Potency assay

Abstract

The present disclosure provides a method for quantifying a target protein in a sample making use of differentially isotopically labeled variants of the target protein.

Description

[0001] "POTENCY ASSAY”

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from United States Patent Application No. 63 / 637,419 filed 23 April 2024 entitled “Potency Assay”, the entire contents of which is hereby incorporated by reference.

[0004] SEQUENCE LISTING

[0005] The present application is filed together with a Sequence Listing in electronic format. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD

[0007] The present disclosure relates methods for quantifying protein expression. The present disclosure also relates to methods for determining the potency of a recombinant nucleic acid, for example an RNA or DNA vaccine.

[0008] BACKGROUND

[0009] Vaccines are one of the critical health interventions to prevent infectious diseases. The recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic has seen the unprecedented development of multiple vaccines in a very short space of time, with RNA vaccines used for the first time as part of a global vaccination strategy. In order to raise an appropriate immune response, these vaccines rely on the intracellular expression of the protein antigen(s) of interest in eukaryotic cells.

[0010] RNA vaccines comprise synthetic mRNA molecules that encode for an antigen that will generate an immune response. Synthetic mRNA has a similar structure to endogenous mRNA and typically includes from 5’ to 3’: a 5’ cap, 5’ untranslated region (5’UTR), an open reading frame encoding the antigen, a 3’UTR and a polyA tail. The use of mRNA for therapeutic purposes, including in vaccines, requires large amounts of mRNA to be synthesized and thoroughly characterized for properties such as antigen expression. At this scale, characterization of mRNA requires techniques that are quantitative, robust, accurate, and able to process many samples quickly. Pre-clinical characterization of the approved RNA vaccines against SARS-CoV-2 (e.g. BNT162b1 and BNT162b2) used flow cytometry or immunoblotting with a mAb against the spike- protein to detect protein expression in transfected cells. Similar approaches have also been used for DNA (e.g. plasmid DNA) vaccines. This approach relies on suitable monoclonal antibodies being available to detect the expressed antigen. However, there remains a need for assays that can be used to characterize mRNA and other nucleic acids for therapeutic purposes, and in particular assays which can be used to quantitate the amount of antigen expressed from nucleic acid vaccines.

[0011] SUMMARY

[0012] The present application provides methods for quantitatively determining the protein expression, for example, protein expression from nucleic acid vaccines.

[0013] The present application provides a method for quantification of a target protein in a sample, the method comprising:

[0014] (a) combining

[0015] (i) a first sample of the target protein to be quantified, wherein the first sample comprises a first variant of the target protein, wherein at least one amino acid in each first variant is isotopically labeled, and

[0016] (ii) a second sample comprising a second variant of the target protein, wherein at least one amino acid in the second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, to form a first mixture

[0017] (b) analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein; and

[0018] (c) quantifying the amount of target protein in the first and / or second sample.

[0019] In some examples, there is provided a method for quantification of a target protein in a sample, the method comprising:

[0020] (a) combining

[0021] (i) a first sample of the target protein to be quantified, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled, and

[0022] (ii) a second sample comprising a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in the second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, to form a first mixture (b) analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; and

[0023] (c) normalizing the amount of the target protein relative to the reference protein; and

[0024] (d) quantifying the amount of target protein in the first sample.

[0025] In some examples, analyzing (e.g. step b) comprises subjecting the first mixture to mass spectrometry to form a spectra comprising one or more sets of matched peptide peaks and comparing relative intensities or peak areas of matched peptide peaks in the spectra to determine the relative amount of the first variant.

[0026] In some examples, normalizing (e.g. step c) comprises multiplying the relative amount of the first variant by a normalization factor. In some examples, the normalization factor is determined by dividing the relative amount of the second variant of the reference protein by the relative amount of the first variant of the reference protein. In some examples, the normalization factor is determined by dividing the relative amount of a control peptide by the relative amount of a test peptide.

[0027] In some examples, the mixture is treated with a protease to prior to analysis. In some examples, the protease is selected from the group consisting of trypsin, endoproteinase GluC, enterokinase light chain, Factor Xa, furin, chymotrypsin, Lys-C, Lys-N, elastase, Asp-N and endoproteinase ArgC.

[0028] In some examples, a known amount of isotopically labeled reference peptide is added to the first mixture prior to analysis.

[0029] In some examples, the reference peptide is a beta actin peptide. In some examples, the beta actin peptide comprises the sequence GYSFTTTAER (SEQ ID NO: 1 ). In some examples, the beta actin peptide comprises the sequence GYSFTTTA(+4)ER(10) (SEQ ID NO: 1 ).

[0030] In some examples, the absolute amount of reference protein in the mixture is determined by comparison to the known amount of the reference peptide.

[0031] In some examples, the isotopically labeled amino acids are labeled with one or more isotopes selected from2D,15N and13C. In some examples, the isotopically labeled amino acids are selected from the group consisting of isoleucine, glutamic acid, aspartic acid, glycine, arginine, lysine and combinations thereof. In some examples, the isotopically labeled amino acids are selected from the group consisting of arginine, lysine and combinations thereof. In some examples, the first sample is isotopically labeled with lysine (K(+4)) and arginine (R(+6)) and the second sample is isotopically labeled with heavy lysine (K(+8)) and heavy arginine (R(+10)). In some examples, the reference protein is an endogenous reference protein. In some examples, the reference protein is a housekeeping protein. In some examples, the reference protein is beta actin.

[0032] In some examples, the reference protein is an exogenous reference protein. In some examples, the exogenous reference protein is encoded by a recombinant nucleic acid. In some examples, the exogenous reference protein is encoded by a recombinant RNA. In some examples, the RNA is a sa-mRNA. In some examples, the RNA forms part of an RNA-LNP. More than one reference protein may be used.

[0033] In some examples, the method further comprises the step of purifying the target protein and the reference protein prior to analysis.

[0034] In some examples, the method further comprises the steps of:

[0035] (e) combining the first and / or second sample with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture;

[0036] (f) analyzing the second mixture to determine relative amounts of the first and / or second variant and third variant of the target protein or peptide thereof;

[0037] (g) determining the absolute amount or concentration of the first and / or second variant in the second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof.

[0038] In some examples, the second mixture is treated with a protease before analysis.

[0039] In some examples, the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled peptide. In some examples, the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled recombinant protein.

[0040] In some examples, a standard curve is used for determining the absolute amount or concentration of the second variant in the second sample. In some examples, the standard curve is produced by combining the second sample with a serial dilution of a known amount of the third, unlabeled variant of the target protein or a peptide thereof

[0041] In some examples, one-point quantification is used for determining the absolute amount or concentration of the second variant in the second sample.

[0042] In some examples, the first sample comprises cells treated with a RNA-LNP.

[0043] In some examples, the second sample comprises cells treated with a RNA-LNP.

[0044] In some examples, the first and second samples comprise cells treated with different amounts of RNA-LNP, different batches of RNA-LNP or different RNA-LNP. In some examples, the RNA-LNP comprise test RNA-LNP, control RNA-LNP or both.

[0045] In some examples, the method is employed to determine the potency of a RNA-

[0046] LNP. In some examples, the target protein is selected from the group consisting of a recombinant protein, a biotherapeutic protein, an antibody, a fusion protein, and a glycosylated protein. In some examples, the target protein is recombinant protein produced from a nucleic acid vaccine.

[0047] The present application also provides an isotopically labeled peptide comprising the sequence GYSFTTTA(+4)ER(+10) (SEQ ID NO: 1 ).

[0048] The present application also provides a method for normalizing an amount of a target protein in a sample based on cell count, the method comprising:

[0049] (a) combining

[0050] (i) a sample comprising a first variant of a reference protein, wherein at least one amino acid in the first variant is isotopically labeled; and

[0051] (ii) a sample comprising a second variant of a reference protein, wherein the least one amino acid is differentially isotopically labeled such that the first variant and second variant have a different molecular weight; to form a mixture;

[0052] (b) analyzing the mixture to determine relative amounts of the first variant and second variant; and

[0053] (c) dividing the amount of the second variant by the amount of first variant to determine a normalization factor, wherein the normalization factor is used for normalizing an amount of a target protein in a sample based on cell count.

[0054] The present application also provides a method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a first sample, wherein the first sample comprises a first variant of the target protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein; optionally normalizing the amount of the target protein relative to a reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

[0055] In some examples, the reference protein is an endogenous reference protein. In some examples, the reference protein is an exogenous reference protein.

[0056] In some examples, the present application also provides a method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a first sample, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; normalizing the amount of the target protein relative to the reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

[0057] In some examples, the reference protein is an endogenous reference protein. In some examples, the reference protein is an exogenous reference protein.

[0058] In some examples, the present application also provides a method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic acid and a control sample of recombinant nucleic acid encoding a reference protein in media comprising an isotopically labeled amino acid to form a first sample, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid and a control sample of recombinant nucleic acid encoding a reference protein in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; normalizing the amount of the target protein relative to the reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

[0059] In some examples, analyzing comprises subjecting the first mixture to mass spectrometry to form a spectra comprising one or more sets of matched peptide peaks and comparing relative intensities or peak areas of matched peptide peaks in the spectra to determine the relative amount of the first variant.

[0060] In some examples, normalizing comprises multiplying the relative amount of the first variant by a normalization factor, wherein the normalization factor is determined by dividing the relative amount of the second variant of the reference protein by the relative amount of the first variant of the reference protein.

