shown.
  • Figure 9 illustrates the broad applicability of the optimized conditions for separating various mRNAs of similar length in a mixture.
  • the optimized conditions were successfully used to separate mRNAs different to the four test mRNAs used in Figures 1-8. Chromatograms of each of the three individual mRNAs that form part of the mixture are shown as an overlay with a representative chromatogram of the mixture.
  • Figure 10 illustrates the broad applicability of the optimized conditions for separating up to eight mRNAs of similar or identical length in a mixture.
  • Figure 10A shows an overlay of two representative chromatograms showing the separation of two mixtures 1 and 2, each containing different sets of four different mRNAs of similar or identical size.
  • Figure 10B shows a representative chromatogram showing the separation of a composition that combined mixtures 1 and 2. Seven peaks were detected. Two of the eight mRNAs in the composition could not be further resolved into individual peaks.
  • Figure 11 illustrates that only minor adjustments to the running conditions are needed to adapt the methods identified herein to a column with a different particle size in order to separate similarly sized mRNAs in a mixture.
  • Figure 11 A shows a representative chromatogram showing the separation of a mixture comprising four similarly sized mRNAs using a column with a particle size of 1000 A.
  • Figure 1 IB shows an overlay of the chromatogram shown in Figure 11 A with a representative chromatogram showing the separation of the same mixture using a column with a particle size of 4000 A.
  • Figure 12 illustrates that columns comprising a plurality of porous particles with a pore size of less than 500 A cannot effectively separate similarly sized mRNAs in a mixture into distinct peaks using RP-HPLC and the optimized separation conditions identified herein.
  • a representative chromatogram showing the incomplete separation of a mixture comprising four similarly sized mRNAs using a column with a particle size of 300 A is shown.
  • mRNA refers to a polyribonucleotide that encodes at least one peptide, polypeptide or protein.
  • mRNA as used herein encompasses both modified and unmodified. mRNA may contain one or more coding and non-coding regions.
  • mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, in vitro transcribed, or chemically synthesized. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5’ to 3’ direction unless otherwise indicated.
  • a typical mRNA comprises a 5’ cap, a 5’ untranslated region (5’ UTR), a protein-coding region, a 3’ untranslated region (3’ UTR), and a 3’ tail.
  • the tail structure is a poly(C) tail. More typically, the tail structure is a poly (A) tail.
  • sequence-optimized is used to describe a nucleotide sequence that is modified relative to a naturally-occurring or wild-type nucleic acid. Such modifications may include, e.g., codon optimization as well as the use of 5’ UTRs and 3’ UTRs which are not normally associated with the naturally-occurring or wild-type nucleic acid.
  • codon optimization and “codon-optimized” refer to modifications of the codon composition of a naturally-occurring or wild-type nucleic acid encoding a peptide, polypeptide or protein that do not alter its amino acid sequence, thereby improving protein expression of said nucleic acid.
  • “codon optimization” may also refer to the process by which one or more optimized nucleotide sequences are arrived at by removing with filters less than optimal nucleotide sequences from a list of nucleotide sequences, such as filtering by guanine-cytosine content, codon adaptation index, presence of destabilizing nucleic acid sequences or motifs, and/or presence of pause sites and/or terminator signals.
  • template DNA (or “DNA template”) relates to a DNA molecule comprising a nucleic acid sequence encoding an mRNA transcript to be synthesized by in vitro transcription.
  • the template DNA is used as template for in vitro transcription in order to produce the mRNA transcript encoded by the template DNA.
  • the template DNA comprises all elements necessary for in vitro transcription, particularly a promoter element for binding of a DNA-dependent RNA polymerase, such as, e.g., T3, T7 and SP6 RNA polymerases, which is operably linked to the DNA sequence encoding a desired mRNA transcript.
  • the “template DNA” in the context of the present disclosure may be a linear or a circular DNA molecule.
  • template DNA may refer to a DNA vector, such as a plasmid DNA, which comprises a nucleic acid sequence encoding the desired mRNA transcript.
  • the term “about” refers to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term indicates a deviation from the indicated numerical value of ⁇ 10%. In some embodiments, the deviation is ⁇ 5% of the indicated numerical value. In certain embodiments, the deviation is ⁇ 1% of the indicated numerical value.
  • the present disclosure relates to a method for separating and detecting multiple mRNAs comprised in a composition, in particular multiple mRNAs of similar length.
  • Each of the mRNAs comprise a different coding sequence, although the coding sequences may be closely related, e.g., may encode homologous proteins of the same or similar length.
  • the present disclosure provides a method for detecting at least a first mRNA and a second mRNA in a composition using reverse phase high pressure liquid chromatography (RP-HPLC), wherein said first mRNA and said second mRNA are of similar length, comprising: (i) providing an RP-HPLC column comprising a stationary phase, wherein the RP-HPLC column has a length of 150 mm or greater and the stationary phase comprises a plurality of porous particles with a pore size of at least about 500 A; (ii) contacting the stationary phase at a flow rate with a mobile phase A comprising an ion pairing reagent; (iii) adding the composition comprising the at least first and second mRNAs to the stationary phase; (iv) applying a gradient of the mobile A with a mobile phase B to the stationary phase, thereby separating the at least first and second mRNAs, wherein the mobile phase B comprises a first organic modifier; and (v) detecting the at least first mRNA and a second m
  • the disclosure provides a method for detecting two or more mRNAs in a composition using reverse-phase high pressure liquid chromatography (RP- HPLC), comprising: (i) providing an RP-HPLC column comprising a stationary phase, wherein the RP-HPLC column is held at a temperature of less than 55°C ; (ii) contacting the stationary phase at a flow rate of less than 0.6 ml/min with a mobile phase A comprising a first ion pairing reagent; (iii) adding the composition comprising the two or more mRNAs to the stationary phase; (iv) applying a mobile phase B comprising the ion pairing reagent and a first organic modifier to the stationary phase, thereby separating the two or more mRNAs; and (v) detecting the two or more mRNAs using UV detection.
  • RP- HPLC reverse-phase high pressure liquid chromatography
  • the inventors demonstrate herein that the disclosed methods can be used to separate three or more (e.g., four or more) mRNAs of similar or identical lengths. More specifically, the inventors demonstrate successful separation and detection of six different mRNAs of similar or identical lengths using RP-HPLC. Thus, in some embodiments, four mRNAs, or more than four mRNAs (e.g., 5, 6, 7 or 8 mRNAs), all of similar length, may be separated using a method in accordance with the disclosure.
  • a composition may comprise two sets of mRNAs, wherein the mRNAs in each set are similar in length.
  • the mRNAs may encode two or more therapeutic peptides, polypeptides or proteins (including, e.g., prophylactic peptides, polypeptides or proteins such as antigens from viruses or bacteria).
  • the mRNAs may encode two or more (e.g., 3 or 4) haemagglutinin (HA) proteins and two or more (e.g., 3 or 4) neuraminidase (NA) proteins, wherein each HA and NA is derived from different, phylogenetically related influenza virus (e.g., from influenza A and influenza B).