[0061] In some examples, the mixture is treated with a protease to prior to analysis. In some examples, the protease is selected from the group consisting of trypsin, endoproteinase GluC, enterokinase light chain, Factor Xa, furin, chymotrypsin, Lys-C, Lys-N, elastase, Asp-N and endoproteinase ArgC.

[0062] In some examples, the isotopically labeled amino acids are labeled with one or more isotopes selected from2D,15N and13C. In some examples, the isotopically labeled amino acids are selected from the group consisting of arginine, lysine and combinations thereof. In some examples, the first sample is isotopically labeled with lysine (K(+4)) and arginine (R(+6)) and the second sample is isotopical ly labeled with heavy lysine (K(+8)) and heavy arginine (R(+10)).

[0063] In some examples, the reference protein is an endogenous reference protein. In some examples, the reference protein is a housekeeping protein. In some examples, the reference protein is beta actin.

[0064] In some examples, the reference protein is an exogenous reference protein. In some examples, the exogenous reference protein is encoded by a recombinant nucleic acid. In some examples, the exogenous reference protein is encoded by a recombinant RNA. In some examples, the RNA is a sa-mRNA. In some examples, the RNA forms part of an RNA-LNP. More than one reference protein may be used.

[0065] In some examples, the first and the second population of cells are cultured in unlabeled media for about 18 to 30 hours prior to transfection, or about 24 hours prior to transfection.

[0066] In some examples, the first and the second population of cells are cultured in labeled media for about 18 to 30 hours prior to transfection, or about 24 hours prior to transfection.

[0067] In some examples, the first and second population of cells are transfected for at least about 12 hours, at least about 18 hours, or about 24 hours.

[0068] The present application also provides a method comprising the steps of obtaining a population of cells that has been treated with the self-amplifying mRNA encoding one or more target proteins; and quantifying the amount of the one or more target proteins expressed from the RNA.

[0069] The present application also provides a method comprising the steps of obtaining a population of cells that has been treated with the self-amplifying mRNA encoding one or more target proteins; and quantifying the potency of the self-amplifying mRNA encoding one or more target proteins.

[0070] BRIEF DESCRIPTION OF DRAWINGS

[0071] The drawings are for illustration purposes only and are in no way limiting.

[0072] Figure 1 illustrates an example method of quantifying a target protein as described herein. In this example, beta actin is used as a reference protein. In this example, the simple direct normalization strategy is used Lot A, B, C, D and E may represent different amounts of RNA or different lots of RNA.

[0073] Figure 2 illustrates an example method of quantifying a target protein as described herein. In this example, beta actin is used as a reference protein, a super heavy beat actin peptide may be added prior to MS and different normalization strategies may be used. Lot A, B, C, D and E may represent different amounts of RNA or different lots of RNA. In this figure, LHC is the amount of light actin peptide or protein in the Heavy cell control; HHC is the amount of heavy actin peptide or protein in the heavy cell control; LM is the amount of light actin peptide or protein in the medium cell sample; MM is the amount of medium actin peptide or protein in the medium cell sample; L is the amount of light actin peptide or protein in the heavy cell sample; HMis the amount of heavy actin peptide or protein in the medium cell sample; LMH is the measured amount of light actin peptide or protein in the combined medium and heavy cell sample; and HMH is the measured amount of heavy actin peptide or protein in the combined medium and heavy cell sample.

[0074] Figure 3 illustrates use of the methods described herein for quantifying the potency of a RNA-LNP vaccine. In this example, unlabeled target protein is used for quantifying the amount of expressed target protein.

[0075] Figure 4 exemplifies one-point quantification.

[0076] Figure 5 exemplifies antigen quantitation using an external calibration curve.

[0077] Figure 6 illustrates example MRM peptide settings.

[0078] Figure 7 illustrates the dose-dependent response of HA and NA expression using beta-actin normalization. Cells were dosed with varying amounts of RNA-LNP encoding HA and NA. A medium / heavy antigen ratio (red) was normalized using either simple direct normalization (green) or total beta-actin normalization (blue). Both normalization strategies eliminated sample variability observed with different doses.

[0079] Figure 8 illustrates quantification of medium HA B / Ya protein using a light and heavy standard curve. The two HA B / Yamagata peptides GVL and SYF were quantified using a standard curve. Samples cultured in medium SILAC media were spiked with cells cultured in heavy SILAC media. The same mixture of heavy cells was spiked into a light peptide serial dilution to generate a standard curve for quantification.

[0080] Figure 9 illustrates quantification of antigens using five exemplified methods. For this analysis three peptides (EQL, VNS and TLD) were monitored using MS. With the first method (A), a standard curve was generated using heavy cells that had been dosed with LNP and are spiked with light peptides of varying concentrations. Heavy cells were then spiked into the target sample as an internal standard for quantification. With the second method (B), a standard curve was generated using recombinant proteins of a single concentration spiked into manufactured heavy peptides of varying concentrations. The recombinant proteins were then added to the target sample as an internal standard for quantification. The third method (B) used single point quantification with recombinant proteins, where light recombinant proteins of a known concentration were spiked into the sample and underwent sample processing along with the target medium and heavy labeled antigens. The relative peak areas and the concentration of the recombinant protein were then used to calculate the corresponding medium and heavy peptide concentrations. The fourth method (C) used a standard curve generated using manufactured light peptides of one concentration (which were also spiked into the sample as an internal standard) and manufactured heavy peptides of varying concentrations. The fifth method (C) used single point quantification with manufactured light peptides of a known amount spiked into the sample.

[0081] Figure 10 illustrates characterization of a multivalent LNP mixture using the exemplified methods. A quadrivalent LNP mixture and the four individual LNP samples were tested using the method described herein. Peptides for each HA subtype were present in the corresponding LNP and the quadrivalent QIV mixture. Four peptides monitored using MS are shown for clarity. The peptide EQL is found in the H1 subtype, STQ in the H3 subtype, GVL in B / Ya, and GILL in B / Vic.

[0082] Figure 11 illustrates characterization of a multivalent LNP mixture using the exemplified methods. Cells were dosed with LNP using either Covid LNP #1 , Covid LNP #2, Influenza Quadrivalent (QIV), or a mixture of both the QIV and Covid LNPs. Each of the HA and NA subtypes, as well as S and NP proteins, were able to be measured with cells that are treated with the respective LNPs, and also when the cells were treated with a combination of drug products.

[0083] Figure 12 illustrates use of labeled beta actin as an indicator of cell health following LNP dosing. Cells were dosed with LNP at various amounts (ng) and the

[0084] concentration of beta actin was quantitated using the methods described herein. Top panel: 0.5M cells. Bottom panel: 1 M cells.

[0085] Figure 13 illustrates peptide selection and calibration curve construction for a potency assay.

[0086] Figure 14 illustrates trypsinization optimization for a potency assay.

[0087] Figure 15 illustrates the correlation between cell number and GAPDH peptide intensity.

[0088] Figure 16 illustrates a potency assay for H5. For this analysis two peptides (IQIIPK and LVLATGLR) were monitored using MS.

[0089] Figure 17 illustrates quantification of antigens with monovalent RNA constructs (H1 N1 , H3N2). For this analysis two or three peptides were monitored using MS.

[0090] Figure 18 illustrates quantification of antigens with monovalent RNA constructs (Byam HA, Bvic HA, NA). For this analysis one or three peptides were monitored using MS.

[0091] Figure 19 illustrates quantification of antigens with monovalent RNA constructs for lipid tests (H1 N1 , H3N2). For this analysis two or three peptides were monitored using MS.

[0092] Figure 20 illustrates quantification of antigens with monovalent RNA constructs for lipid tests (Byam HA, Bvic HA, NA). For this analysis two or three peptides were monitored using MS.

[0093] Figure 21 illustrates quantification of antigens with NS1 modified H5 RNAs.

[0094] Figure 22 illustrates quantification of antigens with 3’ and 5’ UTR modified H1 RNAs. Two different cell lines (HEK 293 and C2C12) were tested.

[0095] Figure 23 illustrates quantification of HA / NA antigens for quadrivalent RNA constructs. Two different cell lines (HEK 293 and BHK 21 ) were tested.

[0096] Figure 24 illustrates comparison of H1 / N1 antigens for monovalent and quadrivalent RNA constructs. Two different cell lines (HEK 293 and BHK 21) were tested.

[0097] Figure 25 illustrates comparison of H3 / N2 antigens for monovalent and quadrivalent RNA constructs. Two different cell lines (HEK 293 and BHK 21) were tested.

[0098] Figure 26 illustrates comparison of Bvic / Byam HA / NA antigens for monovalent and quadrivalent RNA constructs. Two different cell lines (HEK 293 and BHK 21) were tested.

[0099] Figure 27 illustrates ELISA titers for H5 stability tests.

[0100] Figure 28 illustrates use of the exemplified methods for H5 stability tests. Figure 29 illustrates the correlation between the results of the potency assay and serology testing.

[0101] Figure 30 illustrates an example method of quantifying a target protein as described herein using the disclosed in-well cell lysis method.

[0102] KEY TO SEQUENCE LISTING

[0103] DETAILED DESCRIPTION

[0104] General

[0105] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0106] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0107] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0108] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0109] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0110] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, biochemistry, mass spectrometry, analytical chemistry and separation science).

[0111] Unless otherwise indicated, the molecular biology, chemistry, biochemistry, mass spectrometry, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0112] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0113] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0114] As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Similarly, the term “based on” shall be taken to indicate that a specified integer may be developed or used from a particular source albeit not necessarily directly from that source.