  • HA haemagglutinin
  • NA neuraminidase
  • a typical mRNA in accordance with the disclosure comprises a 5’ cap, a 5’ untranslated region (5’ UTR), a protein-coding region, a 3’ untranslated region (3’ UTR), and a 3’ tail.
  • the mRNA of the disclosure comprises a 5’ cap with the following structure:
  • a 5’ cap and/or a 3’ tail may be added after mRNA synthesis.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • the 5’ cap and/or a 3’ tail sequences are included in the DNA template sequences used in in vitro transcription reaction.
  • a 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’5’5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • Examples of cap structures include, but are not limited to, m7G(5’)ppp (5’(A,G(5’)ppp(5’)A and G(5’)ppp(5’)G. Additional cap structures are described in published U.S. Application No. US 2016/0032356 and published U.S. Application No. US 2018/0125989, which are incorporated herein by reference.
  • the tail structure of the mRNA comprises a poly(A) tail. In another specific embodiment, the tail structure of the mRNA comprises a poly(C) tail. In some embodiments, the tail structure comprises at least 50 adenosine or cytosine nucleotides. In a typical embodiment, the tail structure is approximately 100-500 nucleotides in length.
  • a poly(A) or poly(C) tail on the 3’ terminus of mRNA typically includes at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, respectively.
  • a tail structure includes combination of poly(A) and poly(C) tails with various lengths described herein. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.
  • the mRNA may be prepared with nucleotides comprising naturally-occurring or modified nucleosides.
  • the mRNA comprises or consists of naturally- occurring nucleosides (or unmodified nucleosides; i.e., adenosine, guanosine, cytidine, and uridine).
  • the mRNA comprises one or more modified nucleosides, such as nucleoside analogs (e.g., adenosine analog, guanosine analog, cytidine analog, or uridine analog).
  • nucleoside analogs e.g., adenosine analog, guanosine analog, cytidine analog, or uridine analog.
  • the presence of one or more nucleoside analogs may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only naturally-occurring nucleosides.
  • the mRNA comprises both unmodified and modified nucleosides.
  • the one or more modified nucleosides is a nucleoside analog.
  • the one or more modified nucleosides comprises at least one modification selected from a modified sugar, and a modified nucleobase.
  • the mRNA comprises one or more modified intemucleoside linkages.
  • the one or more modified nucleosides is a nucleoside analog selected from the group consisting of 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C 5 -bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8 -oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-l-methyl-pseud
  • the mRNA may be RNA wherein 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine.
  • Teachings for the use of such modified RNA are disclosed in US Patent Publication US 2012/0195936 and international publication WO 2011/012316, both of which are hereby incorporated by reference in their entirety. Length of mRNAs
  • Methods disclosed herein can be used to separate mRNAs of similar lengths.
  • Such mRNAs are typically about 500-5000 nucleotides in length.
  • the two or more mRNAs or the first and second mRNAs may be 1000-3000 nucleotides in length.
  • such mRNA can be separated using the methods disclosed herein, even if the identical or very similar in length, e.g., if each mRNA in a composition is about 1000, about 1500, about 2000, about 2500, or about 3000 nucleotides in length.
  • the at least first and second mRNAs differ in length by no more than 20 nucleotides. In some embodiments, the at least first and second mRNAs differ in length by no more than 15 nucleotides. In some embodiments, the at least first and second mRNAs differ in length by no more than 10 nucleotides. In some embodiments, the at least first and second mRNAs differ in length by no more than 5 nucleotides. In some embodiments, the at least first and second mRNAs are of the same length, i.e., the at least first and second mRNAs have the same number of nucleotides.
  • mRNAs of the disclosure may be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Application No. US 2018/0258423, and can be used to practice the present disclosure, all of which are incorporated herein by reference. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT).
  • IVTT in vitro transcription
  • IVT is typically performed with a linear or circular DNA template or DNA vector containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor.
  • a promoter e.g., a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor.
  • a DNA template or DNA vector may be transcribed in vitro.
  • a suitable DNA template or DNA vector typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal (terminator).
  • mRNA is purified using Tangential Flow Filtration. Suitable purification methods include those described in published U.S. Application No. US 2016/0040154, published U.S. Application No. US 2015/0376220, published U.S. Application No. US 2018/0251755, published U.S. Application No. US 2018/0251754, U.S. Provisional Application No. 62/757,612 filed on November 8, 2018, and U.S. Provisional Application No. 62/891,781 filed on August 26, 2019, all of which are incorporated by reference herein and may be used to practice the present disclosure. It is advantageous to purify the mRNA of the disclosure which may be included in pharmaceutical compositions in some embodiments of the disclosure, as the purity requirements for mRNA products are more stringent for therapeutic applications.
  • the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by filtration. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF).
  • TMF Tangential Flow Filtration
  • LNPs Lipid Nanoparticles
  • mRNAs may be encapsulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • a lipid nanoparticle suitable for use with the present disclosure comprises one or more cationic lipids, one or more non-cationic lipids (e.g., DOPE and/or cholesterol), and one or more PEG-modified lipids (e.g., DMG-PEG2K).
  • a typical lipid nanoparticle for use with the disclosure is composed of four lipid components: a cationic lipid (e.g., a sterol -based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K).
  • a cationic lipid e.g., a sterol -based cationic lipid
  • DOPE DOPE
  • cholesterol-based lipid e.g., cholesterol
  • PEG-modified lipid e.g., DMG-PEG2K
  • the non-cationic lipid is DOPE.
  • the molar ratio of cationic lipid to non-cationic lipid to cholesterol to PEG-modified lipid typically is between about 30-60:25-35:20-30: 1-15, respectively.
  • An exemplary LNP in accordance with the disclosure may be composed of a cationic lipid selected from cKK-E12, cKK-ElO, OF-Deg-Lin and OF-02; a non-cationic lipid selected from DOPE and DEPE; a cholesterol- based lipid such as cholesterol; and a PEG-modified lipid such as DMG-PEG2K.
  • a lipid nanoparticle comprises no more than three distinct lipid components.
  • An exemplary lipid nanoparticle is composed of three lipid components: a cationic lipid (e.g., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE) and a PEG-modified lipid (e.g., DMG-PEG2K).
  • the three distinct lipid components are HGT4002, DOPE and DMG-PEG2K.
  • HGT4002, DOPE and DMG-PEG2K are present in a molar ratio of approximately 60:35:5, respectively.
  • Such LNPs may be particularly suitable for aerosol delivery of the mRNAs of the disclosure.
  • the lipid nanoparticles for use in the disclosure can be prepared by various techniques which are presently known in the art. Such methods are described, e.g., in published U.S. Application No. US 2011/0244026, published U.S. Application No. US 2016/0038432, published U.S. Application No. US 2018/0153822, published U.S. Application No. US 2018/0125989 and U.S. Provisional Application No. 62/877,597, filed July 23, 2019, all of which are incorporated herein by reference.
  • mRNAs encoding different peptides, polypeptides, or proteins may be combined together in a single composition.
  • the at least first mRNA and second mRNA or the two or more mRNAs are combined in a solvent (typically an aqueous solvent, e.g., water) to form a composition.