[0115] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In one example, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0116] Selected Definitions

[0117] As used herein, the term "nucleotide” refers a nucleoside in a phosphorylated form (a phosphate ester of a nucleoside), as a monomer unit or within a polynucleotide polymer. "Nucleotide 5 '-triphosphate" refers to a nucleotide with a triphosphate ester group at the 5' position, sometimes denoted as "NTP", or "dNTP" and "ddNTP" to particularly point out the structural features of the ribose sugar. The triphosphate ester group may include sulfur substitutions for the various oxygen moieties, e.g., alpha-thio- nucleotide 5'- triphosphates. Nucleotides can exist in the mono-, di-, or triphosphorylated forms.

[0118] As used interchangeably herein, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" or "oligonucleotide" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. The nucleotides may be genomic, synthetic or semi-synthetic in origin. Unless otherwise stated, the terms encompass nucleic acid-like structures with synthetic backbones, as well as amplification products. As will be appreciated by one skilled in the art, the length of these polymers (i.e., the number of nucleotides it contains) can vary widely, often depending on their intended function or use. Polynucleotides can be linear, branched linear, or circular molecules. Polynucleotides also have associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+, Na+and the like. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may be composed of internucleotide nucleobase and sugar analogs.

[0119] As used herein, the term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions. As used herein, the term protein includes peptides. The term “peptide” is intended to include compounds composed of amino acid residues linked by amide bonds. A peptide may be natural or unnatural, ribosome encoded or synthetically derived. Typically, a peptide will consist of between 2 and 200 amino acids. For example, the peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within said range(s).

[0120] As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant means” also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using a primer of the invention.

[0121] As used herein "expression" of a polynucleotide or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, "expression" also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene product."

[0122] As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining one or more recombinant nucleic acids within a delivery vehicle, such as a nanoparticle.

[0123] As used herein, the term “analyzing” refers to a process for determining a property of an analyte. Analyzing can determine, for example, physical properties of analytes, such as mass, mass to charge ratio, concentration, absolute abundance, relative abundance, or atomic or substituent composition. In the context of proteomic analysis, the term analyzing can refer to determining the composition (e.g., sequence) and / or abundance of a protein or peptide in a sample.

[0124] As used herein, “isotopically labeled” refer to compounds (e.g., such as isotopically labeled amino acids, isotopically labeled standards, isotopically labeled analyte, isotopically labeled variant, and / or isotopically labeled peptide or proteins) having one or more isotopic labels, such as one or more heavy stable isotopes. An “isotopic label” refers to one or more heavy stable isotopes introduced to a compound, such as such as isotopically labeled amino acids, isotopically labeled standards, isotopically labeled analyte, isotopically labeled variant, and / or isotopically labeled peptide or proteins, such that the compound generates a signal when analyzed using mass spectrometry that can be distinguished from signals generated from other compounds, for example, a signal that can be distinguished from other isotopologues on the basis of mass-to-charge ratio. “Isotopically-heavy” refers to a compound or fragments / moieties thereof having one or more high mass, or heavy isotopes (e.g., stable heavy isotopes such as13C,15N,2H (D),170,180,33S,34S,37CI,81Br,29Si, and30Si.).

[0125] As used herein, the term “mass spectrometry” (MS) refers to an analytical technique for the determination of the elemental composition, mass to charge ratio, absolute abundance and / or relative abundance of an analyte. Mass spectrometric techniques are useful for elucidating the composition and / or abundance of analytes, such as proteins, peptides and other chemical compounds. Mass spectrometry includes processes comprising ionizing analytes to generate charged species or species fragments, fragmentation of charged species or species fragments, such as product ions, and measurement of mass-to-charge ratios of charged species or species fragments, optionally including additional processes of isolation on the basis of mass to charge ratio, additional fragmentation processing, charge transfer processes, etc. Conducting a mass spectrometric analysis of an analyte results in the generation of mass spectrometry data for example, comprising the mass-to-charge ratios and corresponding intensity data for the analyte and / or analyte fragments. Mass spectrometry data corresponding to analyte ion and analyte ion fragments is commonly provided as intensities of as a function of mass-to-charge (m / z) units representing the mass-to-charge ratios of the analyte ions and / or analyte ion fragments. Mass spectrometry commonly allows intensities corresponding to difference analytes to be resolved in terms of different mass to charge ratios. In tandem mass spectrometry (also referred to as MS / MS or MS2), multiple sequences of mass spectrometry analysis are performed. For example, samples containing a mixture of proteins and peptides can be ionized and the resulting precursor ions separated according to their mass-to-charge ratio. Selected precursor ions can then be fragmented and further analyzed according to the mass-to-charge ratio of the fragments. A mass spectrum (plural spectra) is a plot of the ion signal as a function of the mass-to-charge ratio. Many MS techniques are known in the arts. For more information, see the MS Primer (2015) available from Waters Corporation, Milford MA USA. See also, Basiri et al, Bioanalysis 1525-1542 (2014).

[0126] As used herein, the term “mass spectrometer” refers to a device which generates ions from a sample, separates the ions according to mass to charge ratio, and detects ions, such as product ions derived from isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and / or isotopically labeled peptide or proteins. Mass spectrometers include single stage and multistage mass spectrometers. Multistage mass spectrometers include tandem mass spectrometers which fragment the mass-separated ions and separate the product ions by mass once.

[0127] As used herein, the terms “isolation” or an “isolation window” refers to a range of ions, such as precursor ions that is selectively separated and fragmented, manipulated or isolated.

[0128] As used herein, the term “variant” refers to a particular molecule, compound, ion, anion, atom, electron or proton. Variants include isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and / or isotopically labeled peptide or proteins.

[0129] As used herein, the term “mass-to-charge ratio” refers to the ratio of the mass of a species to the charge state of a species. The term “m / z unit” refers to a measure of the mass to charge ratio. The Thomson unit (abbreviated as Th) is an example of an m / z unit and is defined as the absolute value of the ratio of the mass of an ion (in Daltons) to the charge of the ion (with respect to the elemental charge).

[0130] As used herein, “LC” is liquid chromatography, a technique used to separate a sample into its individual parts. This separation occurs based on the interactions of the sample with the mobile and stationary phases. Many LC techniques are known in the arts. For more information, see the Beginners Guide to UPLC (2015) and the HPLC Primer (2015), both available from Waters Corporation, Milford MA USA.

[0131] As used herein, “transfection” refers to the introduction of a nucleic acid, e.g., mRNA, into a cell. In some examples of the methods described herein, cells are transfected with a recombinant nucleic acid encoding a target protein. In some examples of the methods described herein, cells are transfected with an LNP sample encapsulating an mRNA. In some examples, transfecting a population of cells with a recombinant nucleic acid in media comprising isotopically labeled amino acids comprises culturing the population of cells in media comprising isotopically labeled amino acids and the recombinant nucleic acids.

[0132] As used herein, each of “transfection duration”, “duration of transfection”, or “transfection time” refers to the amount of time cells are incubated after the recombinant nucleic acid (e.g. RNA-LNP) is added to the cells seeded in the wells. In one embodiment, transfection time or transfection duration is 48 hours or less. In an embodiment of the present invention, the transfection time is 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6 or 5 hours or less. In another embodiment, the transfection time is about 24 hours. In one embodiment, the transfection time is from 18 hours to 30 hours.

[0133] The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C20 for straight chain, C3-C20 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. In some examples, alkyl comprises straight-chain alkyl groups, for example C1-C20 straight-chain alkyl groups.

[0134] Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylammocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" or an "aralkyl" moiety is an alkyl substituted with an aryl (e.g., phenyhnethyl (benzyl)). The term "alkyl" also includes the side chains of natural and unnatural amino acids. Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbon atoms. In some examples, from one to six carbon atoms. In some examples, from one to four carbon atoms.

[0135] A method for quantifying a protein

[0136] The present application provides methods for quantifying the amount and / or concentration of one or more proteins in one or more samples. The application discloses methods for relatively quantifying the amount and / or concentration of one or more proteins of interest in two or more different samples containing the proteins of interest and methods for absolutely quantifying the amount and / or concentration of one or more proteins of interest in one or more samples.

[0137] Accordingly, the present disclosure provides a method for quantification of a target protein in a first sample, the method comprising:

[0138] (a) combining

[0139] (i) a first sample of the target protein to be quantified, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled, and

[0140] (ii) a second sample comprising a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in the second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, to form a first mixture

[0141] (b) analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein;

[0142] (c) normalizing the amount of the target protein relative to the reference protein; and

[0143] (d) quantifying the amount of target protein in the first sample.

[0144] The reference protein may be an endogenous reference protein or an exogenous reference protein. In some examples, the exogenous reference protein is expressed from an exogenous nucleic acid, for example a control RNA-LNP dosed at the same time as a test RNA-LNP.

[0145] While the method is defined herein by reference to two samples (i.e. a first sample and a second sample), it should be appreciated that further samples (i.e. a third or a fourth sample and so on) may be included step (a) provided the variants from additional samples are differentially isotopically labeled compared to the other variants. For example, there is also provided a method for quantification of a target protein in two or more samples, the method comprising:

[0146] (a) combining

[0147] (i) a first sample of the target protein to be quantified, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled,

[0148] (ii) a second sample comprising a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in the second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, and

[0149] (iii) a third sample comprising a third variant of the target protein and a third variant of the reference protein, wherein at least one amino acid in the third variant is differentially isotopically labeled such that the first variant, second variant and third variant have a different molecular weight, to form a first mixture (b) analyzing the first mixture to determine relative amounts of the first variant, second variant and third variant of the target protein and reference protein;

[0150] (c) normalizing the amount of the target protein relative to the reference protein; and

[0151] (d) quantifying the amount of target protein in two or more of the samples.

[0152] The samples (e.g., first and second samples) each contain or are believed to contain an amount of the same protein (the target protein). The target protein in the first sample (referred to as the first variant of the target protein) is isotopically-labeled such that at least one amino acid in the first variant or a fragment thereof (e.g. a peptide) is isotopically labeled. This results in the first variant of the target protein or a fragment thereof having the same amino acid sequence but a different molecular weight compared to the unlabeled variant or fragment thereof. The target protein in the second sample (referred to as the second variant of the target protein) is isotopically-labeled such that at least one amino acid in the second variant or a fragment thereof (e.g. peptide) is differentially isotopically labeled. This results in the second variant of the target protein or a fragment thereof having the same amino acid sequence but a different molecular weight compared to the unlabeled variant and to the first variant. In other words, when two samples are used, the target protein from the first sample and the target protein from the second sample are differentially isotopically-labeled such that the target protein from the first sample has a different molecular weight than the target protein from the second sample but has the same amino acid sequence as the target protein from the second sample. Both the target protein from the first sample and the target protein from the second sample have the same amino acid sequence but a different molecular weight than the unlabeled variant of the target protein. Similarly, when two samples are used, the reference protein from the first sample and the reference protein from the second sample are differentially isotopically-labeled such that the reference protein from the first sample has a different molecular weight than the reference protein from the second sample but has the same amino acid sequence as the reference protein from the second sample. Both the reference protein from the first sample and the reference protein from the second sample have the same amino acid sequence but a different molecular weight than the unlabeled variant of the reference protein. By labeling each sample with different stable isotope label, identical proteins in separate samples can be distinguished, for example using mass spectrometry.

[0153] As used herein, an “unlabeled variant” of a protein or peptide is not isotopically labeled (other than at the levels that occur in nature). Typically, the methods described herein comprise the step of combining the samples containing the different isotopically labeled variants prior to analysis, for example analysis using mass spectrometry, thereby ensuring each sample undergoes similar sample preparation, purification, ionization, fragmentation and / or detection conditions. Combining the samples prior to preparation for MS analysis is thought to help reduce the level of quantitation bias from processing errors. In some examples, the different isotopically labeled variants for the plurality of samples are analyzed concurrently, for example, via purification steps and mass spectrometric analysis steps of a combination of a plurality of samples. By way of illustration, using a two sample experiment, the first sample and the second sample are combined to form a mixture. In some examples, the first sample and the second sample are combined in equal amounts to form a mixture. In some examples, each sample comprises approximately the same amount of total protein. In some examples, each sample comprises approximately the same concentration of total protein. In some examples, each sample was derived from approximately the same number of cells. In some examples, each sample was derived from approximately the same concentration of cells. In some examples, each sample was derived from the same volume of culture media. In some examples, each sample was derived from the same volume of cell lysate.

[0154] In some examples, the two or more samples may be processed prior to combining although this is not preferred. In some examples, the two or more samples may be combined to form a first mixture prior to processing. In some examples, the first mixture may be processed prior to analysis. Processing includes, but is not limited to, cell lysis, isolation of subcellular compartments, fractionation, concentration, purification, protein digestion, nucleic acid digestion and the like. In some examples, the differentially isotopically-labeled variants are isolated using a substrate having a plurality of isolation agents (e.g., antibodies) immobilized thereon. Each of the isolation agents has a specific affinity or binding specificity for the target protein. Typically, each substrate have only one type of isolation agent immobilized thereon (i.e., it is preferred that all of the isolation agents on a substrate have the same affinity or specificity for the same peptide or protein). However, in some embodiments, isolation agents with an affinity or specificity for different peptides or proteins may be present on the same substrate. When the isolation agents are antibodies, each of the antibodies has a paratope specific to an epitope of the peptide or protein. In some examples, the first and the second variants are not isolated prior to combining to form a mixture.

[0155] In some examples, the samples comprise cells and the cells from each sample are combined prior to cell lysis. In some examples, the mixture comprises intact cells from each sample that are then lysed using techniques known to the person skilled in the art. In some examples, the samples comprise cells and the cells from each sample are lysed and optionally purified prior to combining to form the first mixture. Suitable techniques for lysing cells include, but are not limited to, sonication, freeze thaw, heating vortexing with glass beads, homogenization, detergents and combinations thereof. In some examples, the samples comprise cells and the cells from each sample are combined prior to treatment of the sample with a detergent, such as Rapigest, and optionally heating. Treatment of the sample with a detergent is thought to aid cell lysis and / or protein digestion. Rapigest (also known as sodium 3-[(2-methyl-2-undecyl-1 ,3- dioxolan-4-yl)methoxy]-1 -propanesulfonate) is an acid-cleavable anionic detergent. It is thought to negatively charge the surface of proteins, helping expose important cleavage sites for enzymes such as trypsin, Lys-C, Asp-N and Glu-C. In some examples, the sample is heated to at least 70°C, at least 80°C, at least 90°C, at least 95°C or at about 100°C or more. In some examples, the sample is heated to about 100°C.

[0156] One or more of the samples and / or mixtures containing the differentially isotopically-labeled proteins are typically derived from samples containing proteins. The proteins in such samples are digested to form peptides for analysis. The proteins may be digested by any method, such as, for example, by chemical or enzymatic digestion. In a preferred embodiment, a digestion method is used that will not cleave a continuous epitope of the peptides to be analyzed. Reagents that may be used for chemical or enzymatic digestion of proteins include, but are not limited to proteases such as trypsin, pepsin, protease Lys- C, protease Glu-C, protease V8, protease Arg-C, as well as cyanogen bromide (CNBr). In some examples, the samples or mixture are treated with one or more proteases in order to produce a peptide or peptides for analysis. As described herein, the target protein and reference protein in the first sample and the target protein and reference protein in the second sample are differentially isotopically- labeled. When such differentially isotopically-labeled proteins are digested, one or more of the resulting peptides are also differentially isotopically-labeled. If the differentially isotopically-labeled proteins comprise isotopically labeled lysine and arginine and a protease such as trypsin is chosen which cleaves after lysine and arginine residues, the majority of the peptides that result will contain an isotopically labeled residue. If the differentially isotopically-labeled proteins comprise isotopically labeled lysine and arginine and a protease such as Lys-C or Lys-N is chosen which cleaves N- or C- terminal of lysine residues, the majority of the peptides that result will contain isotopically labeled lysine residues. The differentially isotopically-labeled peptides generated by digestion may be analyzed, for example, by MS to determine the relative abundances, which allows the determination of the relative and / or absolute amounts and / or concentrations of the peptide in the first sample and / or second sample. This then allows the determination of the relative and / or absolute amounts and / or concentrations of the target protein in the first sample and / or second sample

[0157] After combining the first sample and the second sample to form a mixture, the mixture is then analyzed to determine relative amounts of the first variant and second variant of the target protein. The mixture is also analyzed to determine the relative amounts of the first variant and second variant reference protein. In some examples, analysis comprises mass spectrometry, although any method suitable for separating and / or determining relative amounts of isotopically labeled variants may be used. In some examples, the differentially isotopically-labeled variants are analyzed by high resolution mass spectrometry (HRMS) to determine the relative amounts of the peptide in the first sample and the second sample. The present methods are compatible with a wide range of mass spectrometry techniques providing useful resolving powers, including techniques designed to probe the abundances of analytes in a plurality of samples, such as protein and peptide containing samples. Any suitable ionization technique may be used, including Electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). A variety of mass spectrometry systems can be employed in the methods described. Mass analyzers with high mass accuracy, high sensitivity and high resolution include, but are not limited to, matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometers, electrospray ionization time-of-flight (ESI-TOF) mass spectrometers, Fourier transform ion cyclotron mass analyzers (FT-ICR-MS), and ORBITRAP™ analyzer instruments. Other modes of MS include ion trap and triple quadrupole mass spectrometers. In ion trap MS, analytes are ionized by electrospray ionization or MALDI and then moved into an ion trap. Trapped ions can then be separately analyzed by MS upon selective release from the ion trap. Ion traps can also be combined with the other types of mass spectrometers described above. In one example, a triple-quadrupole mass spectrometer is used. In some examples, MALDI-MS is used. Any suitable MALDI matrix may be used for the MALDI- MS including, but not limited to alpha-cyano-4-hydroxycinnamic acid (HCCA) and 2,5- dihydrobenzoic acid (DHB). In some examples, ESI-MS is used. In some examples, EIMS is used. In some examples, the differentially isotopically-labeled peptides are analyzed by tandem MS (also referred to as MS / MS) to determine the relative amounts of the peptide in the first sample and the second sample.

[0158] In some examples, the step of analyzing comprises: generating one or more product ions for each of the isotopically labeled variants, and measuring mass-to-charge ratios for at least a portion of the product ions using mass spectrometry. In some examples, the step of analyzing the isotopically labeled variants for each sample is carried out using a quadrupole ion trap, Fourier transform ion cyclotron resonance ion trap, a linear quadrupole ion trap, an orbitrap ion trap, a quadrupole mass analyzer or a time of flight mass analyzer.

[0159] Mass-spectrometric analysis of the biomolecules in a sample can be targeted, and can include SIM-scans (selected ion monitoring) to increase sensitivity, e.g., 10 to 100 fold or more. In the SIM-scan only a specific mass window is selectively accumulated, therefore increasing the sensitivity. For example, the SIM scan can scan a mass range which includes the predicted mass of the differentially labeled peptide. With the SIM scan, it is then possible to achieve accurate quantification even for biomolecules that are very low in the sample. Other fragmentation methods such as ETD, ECT and EAD may be utilized with or without employing an ion mobility gas separation module.