  • a solvent typically an aqueous solvent, e.g., water
  • the at least first mRNA and second mRNA or the two or more mRNAs are dissolved in the mobile phase.
  • the inventors have found that providing a composition comprising the at least first mRNA and second mRNA or the two or more mRNAs dissolved in an aqueous solution (e.g., water, optionally comprising a divalent cationic chelator such as EDTA) prior to injecting the mRNAs into the RP-HPLC system results in better separation than directly dissolving the at least first mRNA and second mRNA or the two or more mRNAs in the mobile phase (e.g., mobile phase A).
  • an aqueous solution e.g., water, optionally comprising a divalent cationic chelator such as EDTA
  • the composition may also comprise one or more excipients selected from a sugar, a salt, a buffering agent or a chelating agent.
  • the sugar is a disaccharide, e.g., trehalose or sucrose.
  • the salt is NaCl.
  • the buffering agent comprises phosphate, Tris or citrate.
  • the chelating agent is a divalent cation chelator (e.g., ethylenediaminetetraacetic acid or EDTA).
  • the concentration of the chelating agent is from about 0.05 mM to about 1 mM. In some embodiments, the concentration of the chelating agent is from about 0.1 mM to about 0.9 mM. In some embodiments, the concentration of the chelating agent is from about 0.2 mM to about 0.3 mM. In some embodiments, the concentration of the chelating agent is about 0.25 mM. In some embodiments, the chelating agent is EDTA.
  • the methods of the disclosure may be used to assess the quality/integrity of the at least first mRNA and second mRNA or two or more mRNAs in a composition.
  • a composition may be a multi-mRNA composition for use in the prophylaxis or therapy of a disease or disorder.
  • Such therapeutic compositions may include multivalent or multicomponent mRNA vaccines.
  • Such therapeutic compositions may also include compositions comprising two or more mRNAs encoding therapeutic antibodies.
  • the molar ratio of two different mRNAs in the composition is 1 :4, 1 :3, 1 :2 or 1 : 1.
  • the ratio of the different mRNAs in the composition may be equimolar.
  • a composition comprising four different mRNAs may comprise the four mRNAs at a molar ratio of 1 : 1 : 1 : 1.
  • the different mRNAs may be present at different proportions in the composition.
  • a composition comprising four different mRNAs may comprise the four mRNAs at a molar ratio of 1 : 1 :2:2.
  • the mRNA concentration in the composition is about 0.8 mg/ml to 2 mg/ml (e.g., 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, or 2 mg/ml). In some embodiments, the mRNA concentration in the composition is about 1 mg/ml.
  • the mRNAs encoding different peptides, polypeptides, or proteins may be encapsulated in lipid nanoparticles.
  • the at least first and second mRNAs or the two or more mRNAs are encapsulated in lipid nanoparticles in the compositions described herein.
  • the at least first and second mRNAs or the two or more mRNAs are encapsulated in the same lipid nanoparticle.
  • the at least first and second mRNAs or the two or more mRNAs are encapsulated in separate lipid nanoparticles.
  • the separate lipid nanoparticles may have the same lipid composition, or they may have different lipid compositions.
  • the at least first mRNA and second mRNA or the two or more mRNAs of a composition of the present disclosure comprises only two mRNA molecules.
  • the at least first mRNA and second mRNA or the two or more mRNAs comprises a plurality of different mRNA molecules, such as at least three different mRNA molecules, at least four different mRNA molecules, at least different five mRNA molecules, at least six different mRNA molecules, at least seven different mRNA molecules, at least eight different mRNA molecules, at least nine different mRNA molecules, or at least ten different mRNA molecules.
  • a composition of the present disclosure is a multivalent composition (e.g., a multivalent vaccine) comprising a plurality of different mRNA molecules.
  • the composition is a bivalent composition (e.g., a bivalent vaccine) comprising two different species of mRNA molecules.
  • the composition is a trivalent composition (e.g., a trivalent vaccine) comprising three different species of mRNA molecules.
  • the composition is a quadrivalent composition (e.g., a quadrivalent vaccine) comprising four different species of mRNA molecules.
  • the composition is a pentavalent composition (e.g., a pentavalent vaccine) comprising five different species of mRNA molecules.
  • the composition is a hexavalent composition (e.g., a hexavalent vaccine) comprising six different species of mRNA molecules.
  • the composition is a heptavalent composition (e.g., a heptavalent vaccine) comprising seven different species of mRNA molecules.
  • the composition is an octavalent composition (e.g., an octavalent vaccine) comprising eight different species of mRNA molecules.
  • the composition is a nonavalent composition (e.g., a nonavalent vaccine) comprising nine different species of mRNA molecules.
  • the composition is a decavalent composition (e.g., a decavalent vaccine) comprising ten different species of mRNA molecules.
  • the mRNAs may be extracted from the vaccine before being separated and detected using the method of the present disclosure.
  • each of the different at least first mRNA and second mRNA or the two or more mRNAs in a composition of the disclosure shares at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with at least one other mRNA molecule in the composition, including all values and subranges therebetween.
  • the at least first and second mRNAs or the two or more mRNAs encode therapeutic peptides, polypeptides or proteins (including peptides, polypeptides or proteins used in disease prophylaxis), e.g., antigens or antibodies.
  • the at least first and second mRNAs or the two or more mRNAs encode antigens derived from first and second pathogens. Pathogens include bacteria and viruses.
  • each of the at least first mRNA and second mRNA or the two or more mRNAs encodes an antigen derived from a virus including, but not limited to an influenza virus, a coronavirus, a respiratory syncytial virus (RSV), a parainfluenza virus (PIV), a metapneumovirus (MPV), a human immunodeficiency virus (HIV), a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a west Nile virus, a poliovirus, and a rabies virus.
  • a virus including, but not limited to an influenza virus, a coronavirus, a respiratory syncytial virus (
  • the at least first and second mRNAs or the two or more mRNAs encode antigens from a first pathogen and a second pathogen. In some embodiments, the at least first and second mRNAs or the two or more mRNAs encode antigens from two or more pathogens. In some embodiments, the first and second pathogen are phylogenetically related to each other. In some embodiments, the two or more pathogens are phylogenetically related to each other. In some embodiments, the antigens encoded by the first and second mRNAs or the two or more mRNAs are homologs derived from phylogenetically related pathogens (e.g., phylogenetically related viruses).
  • phylogenetically related viruses may be influenza viruses, e.g., circulating influenza viruses.
  • influenza viruses e.g., circulating influenza viruses.
  • the antigens encoded by the first and second mRNAs or the two or more mRNAs may be selected for inclusion in a seasonal influenza vaccine.
  • the at least first and second mRNAs or the two or more mRNAs encode antigens from an influenza A virus and an influenza B virus, respectively.
  • each of the at least first mRNA and second mRNA or the two or more mRNAs encodes hemagglutinin (HA), a nucleoprotein (NP), a neuraminidase (NA) protein, a matrix- 1 (Ml), a matrix-2 (M2), a non-structural protein- 1 (NS1) a non- structural protein-2 (NS2) from influenza A and/or influenza B.
  • HA hemagglutinin
  • NP nucleoprotein
  • NA neuraminidase
  • Ml matrix- 1
  • M2 matrix-2
  • NS1 non-structural protein- 1
  • NS2 non-structural protein-2
  • the influenza protein is of human origin.
  • the influenza protein is of swine or bird origin.
  • the therapeutic protein is from an influenza A strain, such as one or more of HI, H2, H5, H6, H8, H9, Hl 1, Hl 3 and HI6 (phylogenetic group I) and/or one or more of H3, H4, H7, HIO, HI5 and H14 (phylogenetic group 2).
  • the antigens encoded by the first and second mRNAs are hemagglutinin (HA) proteins.
  • the antigens encoded by the first and second mRNAs are neuraminidase (NA) proteins.
  • the therapeutic protein comprises one or more HA proteins from an influenza B strain, such as a Victoria or Yamagata strain.
  • the therapeutic protein comprises one or more HA protein from influenza A and influenza B.
  • the therapeutic protein comprises an HA protein from Group I and Group II influenza A and/or an HA protein from the Victoria and Yamagata.
  • each of the at least first mRNA and second mRNA or the two or more mRNAs encodes an influenza virus protein selected from Hl, H3, HA from a B/Victoria lineage, and/or HA from a B/Yamagata lineage.
  • the composition comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., a quadrivalent vaccine), such as an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B/Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B/Yamagata lineage.
  • a different influenza virus protein e.g., a quadrivalent vaccine
  • each of the at least first mRNA and second mRNA or the two or more mRNAs encodes an influenza virus protein selected from Nl, N2, NA from a B/Victoria lineage, and/or NA from a B/Yamagata lineage.
  • the composition comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., a quadrivalent vaccine), such as a Nl from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, an NA from a third standard of care influenza virus strain from the B/Victoria lineage, and an NA from a fourth standard of care influenza virus strain from the B/Yamagata lineage.
  • a different influenza virus protein e.g., a quadrivalent vaccine
  • each of the at least first mRNA and second mRNA or the two or more mRNAs encodes an antigen derived from an influenza virus, such as a strain of Influenza A, a strain of Influenza B, or combinations thereof.
  • strains of Influenza A include, but are not limited to, A/Califomia/07/2009, A/Japan/305/1957, A/Vietnam/1194/2004, A/Vietnam/1203/2004, A/Netherlands/219/2003, A/HongKong/1073/1999, A/Perth/ 16/2009, A/Wisconsin/588/2019 and/or A/Tasmania/503/2020.
  • the source of the at least one mRNA molecule in a composition of the present disclosure is from a strain of Influenza A, such as A/Wisconsin/588/2019 and/or A/Tasmania/503/2020.
  • strains of Influenza B include, but are not limited to, B/Brisbane/2008, B/Malaysia/2004, B/Victoria/1987, and/or B/Washington/02/2019 (Victoria lineage) and/or B/PHUKET/3073/2013, B/Florida/2006, B/Mass/2012, and/or B/Wisconsin/2010 (Yamagata lineage).
  • the source of the at least first mRNA and second mRNA or the two or more mRNAs molecule in a composition of the present disclosure is a strain of Influenza B, such as B/Washington/02/2019 and/or B/PHUKET/3073/2013.
  • the at least first mRNA and second mRNA or the two or more mRNAs encodes one or more antibodies or fragments thereof.
  • antibody includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments.
  • immunoglobulin Ig is used interchangeably with “antibody” herein.
  • an “antibody fragment” comprises a portion of an intact antibody, typically the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • the proteins encoded by the first and second mRNAs or the two or more mRNAs are heavy chains of an antibody. In some embodiments, the proteins encoded by the first and second mRNAs are lights chains of an antibody. In some embodiments, the first and second mRNAs or the two or more mRNAs encode two or more full-length antibodies, wherein the heavy chains and light chains of each of the two or more full-length antibodies are encoded by separate mRNAs.
  • the composition comprising the at least first mRNA and second mRNA or the two or more mRNAs are encapsulated in one or more lipid nanoparticles.
  • the lipid nanoparticle is a liposome.
  • the mRNAs are extracted from the one or more lipid nanoparticles before they can be separated by a method of the present disclosure.
  • the extraction of the mRNAs from the lipid nanoparticle is performed using phenol-chloroform.
  • the extraction of the mRNAs from the lipid nanoparticle is performed using a mixture of ammonium acetate and isopropanol.
  • the extraction of the mRNAs from the lipid nanoparticle is performed using a detergent.
  • Suitable detergents include, e.g., a non -ionic detergent such as Triton X-100, Brij-35 and Brij-58.
  • the mRNA is purified after extraction from the lipid nanoparticles.
  • the detergent and/or lipids can be removed, e.g., by precipitating the mRNAs.
  • the purified mRNA can then be resuspended in a suitable solution, e.g., water.
  • a detergent is used for extracting the mRNAs which does not have to be removed from the extracted mRNA prior to analyzing it in a method of the disclosure.
  • Prior art methods to separate mRNAs have utilized columns with a length of about 100 mm.
  • the inventors of the present disclosure surprisingly found that a mixture of different mRNAs (e.g., at least first mRNA and second mRNA or the two or more mRNAs) of similar length or the same length can be separated more effectively when a longer RP- HPLC column is used.
  • the use of stationary phase composed of porous particles with a large pore size e.g., at least about 500 A was also found to be advantageous.
  • the column has a length of about 150 mm or greater. In some embodiments, the column has a length greater than about 150 mm. In some embodiments, the column has a length greater than about 200 mm. In some embodiments, the column has a length between about 150 mm and about 300 mm. In some embodiments the column has a length of about 150 mm, about 200 mm, about 250 mm, or about 300 mm.
  • a column for use with a method of the disclosure has a length between about 200 mm and about 300 mm. In a specific embodiment, the column has a length of about 250 mm.
  • [Hl] Standard RP-HPLC columns come in a range of diameters.
  • the column has an inner diameter less than about 5 mm.
  • the column has an inner diameter greater than 2 mm.
  • the column has an inner diameter between about 2 mm and about 5 mm.
  • the column has an inner diameter of about 2 mm, about 3 mm, about 4 mm, or about 5 mm.
  • the inventors usefully employed RP-HPLC columns with an inner diameter of 4.6 mm and 2.1 mm. Accordingly, in one specific embodiment, the column has an inner diameter of 4.6 mm. In another specific embodiment, the column has an inner diameter of 2.1 mm.
  • the method of the disclosure uses an RP-HPLC column with a length of between about 150 mm and about 300 mm and an inner diameter of between about 2 mm and about 5 mm.
  • the column has a length of about 150 mm and an inner diameter of about 5 mm (e.g., 4.6 mm).
  • the column has a length of about 250 mm and an inner diameter of about 2 mm (e.g., 2.1 mm).
  • the inner volume of a RP-HPLC column for use with the disclosure is typically approximately cylindrical in shape.
  • the inner volume is between about 800 mm 3 and about 2600 mm 3 .