[0160] It will be appreciated by those skilled in the art, that in the same way, targeted analysis by Selected / Multiple Reaction Monitoring (SRM / MRM) techniques known in the art (see for example, Picotti, P., Bodenmiller, B., Mueller, L. N., Domon, B., and Aebersold, R. (2009): Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795-806) can be used to increase the sensitivity of the measurement. Instead of monitoring a narrow mass range, specific transitions from the precursor to specific fragments may be monitored.

[0161] The mass spectrometry data can be acquired and processed using any suitable software, for example the vendor’s software programs, such as Chromeleon and TraceFinder.

[0162] In some examples, the method described herein further comprise purifying proteins or peptides of the samples prior to or during analysis (e.g. prior to mass spectrometry), for example, via liquid phase chromatography (e.g., HPLC), gas phase chromatography, and / or capillary electrophoresis. In some examples, the methods comprise fractionating proteins or peptides of the samples prior to or during the step of analyzing the isotopically labeled variants for each sample. In some examples, the methods comprise fractionating proteins or peptides of the samples by LC, gas phase chromatography, and / or capillary electrophoresis. In some examples, the methods comprise fractionating proteins or peptides of the samples by LC.

[0163] In some examples, the differentially isotopically-labeled proteins are analyzed by LC-MS (liquid chromatography-mass spectrometry) to determine the relative amounts of the peptide in the first sample and the second sample. For high resolution separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS, which utilizes capillary reverse phase chromatography as the separation method, can be used. Other separation systems known to the person skilled in the art may be employed to separate the product of interest prior to MS analysis. Systems that can be linked to MS include, but are not limited to, capillary electrophoresis and gas chromatography.

[0164] Using Reverse Phase - High Performance Liquid Chromatography (RP-HPLC) as an example, under standard conditions, labeled and unlabeled peptides have nearly identical retention times and, therefore, migrate together. While not resolvable by chromatography, isotopically labeled and unlabeled peptides are distinguishable by MS detection, yielding differences in Daltons (Da) based on the number of labeled atoms in the particular peptide. Accordingly, when the differentially isotopically labeled samples are combined to form a mixture, each differentially labeled peptide derived from the target protein and reference protein will appear as a multiplet in a mass spectrum. These multiplets are also referred to as matched peptide peaks. In the case of an exogenous reference peptide (e.g. from a control RNA-LNP dosed at the same time as the test RNA-LNP), each peptide will typically appear as a medium labeled peptide and a heavy labeled peptide (both representing the reference protein expressed after treatment in the medium and heavy samples). In the case of an endogenous reference peptide, each peptide will typically appear as an unlabeled peptide (for example, representing the reference protein expressed before the sample was transferred to the labeled media) and a medium labeled peptide and a heavy labeled peptide (both representing the reference protein expressed after treatment in the medium and heavy samples). Without wishing to be bound by theory it is thought that the unlabeled peptide can be used to control for the number of cells in the sample when treated and / or as an internal standard for quantitation of expressed antigen peptides. It is also thought that the labeled peptide can be used to control for the health of the cells following treatment. In the case of the target protein which is only expressed after treatment of the sample (e.g. with an RNA encapsulated in an LNP), each peptide will typically appear as a medium labeled peptide and heavy labeled peptide.

[0165] In some examples, the one or more of the methods described herein may be used to determine the relative (or absolute) amounts or concentrations of the protein in the protein-containing sample(s). As would be understood by the skilled addressee, the relative or absolute amounts or concentrations of the measured peptide(s) will directly reflect the relative or absolute amounts or concentrations of the corresponding protein in the protein-containing sample, subject, of course, to any changes in volume or concentration between the protein-containing sample and the peptide-containing sample. The relative amounts or concentrations of the peptide in each sample may be determined by comparing the abundance (i.e., the mass spectrometric signal intensity and / or area) of the ions corresponding to the differentially isotopically-labeled peptides. In some examples, the relative abundance of the proteins is measured based on the intensities of the medium and heavy peptides. In some examples, the relative abundance of the proteins is measured based on the peak area of the medium and heavy peptides. In one example, the determining the relative amounts or concentrations of the peptide comprises determining the area of the peak or the peaks of the ions corresponding to the differentially isotopically-labeled peptides (e.g. the medium labeled peptide and the heavy labeled peptide). For example, the relative amounts or concentrations of the peptide may be obtained by first integrating the signal of the medium labeled peptide prior to integrating the signal of the heavy labeled peptide, and then determining their ratio by dividing one signal by the other or vice versa.

[0166] As would be appreciated by the person skilled in the art, seeding cell number and / or growth rate may vary from well to well and plate to plate. These factors (and others) can cause variability in MS peptide quantification. Accordingly, it can be difficult to determine if the observed difference between the first and second sample is due to cell number, cell health or to the treatment (e.g. differences in LNP lots). For example, when the number of cells in the samples combined in step (a) are not equal, the abundance determined for each peptide for each sample may not reflect the potency of the treatment. It is thought that this can be corrected for by using the relative abundance of peptides derived from the reference protein to calculate a cell normalization factor. In some examples, the reference protein (e.g. an endogenous reference protein) is produced by all cells so it may be used as an indicator of cell number. Cell count normalization is possible as the cells have been grown in medium and heavy media at the same time as treatment or immediately after the cells have been treated. This means that any labeled endogenous reference protein produced is an indicator of cell count and / or growth rate following treatment.

[0167] In some examples, the reference protein may be used as a control for the LNP dosing process. For example, the reference protein may be an exogenous reference protein, such as one expressed from a control sample (e.g. control RNA-LNP) dosed at the same time as the test sample (e.g. test RNA-LNP). It is thought that use of an exogenous reference protein can help correct for inherent well-to-well cell variability and / or LNP-induced cell toxicity (causing decreased protein expression) at higher LNP doses. In some examples, use of the exogenous reference protein corrects for LNP- induced cell toxicity. This normalization strategy can be used as an alternative to (or even in addition to) normalization using an endogenous reference protein. In some examples, normalization using an exogenous reference protein test improves the linearity (R2) across a range of doses and brings the potency slope ratio between two replicates of the same LNP closer to 1 (see Example 12).

[0168] Accordingly, in some examples, the method further comprises normalizing the amount of the target protein relative to the reference protein and quantifying the amount of target protein in the first sample. In some examples, the ratio of target protein or peptide is normalized using the reference protein or peptide signal. In some examples, the reference protein is an endogenous reference protein. In some examples, the reference protein is an exogenous reference protein.

[0169] In some examples, the ratio of “heavy” to “medium” reference peptide or protein is used as a normalization factor (see Figure 1 ). In some examples, the abundance of one or more medium labeled reference peptides represents the cell count in the medium labeled cell culture and the abundance of one or more heavy labeled reference peptides represents the cell count in the heavy labeled cell culture. In this example, the normalization factor (NF) is calculated as follows:

[0170] In some examples, the second variant reference protein signal is the “heavy” reference protein or peptide signal and the first variant reference protein signal is the “light” reference protein or peptide signal. In examples where the reference peptide is beta actin, the normalization factor for each sample is calculated by dividing the second variant beta actin protein or peptide (e.g. heavy beta actin peptide) peak area by the first variant beta actin protein or peptide (e.g. medium beta actin peptide) peak area. In some examples, the beta actin peptide is the GYS peptide as defined herein. The normalized amount of target protein, for example the normalized amount of protein (e.g. medium vs heavy) which takes into account cell count variations between medium isotopically- labeled cells and heavy isotopically-labeled cells may be calculated as follows:

[0171] Normalized amount of target protein first variant target protein or peptide signal

[0172] = NF x - - - ■. - ■. - — -:- - second variant target protein or peptide signal

[0173] In some examples, the second variant target protein signal is the “heavy” target protein or peptide signal and the first variant target protein signal is the “light” target protein or peptide signal. In some examples, the ratio of total reference peptide or protein in the first (e.g. medium) and second (e.g. heavy) samples is used as a normalization factor (see Figure 2). In this example, the amount of unlabeled (e.g. light) reference peptide in a control sample of the first or second sample is determined. For the purpose of illustration only, the following discussion assumes that the second sample is used as the internal control in the first mixture. In this example, the control sample comprises cells from the second sample (e.g. heavy sample) only. Typically, the amount of unlabeled reference peptide in a control sample of the second (e.g. heavy) cells is determined, for example, using any suitable technique known to the skilled addressee. In some examples, the amount of unlabeled reference peptide in the control sample is determined by comparison to a labeled peptide standard (e.g. superheavy peptide standard as defined herein). Using the amount of unlabeled reference peptide, a ratio (R) of unlabeled (L2nd, con) to labeled (H2nd,con) reference protein in the control sample is calculated, for example, using the formula below:

[0174] In the above, l_2nd, con is the amount of unlabeled reference protein measured in the control sample (e.g. heavy sample); H2nd, con is the amount of labeled reference protein measured in the control sample (e.g. heavy sample). Accordingly, the amount of unlabeled reference protein in the first mixture can then be estimated, for example using the formula below:

[0175] ^2nd, mix — R * f^2nd, mix

[0176] In the above, R is calculated as above, l_2nd, mix is the amount of unlabeled reference protein contributed to the first mixture from the second sample; and H2nd, mix is the amount of labeled reference protein contributed to the first mixture from the second sample (e.g. heavy labeled reference protein). In this example, the second sample (which comprises a second variant of the target protein and a second variant of the reference protein) functions as an internal standard in the first mixture. The calculated unlabeled reference peptide from the second (control) sample ( _2nd, mix) can then be used to determine the amount of unlabeled reference protein in the first sample (List, mix), for example, using the equation below:

[0177] In the above, L2nd, mix is the amount of unlabeled reference protein contributed to the first mixture from the second sample; List,mixis the amount of unlabeled reference protein contributed to the first mixture from the second sample; 1— total, mix is the total amount of unlabeled reference protein contributed to the first mixture from the first sample;

[0178] The total reference peptide in the first mixture from the first and second samples can then be calculated, for example, using the equation below.