  • the inner volume is about 800 mm 3 , about 900 mm 3 , about 1000 mm 3 , about 1100 mm 3 , about 1200 mm 3 , about 1300 mm 3 , about 1400 mm 3 , about 1500 mm 3 , about 1600 mm 3 , about 1700 mm 3 , about 1800 mm 3 , about 1900 mm 3 , about 2000 mm 3 , about 2100 mm 3 , about 2200 mm 3 , about 2300 mm 3 , about 2400 mm 3 , about 2500 mm 3 , or about 2600 mm 3 .
  • the inner volume of an RP-HPLC column for use with the disclosure is about 2400 mm 3 to about 2500 mm 3 . More typically, however, a smaller volume is sufficient, in particular when a long, thin column is used for the separation of multiple mRNAs of similar length.
  • the inventors have found an RP-HPLC column with an inner volume of about 800 mm 3 to about 900 mm 3 particularly suitable for use with the methods of the disclosure.
  • a chromatography column comprises a stationary phase that separates molecules suspended in a mobile phase by size as it traverses the column.
  • a reverse-phase high pressure liquid chromatography (RP-HPLC) column typically comprises a non-polar stationary phase that includes aromatic hydrocarbons. As mRNAs traverse the column, aromatic rings of the mRNA nucleotides can interact with the aromatic hydrocarbons of the stationary phase.
  • the stationary phase comprises a plurality of identical particles. Each particle is typically only a few pm in size. In some embodiments, the stationary phase comprises porous particles.
  • the particles may be made from various materials.
  • the particles are macro-porous and made from a non-polar (e.g., hydrophobic) material. Suitable non-polar or hydrophobic materials for making such particles may include but are not limited to a synthetic aromatic hydrocarbon polymers such as polystyrene or poly(divinylbenzene).
  • the stationary phase comprises porous particles made of divinylbenzene.
  • the stationary phase comprises porous particles made of styrene.
  • stationary phase comprises porous particles made of styrene and divinylbenzene (e.g., polystyrenedi viny lb enzene)) .
  • An RP-HPLC column for use with the disclosure typically comprises a plurality of porous particles.
  • the particles are typically spherical in size.
  • the term “particle size” refers to the average diameter of the plurality of particles.
  • the particles have a size from about 4 pm to about 8 pm.
  • the particles have a size greater than about 4 pm.
  • the particles have a size less than about 8 pm.
  • the particles have a size of about 4 pm, about 5 pm, about 6 pm, about 7 pm, or about 8 pm.
  • the particles have a size from about 5 pm to about 8 pm.
  • an RP-HPLC with a stationary phase composed of a plurality of particles with a size of about 5 pm could be used to effectively separate similarly sized mRNAs in a mixture. Accordingly, in a specific embodiment, the particles have a size of about 5 pm.
  • the porosity of a particle is typically described by reference to an average pore size.
  • Porous particles for use with the present disclosure typically have wide pores (i.e., the particles are macro-porous).
  • the pore size is at least about 500 A.
  • the pore size is at least about 1000 A.
  • the pore size is from about 500 A to about 5000 A.
  • the pore size is greater than about 2000 A.
  • the pore size is greater than about 3000 A.
  • the pore size is from about 2000 A to about 5000 A.
  • the pore size is from about 3000 A to about 5000 A.
  • the pore size about 500 A, about 1000 A, about 2000 A, about 3000 A, about 4000 A, or about 5000 A, including all values and subranges therebetween.
  • RP-HPLC columns comprising porous particles with a pore size from about 1000 A to about 5000 A were shown to be particularly suitable for use in the methods of the disclosure. Accordingly, in some embodiments, suitable particles have a pore size between about 1000 A and about 5000 A. In a specific embodiment, suitable particles have a pore size of about 1000 A or about 4000 A.
  • particles for use in the methods of the disclosure have a particle size from about 4 pm to about 8 pm and a pore size from about 500 A to about 5000 A (e.g., about 1000 A to about 4000 A).
  • the methods disclosed herein use RP-UHPLC.
  • UHPLC typically uses columns packed with particles smaller than 2 pm (e.g., 1.7 pm).
  • UHPLC systems usually employ a pressure higher than 6000 psi (e.g., up to 15,000 psi), resulting in high flow rates for increased speed.
  • UHPLC can provides superior resolution and sensitivity relative to conventional HPLC systems (see, e.g., Swartz, M.E., “Ultra Performance Liquid Chromatography (UPLC): an introduction”, SEPARATION SCIENCE REDEFINED (2005)).
  • RP-UHPLC can advantageously be combined with columns having a length from about 150 mm to about 300 mm and an internal diameter from about 2 mm to about 5 mm and comprising a stationary phase comprising porous particles having (i) a size from about 4 pm to about 8 pm, and (ii) a pore size from about 500 A to about 5000 A (e.g., about 1000 A to about 4000 A), were shown to be particularly suitable for use in the method of the disclosure.
  • the methods of the disclosure employ a column that has a length from about 200 mm to about 300 mm and an internal diameter from about 2 mm to about 5 mm and comprises a stationary phase comprising porous particles having (i) a size from about 5 pm to about 8 pm, and (ii) a pore size from about 500 A to about 5000 A (e.g., about 1000 A to about 4000 A).
  • the methods of the disclosure employ columns that have a length of about 250 mm and an internal diameter of about 2.1 mm and comprise a stationary phase comprising porous particles having (i) a size of about 5 pm, and (ii) a pore size from about 1000 A to about 4000 A.
  • the separation conditions may be adjusted. Specific adjustments may depend on the particular RP-HPLC column that is employed in a method of the disclosure. Parameters that may be adjusted include the column temperature, the use of particular ion pairing reagents and organic modifiers in the mobile phase.
  • the RP-HPLC column is typically heated.
  • the RP-HPLC column is held at a temperature from about 40°C to about 65°C.
  • the RP-HPLC column has a temperature from about 45 °C to about 60 °C.
  • the temperature of the RP-HPLC column is about 60 °C or less.
  • the temperature of the RP-HPLC column is about 45 °C or greater.
  • the temperature of the RP-HPLC column is about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, or about 60 °C, including all values and subranges therebetween.
  • the inventors of the present disclosure found that keeping the RP-HPLC column at a temperature from about 45 °C to about 55 °C (e.g., about 48 °C to about 55 °C) is particularly useful for the separation of a mixture of multiple mRNAs of similar length. Accordingly, in some embodiments, the column of the RP-HPLC column is held at a temperature of about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, or about 55 °C, including all values and subranges therebetween. In a specific embodiment, the RP-HPLC column is held at a temperature of about 50 °C (e.g., ⁇ 2 °C).
  • Reverse-phase High-Performance Liquid Chromatography uses a nonpolar stationary phase and a polar mobile phase for separating analytes from each other.
  • the mobile phase is an aqueous solution comprising a first ion pairing reagent.
  • the ion pairing reagent facilities hydrophobic interactions with the non-polar stationary phase and electrostatic interactions with the negatively charged mRNA molecules, resulting in the formation of neutral complexes between ion pairing reagent and mRNA. As a results of these interactions, the neutral complexes can bind to the non-polar stationary phase (retention).
  • the polarity of a mobile phase may be adjusted by the addition of a less polar or non-polar first organic modifier, which results in the elution of the bound mRNA molecules.
  • the mobile phase may also comprise additional components such an acid, a base or a buffer to adjust and/or maintain the pH of the mobile phase.
  • the adjustment of polarity is achieved by mixing a mobile phase A (or first mobile phase) with a mobile phase B (or second mobile phase). Gradual mixing is used to form a gradient of the non-polar mobile phase B, resulting in the elution of the bound mRNA molecules at different times, depending on their nucleotide composition.
  • mobile phase A comprises the first ion pairing reagent (e.g., in water)
  • mobile phase B comprises the first organic modifier (e.g., in water).
  • Separation of the at least first and second mRNAs or the two more mRNAs occurs because the first mRNA and the second mRNA, or the two or more mRNAs, are eluted from the stationary phase at different concentrations of the first ion pairing reagent and the first organic modifier.
  • hexylammonium acetate has been found to be a particularly useful first ion pairing reagent.
  • the first ion pairing reagent is HAA.
  • the first ion pairing reagent is triethylammonium acetate (TEAA), dibutylammonium acetate (DBAA) or hexafluoroisopropanol (HFIP).
  • the concentration of the first ion pairing reagent in the mobile phase A is from about 75 mM to about 500 mM, including all values and subranges therebetween.
  • the first ion pairing reagent has a concentration of about 100 mM.
  • the first ion pairing reagent has a concentration of about 150 mM.
  • the first ion pairing reagent has a concentration of about 200 mM.
  • the first ion pairing reagent has a concentration of about 250 mM.
  • the first ion pairing reagent has a concentration of about 300 mM.
  • the first ion pairing reagent has a concentration of about 350 mM. In some embodiments, the first ion pairing reagent has a concentration of about 400 mM. In some embodiments, the first ion pairing reagent has a concentration of about 450 mM.
  • the first ion pairing reagent has a concentration of about 100 mM to about 400 mM. In some embodiments, the first ion pairing reagent has a concentration of about 150 mM to about 400 mM, e.g. about 200 mM to about 400 mM. In one specific embodiment, the first ion pairing reagent has a concentration of about 150 mM. In another specific embodiment, the first ion pairing reagent has a concentration of about 200 mM. In another specific embodiment, the first ion pairing reagent has a concentration of about 250 mM.
  • the first ion pairing reagent has a concentration of about 400 mM.
  • Acetonitrile (ACN) and methanol are the most commonly used organic modifiers in RP-HPLC. Acetonitrile and methanol may provide different selectivity. In some embodiments, acetonitrile may be used interchangeably, or in combination, with methanol in the methods of the disclosure. Acetonitrile has a lower UV cut-off than methanol (190 nm vs 205 nm), making it more suitable for use in applications requiring low UV detection wavelengths. For example, mRNA can be detected by using a wavelength of 260 nm. Accordingly, in some embodiments, the first organic modifier is acetonitrile. In other embodiments, the first organic modifier may be an alcohol, e.g., methanol.
  • the organic modifier is about 40% (v/v) to about 100% (v/v) of the mobile phase B. In some embodiments, the organic modifier is about 50% (v/v) of the mobile phase B. In some embodiments, the organic modifier is about 70% (v/v) to about 80% (v/v) of the mobile phase B. In some embodiments, the organic modifier is about 75% (v/v) of the mobile phase B.
  • mobile phase B is added to mobile phase A at different percentages, resulting in a gradient of mobile phase B in mobile phase A.
  • Adjustments made to the components of the mobile phase e.g., the additional presence of a first ion pairing reagent in mobile phase B; and/or the additional presence a first organic modifier in mobile phase A as well as in mobile phase B) can alter the retention time of each mRNA of the at least first mRNA and second mRNA or two or more mRNAs in the stationary phase. Alteration of the gradient can be used to optimize separation of the first and second or two or more mRNAs in a composition.
  • mobile phase A can additionally include the first organic modifier and/or mobile phase B can additionally include the first ion pairing reagent. If the first organic modifier is added to mobile phase A, then the concentration of the first organic modifier in mobile phase B can be reduced. The skilled person understands that the concentration of the first organic modifier in mobile phase A is selected to avoid premature elution of the mRNAs.
  • the mobile phase A further comprises a first organic modifier.
  • the first organic modifier is at a concentration of about 20% (v/v) to about 60% (v/v), including all values and subranges therebetween. In some embodiments, the organic modifier is at a concentration of about 40% (v/v).
  • the mobile phase B further comprises the first ion pairing reagent.
  • the mobile phase B comprises the first ion pairing reagent at the same concentration as the mobile phase A.
  • the mobile phase B comprises the first ion pairing reagent at a concentration of about 150 mM.
  • the mobile phase B comprises the first ion pairing reagent at a concentration of about 200 mM.
  • a mobile phase A comprising a first ion pairing reagent at a concentration of about 200 mM was shown to be particularly useful for use in the method of the disclosure, when used with a mobile phase B comprising the first ion pairing reagent at a concentration of about 200 mM.
  • the first ion pairing reagent is hexylammonium acetate (HA A).
  • the retention time of the mRNA in the stationary phase is adjusted by the addition of a second organic modifier to mobile phase B.
  • a second organic modifier can be used to modify the resolution of peaks for the first and second mRNAs or at least two mRNAs in the resulting chromatogram.
  • the first organic modifier is acetonitrile and the second organic modifier is methanol.
  • the pH of the mobile phase is about pH 8 or less. In some embodiments, the pH of the mobile phase is about pH 6 or greater. In some embodiments, the pH is from about pH 6 to about pH 8. In some embodiments, the pH is from about pH 7.0 to about pH 8.0. In some embodiments, the pH is about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, or about pH 8.0.
  • a mobile phase comprising a pH from about pH 7 to about pH 8 was shown to be particularly useful for use in the method of the disclosure.
  • the pH of the mobile phase is about pH 7.
  • the mobile phase comprises a chelating agent.
  • the chelating agent is a divalent cation chelator.
  • the divalent cation chelator is ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • any divalent cation chelator that is suitable for the present disclosure can be used.
  • the concentration of the chelating agent in the mobile phase is from about 0.1 mM to about 0.9 mM, including all values and subranges therebetween. In some embodiments, the concentration of the chelating agent in the mobile phase is from about 0.2 mM to about 0.3 mM. In some embodiments, the concentration of the chelating agent in the mobile phase is about 0.25 mM. In some embodiments, the mobile phase does not comprise an agent for chelating divalent cations.
  • the at least first mRNA and the second mRNAs or the two or more mRNAs separate at a gradient ranging from about 50% to about 70% of mobile phase B. In some embodiments, the at least first and second mRNAs or the two or more mRNAs separate at a gradient ranging from about 54% to about 68% of mobile phase B. In some embodiments, the at least first and second mRNAs or the two or more mRNAs separate at a gradient ranging about 56% to about 64% of mobile phase B.
  • the mobile phase traverses the stationary phase at a certain flow rate.
  • the inventors of the present disclosure found that adjusting the flow rate can improve separation of the at least first and second mRNAs.