[0179] The normalization factor (NF) can then be calculated using the equation below:

[0180] In the above, H2nd, mix is the amount of labeled reference protein contributed to the first mixture from the second sample (e.g. heavy labeled reference protein); Mist, mix is the total amount of labeled reference protein contributed to the first mixture from the first sample (e.g. medium labeled reference protein). The target protein abundance in the first mixture can be calculated and normalized using NF:

[0181] In some examples, the peptides from the first and second samples (e.g. the medium and heavy peptides” are quantified using the respective light peptides from recombinant protein standards of a known concentration. The concentration of medium or heavy protein may then be normalized against the concentration of the reference protein (e.g. the exogenous reference protein).

[0182] In some examples, the method comprises combining the one or more samples with a super heavy labeled peptide prior to analysis, for example a super heavy labeled peptide as described herein, to form a second mixture. In some examples, the method comprises combining the first mixture with a super heavy labeled peptide prior to analysis, for example a super heavy labeled peptide as described herein, to form a second mixture. As would be understood by the person skilled in the art, the order of addition of the super heavy peptide is not important and the super heavy peptide may be added at any stage prior to analysis, for example, it may be combined with the first sample and second sample to form a mixture, may be added after the first mixture has been formed, it may be added after the first mixture has been processed or it may be added just prior to analysis. Typically, the amount of super heavy labeled peptide added to each mixture is known. In this example, the reference protein (or a peptide derived from the reference protein) is observed as a quartet in the mass spectrum comprising a peak representing the unlabeled reference peptide (A+0), the medium labeled peptide (A+3), the heavy labeled peptide (A+5) and the super heavy peptide (A+7). In some examples, the super heavy labeled peptide is used to estimate the amount of the first variant of the reference peptide and / or the amount of the second variant of the reference peptide. In some examples, the super heavy labeled peptide is used to normalize the amount of the first variant of the reference peptide and / or the amount of the second variant of the reference peptide.

[0183] In some examples, the method further comprises absolute quantitation of the amount or concentration of the target protein one or more samples. In some examples, absolute quantitation of the amount or concentration of the target protein in a sample may be determined by using a known amount of a reference protein or a peptide thereof. In some examples, absolute quantitation of the amount or concentration of the target protein in a sample may be determined by using a known amount of the target protein or a peptide thereof. For example, a known amount of a third, unlabeled variant of the target protein or a peptide thereof may be combined with one of the samples (e.g. the first or second sample) to form a second mixture. In one example, the isotopically labeled variant and the unlabeled variant are the same target protein (i.e., they have the same amino acid sequence) but they have a different molecular weight. In one example, the unlabeled peptide corresponds to an isotopically labeled peptide that can produced by digestion of the isotopically labeled target protein such that the unlabeled peptide has the same amino acid sequence as the isotopically labeled peptide but has a different molecular weight. In some embodiments, the unlabeled peptide or protein could be chemically synthesized and produced by recombinant techniques. The unlabeled protein or peptide may also originate from other sources, such as, for example, biological protein-containing samples. In some examples, the second mixture is analyzed to determine the relative amounts or concentrations of the isotopically labeled variant and the unlabeled variant. In some examples, the second mixture is analyzed using any suitable technique known to the person skilled in the art for determining relative amounts or concentrations or using the methods (e.g. mass spectrometry) as described herein. In some examples, the second mixture is analyzed by mass spectrometry (e.g. LC-MS). In some examples, the second mixture is treated with a protease prior to analysis. In some examples, the sample comprising the isotopically labeled variant is processed prior to combining with the unlabeled protein or peptide, for example any cells contained within the sample may be lysed and optionally cell debris removed. In some examples, the sample comprising the isotopically labeled variant is treated with a protease prior to combining with the unlabeled peptide or protein, preferably unlabeled peptide. In some examples, the sample comprising the isotopically labeled variant is combined with the unlabeled peptide or protein, preferably unlabeled protein, prior to treatment with a protease. In some examples, the resulting peptides may then be analyzed by MS to determine the relative amounts or concentrations of the isotopically labeled peptide in the sample and the corresponding unlabeled peptide. Because a predetermined amount / concentration of the unlabeled protein or peptide is used, the absolute amount and / or concentration of the peptide in the first sample may be calculated.

[0184] Accordingly, in some examples the methods described herein comprise the steps of:

[0185] (d) combining the first and / or second sample with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture;

[0186] (e) analyzing the second mixture to determine relative amounts of the first and / or second variant and third variant of the target protein or peptide thereof;

[0187] (f) determining the absolute amount or concentration of the first and / or second variant in the second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof.

[0188] In some examples the methods described herein comprise the steps of:

[0189] (d) combining the second sample with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture;

[0190] (e) analyzing the second mixture to determine relative amounts of the second variant and third variant of the target protein or peptide thereof;

[0191] (f) determining the absolute amount or concentration of the second variant in the second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof. An example of standardization is illustrated in Figure 3.

[0192] In some examples, second mixture is treated with a protease before analysis. In some examples, second sample is treated with a protease before analysis.

[0193] In some examples, the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled peptide (e.g. a light peptide, e.g. a light tryptic peptide). In some examples, the unlablled peptide is a synthetic peptide. In some examples, the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled recombinant protein.

[0194] In some examples, a standard curve is used for determining the absolute amount or concentration of the first and / or second variant in the first and / or second sample. In some examples, a standard curve is used for determining the absolute amount or concentration of the second variant in the second sample. In some examples, the standard curve is produced by combining the second sample with a serial dilution of a known amount of the third, unlabeled variant of the target protein or a peptide thereof.

[0195] In some examples, a standard curve is used for determining the absolute amount or concentration of the recombinant protein (e.g. using a heavy manufactured peptide of varying concentration as the standard). In this example, the recombinant protein is digested into peptides using a protease such as trypsin. A single, known concentration of the recombinant proteins is then combined with the first and / or second samples as an internal standard to form the second mixture. In this example, third, unlabeled variant of the target protein or a peptide thereof is a recombinant protein.

[0196] In some examples, quantitation of the amount or concentration of the target protein in a sample may be determined using a one-point quantification approach (see Figure 4). In some examples, a known amount of a third, unlabeled variant of the target protein or a peptide thereof may be combined with the first mixture to form a second mixture. In one example, the isotopically labeled variant and the unlabeled variant are the same target protein (i.e., they have the same amino acid sequence) but they have a different molecular weight. In one example, the third, unlabeled variant corresponds to a peptide that can produced by digestion of the isotopically labeled target protein such that the unlabeled peptide has the same amino acid sequence as the isotopically labeled peptide but has a different molecular weight. In one example, the third, unlabeled variant corresponds to a protein (e.g. recombinant protein) that has the same amino acid sequence as the isotopically labeled variant but has a different molecular weight. In some embodiments, the unlabeled peptide or protein could be chemically synthesized and produced by recombinant techniques. The unlabeled protein or peptide may also originate from other sources, such as, for example, biological protein-containing samples. In some examples, the second mixture is analyzed using any suitable technique known to the person skilled in the art for determining relative amounts or concentrations or using the methods (e.g. mass spectrometry) as described herein. In some examples, the second mixture is analyzed by mass spectrometry (e.g. LC-MS). In some examples, the second mixture is purified prior to analysis with MS (e.g. chloroform or other solvent extraction). In some examples, the second mixture is treated with a protease prior to analysis. In some examples, the samples comprising the isotopically labeled variants are processed prior to combining with the unlabeled protein or peptide, for example any cells contained within the sample may be lysed and optionally cell debris removed. In some examples, the first mixture is processed prior to combining with the unlabeled protein or peptide, for example any cells contained within the sample may be lysed and optionally cell debris removed. In some examples, the first mixture is treated with a protease prior to combining with the unlabeled peptide or protein, preferably unlabeled peptide. In some examples, the first mixture is combined with the unlabeled peptide or protein, preferably unlabeled protein, prior to cell lysis. In some examples, the first mixture is combined with the unlabeled peptide or protein, preferably unlabeled protein, prior to treatment with a protease. In some examples, the resulting peptides may then be analyzed by MS to determine the relative amounts or concentrations of the isotopically labeled peptide in the sample and the corresponding unlabeled peptide. Because a predetermined amount / concentration of the unlabeled protein or peptide is used, the absolute amount and / or concentration of the peptide in the first sample may be calculated. In some examples, the concentration is calculated using the following equation:

[0197] CtrestOT.. On?, concentrations for medium, heavy, and recombinant antigen -concentrations.

[0198] Accordingly, in some examples the methods described herein comprise the steps of:

[0199] (d) combining the first mixture with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture;

[0200] (e) analyzing the second mixture to determine relative amounts of the first variant, second variant and third variant of the target protein or peptide thereof;

[0201] (f) determining the absolute amount or concentration of the first and / or second variant in the first and / or second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof.