  • the flow rate is about 1 ml/min or less. In some embodiments, the flow rate is about 0.1 ml/min or greater. In some embodiments, the flow rate is from about 0.1 ml/min to about 1 ml/min. In some embodiments, the flow rate is from about 0.1 ml/min to about 0.7 ml/min.
  • the flow rate is about 0.1 ml/min, about 0.2 ml/min, about 0.3 ml/min, about 0.4 ml/min, about 0.5 ml/min, about 0.6 ml/min, or about 0.7 ml/min.
  • a flow rate from about 0.4 ml/min to about 0.6 ml/min is particularly suitable to separate mRNAs of similar length using the methods of the disclosure.
  • the flow rate is about 0.6 ml/min.
  • the flow rate is about 0.5 ml/min.
  • the flow rate is about 0.4 ml/min.
  • the flow rate is about 0.3 ml/min.
  • a method for detecting two or more mRNAs of similar length or of the same length in a composition using reverse phase ultrahigh pressure liquid chromatography comprising providing an RP- HPLC column comprising a stationary phase, wherein the RP-UHPLC column is held at a temperature of 48-52°C (e.g., about 50°C); contacting the stationary phase at a flow rate of about 0.2-0.4 ml/min (e.g., about 0.3 ml/min) with a mobile phase A comprising a first pairing reagent (e.g., HAA) at a concentration of 100-400 mM; adding the composition comprising the two or more mRNAs to the stationary phase; applying a gradient of the mobile phase A with a mobile phase B to the stationary phase, wherein the mobile phase B comprises a first organic modifier (e.g., acetonitrile) at a concentration of 50-80% (v/v), thereby
  • RP-UHPLC reverse phase ultrahigh pressure liquid chromatography
  • a method for detecting two or more mRNAs of similar length or of the same length in a composition using reverse phase ultrahigh pressure liquid chromatography comprising providing an RP-UHPLC column comprising a stationary phase, wherein the RP-UHPLC column has a length of 150 mm or greater and the stationary phase comprises a plurality of porous particles with a pore size of about 500-5000 A (e.g., about 1000- 4000 A), wherein the RP- UHPLC column is held at a temperature of about 45-55°C (e.g., about 50°C); contacting the stationary phase at a flow rate of 0.2-04 ml/min (e.g., about 0.3 ml/min) with a mobile phase A comprising a first ion pairing reagent (e.g., HAA); adding the composition comprising the two or more mRNAs to the stationary phase; applying a gradient of the mobile phase A with RP-UHPLC
  • the mobile phase A further comprises the first organic modifier present in the mobile phase B.
  • the mobile phase B comprises the first ion pairing reagent present in the mobile phase A.
  • the mobile phase A comprises the first ion pairing reagent only. In some embodiments, the mobile phase B comprises the first organic modifier.
  • the mobile phase B comprises a second organic modifier (e.g., methanol).
  • the mobile phase A comprises a second ion pairing reagent (e.g., triethylammonium acetate (TEAA), dibutylammonium acetate (DBAA) or hexafluoroisopropanol (HFIP)).
  • TEAA triethylammonium acetate
  • DBAA dibutylammonium acetate
  • HFIP hexafluoroisopropanol
  • the mobile phase A and the mobile phase B include water as the solvent.
  • the mobile phase A and the mobile phase B further comprise a buffering agent (e.g., triethyl amine (TEA)).
  • a buffering agent e.g., triethyl amine (TEA)
  • the pH of the mobile phase A and the mobile phase B is about 7-7.5 (e.g., about 7).
  • the RP-UHPLC column has a length of about 200-300 mm (e.g., about 250 mm) and an inner diameter between about 2-5 mm (e.g., 2.1 mm).
  • the stationary phase comprises a plurality of porous particles with a size between about 4-8 pm (e.g., about 5 pm) and a pore size of about 500-5000 A (e.g., about 1000 A or 4000 A).
  • the stationary phase comprises polystyrene and divinylbenzene polymers.
  • the gradient is about 54%-64% of the mobile phase B.
  • detectors or detection systems include but are not limited to absorbance detectors (e.g., UV/VIS detectors), fluorescence detectors, electrochemical detectors, and mass spectrometric detectors.
  • absorbance detectors e.g., UV/VIS detectors
  • fluorescence detectors e.g., fluorescence detectors
  • electrochemical detectors e.g., electrochemical detectors
  • mass spectrometric detectors e.g., mass spectrometric detectors.
  • methods of the disclosure use an absorbance detector to detect the mRNAs after separation. mRNA can readily be detected at 260 nm using UV detection.
  • a method for detecting at least a first mRNA and a second mRNA in a composition using reverse phase high pressure liquid chromatography (RP-HPLC), wherein said first mRNA and said second mRNA are of similar length, comprising:
  • porous particles have a size from about 4 pm to about 8 pm, optionally wherein the porous particles have a size of about 5 pm.
  • porous particles have a pore size from about 1000 A to about 4000 A.
  • porous particles have a pore size of about 1000 A or about 4000 A.
  • the RP-HPLC column has a length of greater than 200 mm. 12. The method of any one of the preceding embodiments, wherein the RP-HPLC column has a length from about 200 mm to about 300 mm, optionally wherein the length is about 250 mm.
  • the mobile phase A further comprises an organic modifier. 22. The method of embodiment 21, wherein the organic modifier is at a concentration of about 20% (v/v) to about 60% (v/v).
  • composition comprising the at least first and second mRNAs, the mobile phase A and/or the mobile phase B comprise(s) an agent for chelating divalent cations.
  • composition comprises the at least first and second mRNAs encapsulated in the same lipid nanoparticle.
  • composition comprises the at least first and second mRNAs encapsulated in different lipid nanoparticles.
  • composition comprises the at least first and second mRNAs in purified form.
  • step (v) yields a chromatogram providing information about characteristics of the at least first and second mRNAs.
  • the method any one of the preceding embodiments, wherein the at least first and second mRNAs encode homologous proteins. 52. The method of any one of the preceding embodiments, wherein the at least first and second mRNAs encode antigens from at least a first virus and a second virus, optionally wherein the first and second viruses are phylogenetically related to each other.
  • antigens are hemagglutinin (HA) or neuraminidase (NA) proteins.
  • porous particles are made of styrene and/or divinylbenzene.
  • a method for detecting two or more mRNAs in a composition using reverse phase high pressure liquid chromatography comprising:
  • composition comprises the two or more mRNAs encapsulated in the same lipid nanoparticle.
  • composition comprises the two or more mRNAs in purified form.
  • step (v) yields a chromatogram providing information about characteristics of the two or more mRNAs.
  • antigens are hemagglutinin (HA) or neuraminidase (NA) proteins.
  • mRNAs were synthesized as described in published U.S. Application No. US 2018/0258423, which is incorporated herein by reference. Briefly, 4 different mRNAs were synthesized via in vitro transcription (IVT) in separate reactions. Each reaction comprised a circular DNA template, a pool of ribonucleotide triphosphates, an SP6 RNA polymerase and a suitable buffer. The IVT reactions were terminated by the addition of DNase I.
  • IVTT in vitro transcription
  • Each of the DNA templates included an mRNA sequence operationally linked to an SP6 promoter.
  • the coding sequence of each of the 4 mRNAs encoded a different influenza HA antigen. Accordingly, the nucleic acid composition of each coding sequence is different, as can be seen from Table 1 :
  • Example 3 Separation conditions
  • Figure 1 shows an illustrative chromatogram using condition I, which employed HAA as the ion pairing reagent and acetonitrile as the organic modifier.
  • the chromatogram labelled “mixture” was obtained by separating 4 HA mRNAs (HA mRNA 1, HA mRNA 2, HA mRNA 3, and HA mRNA 4) shown in Table 1 using column 4 (see Table 2) and condition I (see Table 3). Under these conditions, HA mRNA 1 and HA mRNA 2 could be resolved as separate peaks (at 14.435 minutes and 14.648 minutes), whereas HA mRNA 3 was visible as a shoulder only (at 15.99 minutes) in a larger peak (at 15.625 minutes) also including HA mRNA 4. Each mRNA was also run separately, as indicated in the figure.
  • FIG. 2 shows an illustrative chromatogram using conditions Ila and lib (see Table 3) to separate the 4 HA mRNAs shown in Table 1 using column 4 (of Table 2).
  • the only difference between these conditions (Ila and lib) was that for condition lib the mixture was pre-heated prior to injection for 10 minutes at 70°C prior to injection to denature the HA mRNAs.
  • the preheating step resulted in longer retention times for each HA mRNA, but did not increase resolution. Under these conditions, only two clearly distinguishable peaks were identified in each of these conditions: in condition Ila, at 18.014 minutes and 19.041 minutes; in condition lib, at 18.352 minutes and 19.486 minutes.
  • condition III resulted in a similar separation pattern of the 4 HA mRNAs (see Table 1) as conditions I, Ila and lib.
  • the chromatogram was obtained using column 4 (see Table 2).
  • condition III HFIP was used as the ion pairing reagent and methanol as the organic modifier. Only two clearly distinguishable peaks were identified (at 5.904 minutes and 9.145 minutes), each with a small shoulder.
  • Example 3 two ion pairing reagents (HAA in water and HFIP in TEA buffer) were tested. These experiments were repeated. The use of TEA-buffered HFIP did not yield a better peak resolution, and therefore subsequent experiments were performed with HAA as the ion paring reagent. Using 100 mM HAA resulted in a better resolution of peaks than using 75 mM HAA.
  • the peaks representing mRNAs 3 and 4 could not be completely separated under the test conditions described in Table 6. However, to determine the presence of all 4 mRNAs in a mixture, a complete separation of the peaks is not required.
  • This Example demonstrates that the separation conditions used in the preceding examples can be adapted for use with a column that has a length greater than 150 mm.
  • the identified conditions were able to separate 4 similarly sized mRNAs (2 being of the same size and two differing by about 50 nucleotides) in a mixture into distinct peaks using RP- HPLC.
  • This Example illustrates that the presence of an ion pairing reagent in mobile phase B is not required to achieve effective separation of similarly sized mRNAs in a mixture. This Example also demonstrates that better resolution can be obtained when the same ion paring reagent is present in both mobile phase A and mobile phase B.
  • mobile phase A and mobile phase B each comprised an ion pairing reagent. It was hypothesized that the presence of an ion pairing reagent in mobile phase B is not required for effective separation. Therefore, experiments were performed in which only mobile phase A comprised an ion pairing reagent. These experiments employed the column listed in Table 5. The flow rate and column temperature were as described in Table 6. The test conditions are shown in Table 7. Table 7
  • Example 2 illustrates the effects of column temperature on the separation of the mRNAs.
  • Mobile phase A comprised 200 mM HAA in water
  • mobile phase B comprised 75% acetonitrile (v/v) and 200 mM HAA in water, as shown in Table 8.
  • This Example illustrates that a narrow gradient at 57-61% of mobile phase B can result in effective separation of similarly sized mRNAs in a mixture.
  • Mobile phase A was composed of 200 mM HAA and 40% (v/v) acetonitrile in water at pH 7.
  • Mobile phase B was composed of 200 mM HAA and 50% (v/v) acetonitrile in water at pH 7.
  • the mobile phase compositions are summarized in Table 9. Providing the organic modifier in both mobile phases provides greater flexibility for adjusting the gradient.
  • Example 10 illustrates that addition of an organic modifier such as acetonitrile to both mobile phase A and mobile phase B can further improve separation by allowing greater flexibility for gradient adjustments. For example, using a gradient of 50%-64% of mobile phase B, further improved peak separation was observed.
  • Example 10 Separation of mRNAs using two organic modifiers
  • This Example illustrates that addition of a second organic modifier to mobile phase B also allows for adjustments of the sample retention time and allows for effective separation of similarly sized mRNAs.
  • Figure 9 shows the separation and detection of each mRNA individually, overlaid with the separation and detection of the three mRNAs from the mixture.
  • the stationary phase was formed by rigid macroporous particles made from aromatic hydrocarbon polymers (polystyrene-divinylbenzene).
  • FIG. 1 IB shows an overlay of the chromatogram shown in Figure 11 A with a previously obtained chromatogram of the same mixture of mRNA using the same column loaded with a stationary phase comprising particles of the same composition with a pore size of 4000 A.
  • the smaller pore size of 1000 A resulted in slightly wider, well-separated peaks.
  • This Example illustrates that the use of a column comprising a plurality of porous particles with a pore size less than 500 A cannot effectively separate similarly sized mRNAs from a mixture using optimized separation conditions identified in preceding examples.
  • a mixture of four mRNAs was encapsulated in liquid nanoparticles.
  • the coding sequence of each of the 4 mRNAs encoded an influenza HA antigen derived from H1-A/H1N1 (A/Wisconsin/588/2019), B Yamagata (B/Phuket/3073/2013), H3-A/H3N2 (A/Darwin/6/2021), and B-Victoria (B/Austria/1359417/2021), respectively.
  • RNAs comprising multiple different RNAs
  • the methods described herein can be used for quality assurance.
  • a standard can be prepared that contains each mRNA at the ratios used for the manufacturing of the LNPs encapsulating the different mRNAs.

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Abstract

La divulgation concerne des procédés de détection d'au moins un premier ARNm et d'un second ARNm, ou d'au moins deux ARNm dans une composition multi-ARNm à l'aide d'une chromatographie en phase liquide sous haute pression en phase inverse (CLHP-PI). Les procédés sont particulièrement appropriés pour analyser des compositions multi-ARNm comprenant des premier et second ARNm ou au moins deux ARNm de longueurs similaires. Les procédés selon la divulgation sont également utiles pour l'évaluation de la qualité et/ou de l'intégrité de chacun desdits premier et second ARNm ou desdits ARNm dans une composition multi-ARNm. De plus, les procédés selon la divulgation peuvent être utilisés pour calculer la proportion de chaque ARNm dans une composition multi-ARNm.
PCT/EP2024/082760 2023-11-17 2024-11-18 Dosages par clhp pour détecter de multiples constructions d'arnm Pending WO2025104351A1 (fr)

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