[0202] In some examples, one-point quantification is used for determining the absolute amount or concentration of the first and / or second variant in the first and / or second sample. In some examples, one-point quantification is used for determining the absolute amount or concentration of the second variant in the second sample.

[0203] In some examples, absolute quantitation of the amount or concentration of the target protein in a sample may be determined by using a known amount of an isotopically labeled peptide. In some examples, absolute quantification using isotopically labeled peptides typically comprises spiking known concentrations of synthetic, heavy isotopologues of the peptide being monitored into an experimental sample and then analyzing the sample using mass spectrometry. In some examples, the abundance of the target peptide being monitored in the experimental sample is compared to that of the heavy peptide and back-calculated to the initial concentration of the standard using a standard curve to yield the absolute quantification of the target peptide and therefore protein.

[0204] In some examples, a standard curve is used for absolute quantification (e.g. copy number per cell) of the target protein. A standard curve may be generated using a suitable standard. In some examples, the standard is a recombinant, unlabeled protein corresponding to the target protein or reference protein. In some examples, the standard is an unlabeled peptide corresponding to a portion of the target protein. In some examples, the unlabeled peptide is used as the standard to determine the concentration or amount of target protein expressed by the second sample. The second sample is then combined with the first sample to form the first mixture and used as an internal standard. In some examples, the standard curve is constructed using a recombinant, unlabeled protein corresponding to the target protein or reference protein which is digested with a protease (e.g. trypsin) (see Figure 5). In some examples, the standard is an isotopically labeled peptide corresponding to a portion of the target protein. In some examples, the isotopically labeled peptide is used as the standard to determine the concentration of a recombinant, unlabeled protein corresponding to the target protein or reference protein. The recombinant, unlabeled protein is then spiked into the first and / or second samples or the first mixture and used as an internal standard. In some examples, the isotopically labeled peptide is used as the standard to determine the concentration of an unlabeled peptide corresponding to a portion of the target protein or reference protein. The unlabeled peptide corresponding to a portion of the target protein or reference protein is then spiked into the first and / or second samples or the first mixture and used as an internal standard.

[0205] The present inventors have found that by isotopically labeling both the first and the second samples (e.g. by differentially isotopically labeling the targeted and reference samples)- light (unlabeled, no isotopic labeling) peptides or recombinant proteins that match the targeted protein can be directly added to the sample for simultaneous sample processing & quantification.

[0206] The methods described herein are useful for quantification of protein expression, for example expression of one or more antigens from a nucleic acid vaccine. As discussed herein, the samples containing the target protein (or believed to contain the target protein) and reference protein may originate from any source and the target protein or proteins may be differentially isotopically-labeled by any suitable method known to the person skilled in the art. In some examples, the samples comprise cells, originate or are derived from cells. In some examples, the cells are contacted with a nucleic acid encoding a target protein. In some examples, the cells are contacted with a RNA encoding the target protein. In some examples, the cells are contacted with a delivery vehicle comprising an RNA encoding the target protein. In some examples, the cells are contacted with a LNP comprising an RNA encoding the target protein. In some examples, the cells are cultured in a medium supplemented with stable isotopically labeled amino acids. In some examples, the cells in the first sample are cultured in a medium supplemented with a first set of isotopically labeled amino acids (e.g. medium isotopically labeled amino acids) and the cells in the second sample are cultured in a medium supplemented with a different set of isotopically labeled amino acids (e.g. heavy isotopically labeled amino acids). Typically, the cells are only cultured with the labeled medium while being treated with the nucleic acid encoding the target protein. The labeled amino acids are metabolically incorporated into the proteins of the cells through protein synthesis resulting in labeled proteins. When the first and the second samples are analyzed using the methods described herein, the source of the sample can be easily distinguished by the mass difference caused by the differential labeling. Unlike, traditional SILAC (stable isotope labeling with amino acids in cell culture) which uses unlabeled and labeled samples, the methods described herein use two, differentially labeled samples for relative quantitation. In addition, the cells are only cultured in a medium supplemented with stable isotopically labeled amino acids during treatment with the nucleic acid encoding the target protein.

[0207] In some examples, there is provided a method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic in media comprising an isotopically labeled amino acid to form a first sample, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; normalizing the amount of the target protein relative to the reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

[0208] As used herein, “in-vitro potency” or “potency” refers to a measure of the ability of the transfected cells to express the target protein encoded by the recombinant nucleic acid in the cell-based potency assay described herein. As used herein, “relative in-vitro potency” or “relative potency” as used herein is the comparison of the potency of a test sample to the potency of a reference standard. In some examples, the relative potency of the test sample is represented by the normalized medium / heavy ratio for one or more peptides:

[0209] Medium peptide peak area heavy beta actin peak area - * - heavy peptide peak area medium beta actin peak area

[0210] In some examples, the normalized protein concentrations are plotted against the dose (e.g. RNA-LNP dose) to create a linear dose response curve. The linear slope of test sample (e.g. test RNA-LNP) can be compared to the linear slope of reference sample (e.g. reference RNA-LNP dose) to generate a relative potency value slope ratio.

[0211] The method described herein enables simultaneous processing of two different LNP sample lots using medium and heavy isotopically labeled growth media. This allows for the generation of two different linear dose-response curves from the same sample (one medium and one heavy), from which a slope ratio can be calculated. The slope ratio is a relative potency value, where the potency of a test LNP can be directly compared to the potency of a reference LNP. In some examples, the relative potency of the test sample (e.g. test RNA-LNP) is represented by the slope ratio of the dose response curve. In some examples, the antigen expression results are normalized to a dosing control sample (e.g. control RNA-LNP) to produce a normalized slope ratio relative potency value. The relative potency value (e.g. slope ratio) and its related parameters (such as slope, R2) can be computed from the processed mass spectrometry data using any suitable software, for example a customized report template in the vendor’s software (such as Chromeleon and TraceFinder) or using an in-house script (e.g. R script).

[0212] In some examples, the recombinant nucleic acid is a RNA. In some examples, the recombinant nucleic acid is a mRNA. In some examples, the recombinant nucleic acid is an mRNA encapsulated in a delivery vehicle. In some examples, the recombinant nucleic acid is an mRNA encapsulated in a LNP. In some examples, the recombinant nucleic acid is an RNA vaccine. In some examples, the recombinant nucleic acid is DNA. In some examples, the recombinant nucleic acid is plasmid DNA. In some examples, the recombinant nucleic acid is DNA encapsulated in a delivery vehicle. In some examples, the recombinant nucleic acid is a DNA vaccine.

[0213] In some examples, the method further comprises, prior to transfecting the population of cells, seeding a population of cells on a cell culture plate. In one embodiment, the cell culture plate comprises at least 6, 12, 24, 48, 96, 384 or 1536 wells. In some examples, the plate comprises at least 6 wells. In some examples, the plate comprises at least 12 wells. In some examples, the plate comprises at least 24 wells. In some examples, the plate comprises at least 48 wells. In some examples, the plate comprises at least 96 wells. In some examples, the plate comprises at least 384 wells. In some examples, the plate comprises at least 1536 wells.

[0214] In some examples, prior to transfection, the seeded cells are grown in unlabeled media. In some examples, the growth time of the seeded cells prior to transfection is up to 32 hours. In some examples, the growth time of the seeded cells prior to transfection is about 16 to about 32 hours. In some examples, the growth time is about 20 to about 28 hours prior to transfection. In some examples, the growth time is about 22 to 26 hours prior to transfection. In some examples, the growth time is about 24 hours prior to transfection. In a further embodiment, the growth time is about 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , or about 32 hours prior to transfection. In some examples, the seeded cells are grown to greater than 70% confluence prior to transfection. In some examples, the seeded cells are grown to between about 70% and 90% confluence prior to transfection, for example between about 75% and 85% confluence prior to transfection. In some examples, the seeded cells are grown to about 80% confluence prior to transfection. The unlabeled media is then removed from the wells prior to transfection.

[0215] In some examples, the recombinant nucleic acid is combined with the media comprising an isotopically labeled amino acid and the media comprising the isotopical ly labeled amino acid and the recombinant nucleic acid is added to the seeded cells. This means that the cells are transfected with the recombinant nucleic acid in the presence of the isotopical ly labeled amino acids.

[0216] In some examples, the recombinant nucleic acid is a RNA or RNA. In some examples, the recombinant nucleic acid is an RNA. In some examples, the recombinant nucleic acid is an mRNA (e.g. sa-mRNA). In some examples, the recombinant nucleic acid is comprised within an LNP or other delivery vehicle. In some examples, the cells are transfected with a RNA-LNP. Any suitable amount of recombinant nucleic acid may be used. In some examples, the amount of nucleic acid (e.g RNA) is less than about 50 ng, less than about 25 ng, less than about 12.5 ng, less than about 3.125 ng or less than about 1 .6 ng. In some examples, the amount of nucleic acid (e.g RNA) is less than about 25ng. In some examples, the amount of nucleic acid (e.g RNA) is less than about 12.5 ng. In some examples, the amount of nucleic acid (e.g RNA) is between about 0.1 ng and 50 ng. In some examples, the amount of nucleic acid (e.g RNA) is between about 0.3 ng and 25 ng. In some examples, the amount of nucleic acid (e.g RNA) is between about 0.3 ng and 12.5 ng.

[0217] In some examples, the cell populations are transfected with the recombinant nucleic acid in the presence of isotopically labeled amino acids for at least 4 hours at 35 - 39°C, with 4-6% CO2. In some examples, the duration of the transfection is up to 48 hours. In some examples, the duration of the transfection is up to 32 hours. In some examples, the duration of the transfection is about 16 to about 32 hours. In some examples, the duration of the transfection is about 20 to about 28 hours. In some examples, the duration of the transfection is about 22 to 26. In some examples, the duration of the transfection is about 24 hours. In some examples, the growth time is about 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , or about 32 hours. In some examples, the cells reach about 90% confluence after transfection. In some examples, the cells reach greater than 90% confluence after transfection. In some examples, the cells reach 95% or higher 90% confluence after transfection.

[0218] As used herein, the term “confluency” with respect to HEK293 and BHK21 means the percentage of the surface of a cell culture plate that is covered by adherent cells.

[0219] In some examples, the method further comprises adding Apolipoprotein E (ApoE) during the transfection step. Any suitable concentration of ApoE may be used. In some examples, ApoE is added in an amount of 2 pg / mL. The addition of ApoE to certain cell lines allows for complete transfections. In some examples, ApoE is not added during the transfection step. In some examples, the labeled media is removed and the cells are removed from the wells. In some examples, the cells are treated to enable dissociation of the adherent cells from the plastic. In some examples, the cells are treated to with TrypLE. In some examples, the cells are collected and optionally washed to remove any remaining media. In some examples, the heavy labeled cells are combined in a single tube and the medium labeled cells are collected using an individual tube per well. In some examples, an equal amounts of the heavy labeled cells are added to the individual tubes containing the medium labeled cells. In some examples, the combined cells are pelleted and washed to remove any remaining media. The cells are lysed and the proteins digested as described herein. The digested samples are then analyzed as described herein.

[0220] In some examples, the methods described herein are performed on a series of dilutions of the test sample. In some examples, the methods described herein are performed on a single dilution of the test sample. In some examples, the methods described herein are performed on a series of dilutions of the reference sample. In some examples, the methods described herein are performed on a single dilution of the reference sample. In some examples, the methods described herein are performed on a series of dilutions of the test sample and a single dilution of the reference sample. In some examples, the methods described herein are performed on a series of dilutions of the test sample and a series of dilutions of the reference sample. In some examples, a does response curve is generated for the test sample. In some examples, the dose response curve may be used to calculate an EC5o value for the test sample.

[0221] An advantage of the methods described herein is that shorter labeling times are required. Traditional SILAC often requires a growth of 5-10 doubling times before analysis to allow full incorporation of the labeled amino acids. In contrast, and as demonstrated in the Examples described herein, the current methods do not require the cells to be cultured for a sufficient period of time to allow full incorporation. The use of only isotopically labeled amino acids eliminates the need for 100% incorporation of amino acids needed for SILAC.

[0222] The present application also provides a method comprising the steps of obtaining a population of cells that has been treated with the self-amplifying mRNA encoding one or more target proteins; and quantifying the amount of the one or more target proteins expressed from the RNA. The present application also provides a method comprising the steps of obtaining a population of cells that has been treated with the self-amplifying mRNA encoding one or more target proteins; and quantifying the potency of the selfamplifying mRNA encoding one or more target proteins. In some examples, the RNA is self-amplifying mRNA. In some examples, the population of cells that has been treated with one or more self-amplifying mRNA. The RNA may be naked RNA (i.e. not associated with a delivery vehicle) or associated with a delivery vehicle (e.g. an lipid nanoparticle). In some examples, the RNA is naked RNA. In some examples, the RNA is an RNA-LNP. In some examples, the RNA-LNP is a monovalent (i.e. comprises one type of RNA). In some examples, the RNA-LNP is a bivalent (i.e. comprises two different types of RNA). In some examples, the RNA-LNP is a trivalent (i.e. comprises three different types of RNA). In some examples, the RNA-LNP is a quadrivalent (i.e. comprises four different types of RNA). In some examples, the RNA-LNP is a multivalent (i.e. comprises multiple (e.g. 2, 3, 4, or more) different types of RNA). The RNA may be mono-cistronic, or multi-cistronic. In some examples, quantification of the one or more target proteins is performed by stable isotope dilution mass spectrometry (ID-MS). In some examples, quantification of the one or more target proteins is performed by stable isotope dilution mass spectrometry (ID-MS) using a light (unlabeled) peptide as the internal standard. In some examples, the light (unlabeled) peptide corresponds to one or more of the peptides produced by digestion (e.g. protease digestion) of the target protein. In some examples, the light (unlabeled) peptide corresponds to one the peptides produced by digestion the target protein using trypsin (e.g. a tryptic peptide). In some examples, the light (unlabeled) peptide corresponds to a peptide comprising the amino acid sequence of any one of SEQ ID NO: 1 -38. In some examples, the light (unlabeled) peptide corresponds to a peptide comprising the amino acid sequence of any one or more of SEQ ID NO: 3-38

[0223] In some examples, the method may be used to determine the potency of an RNA vaccine (e.g. a naked RNA or RNA-LNP). As illustrated in the Examples, the present inventors have demonstrated that there is a surprising correlation between the potency of the RNA vaccine measured using the methods described herein and more traditional animal studies. Accordingly, it is thought that the methods described herein provide an in vitro alternative to in vivo potency assays. In some examples, the potency of an RNA is calculated from a standard curve of serial dilutions of the RNA. For example, a series of two-fold dilutions of the RNA (e.g. comprising RNA encoding each antigen formulated in LNPs at varying concentrations of RNA) is performed. An exemplary 2-fold dilution series includes 800 ng RNA, 400 ng RNA, 200 ng RNA, 100 ng RNA, 50 ng RNA, 25 ng RNA, 12.5 ng RNA, 6.25 ng RNA, 3.125 ng RNA, 1.56 ng RNA, 0.78 ng RNA, and / or 0.39 ng RNA. Each dilution is incubated with a population of cells under culture conditions and the treated cells used in the methods described herein to quantify the amount of target protein produced. The amount of target protein produced can then be plotted against the amount of RNA and a linear regression analysis can be performed on the curve to calculate its slope which is related to the potency of the RNA. In some examples, the specific potency can then be calculated by multiplying the value of the slope by a constant (linear regression slope x 105). In an example, specific potency can be calculated according to the following formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error.

[0224] In one example, potency is the amount of protein translated from one million cells per nanogram of RNA provided.

[0225] As demonstrated in Example 14, the methods described herein are suitable for use in a high-throughput format. In some examples, the methods may be used in a multiwell format, for example, a six-well, a 12-well, a 24-well, a 48-well, a 96-well or more format. In some examples, the methods may be used in a 96-well format. In some examples, the methods may be used in a six-well format. This is thought to maximize the assay throughput. In addition, it is possible to utilize the same multi-well plate format from cell pre-seeding through MS sample injection.

[0226] In some examples, there is provided a method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic and a control sample in media comprising an isotopically labeled amino acid to form a first sample, wherein the first sample comprises a first variant of the target protein and a first variant of a exogenous reference protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid and a control sample in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein and a second variant of the exogenous reference protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and exogenous reference protein; normalizing the amount of the target protein relative to the exogenous reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

[0227] In some examples, the test sample is a test RNA-LNP. In some examples, the control sample is a control RNA-LNP.

[0228] In some examples, the labelled test and / or control peptides are quantified using light peptides from a matched recombinant protein standard of a known concentration.

[0229] In some examples, the concentration of labelled protein is normalized against the concentration of the exogenous reference protein. In some examples, the normalized protein concentrations are plotted against sample dose to create a linear dose response curve. In some examples, the linear slope of the test sample can be compared to the linear slope of reference sample to generate a relative potency value slope ratio.

[0230] In some examples, the method is a high-throughput method. For example, the method may be executed in a high-throughput manner with the utilization of 12-well, 24- well, 96-well, or 384 well format.

[0231] Samples

[0232] The samples used in the methods described herein typically comprise, or are derived from samples containing, proteins. The samples may originate from various sources. For example, the samples may be chemically synthesized (i.e. comprises proteins or peptides that are chemically synthesized) and / or may originate from biological samples such as body fluids (e.g., blood, urine, etc.), from cultured cells, from organ or tissue specimens, or from any other biological source or sources. In some examples, the samples are from cultured cells. The samples may contain a plurality of proteins and / or peptides or only a single protein or peptide. The samples may have been treated with a pharmaceutical, chemical, biologic or vaccine of interest. In some examples, the proteins and peptides may originate from cultured cells that have been transfected with a nucleic acid. In some examples, the proteins and peptides may originate from cultured cells that have been transfected with a DNA vaccine or an RNA vaccine. In some examples, the proteins and peptides may originate from cultured cells that have been transfected with a composition comprising a delivery vehicle and a nucleic acid. In some examples, the proteins and peptides may originate from cultured cells that have been transfected with a composition comprising a lipid nanoparticle and RNA. In some examples, the proteins and peptides may originate from cultured cells that have been transfected with a composition comprising a lipid nanoparticle and selfamplifying RNA.

[0233] In some examples, the cells are cultured in a medium supplemented with amino acids containing either medium or heavy stable isotopes. The amino acids are metabolically incorporated into the proteins of the cells through protein synthesis. When the combined medium and heavy isotope labeled proteins are analyzed by MS, the source of the sample can be easily distinguished by the mass difference caused by the differential labeling. The relative abundance of the proteins is measured based on the intensities of the light and heavy peptides.

[0234] The methods described herein may be used to quantify the potency of a recombinant nucleic acid. In some examples, the methods described herein may be used to quantify the potency of a nucleic acid vaccine (e.g. a DNA vaccine or an RNA vaccine). The methods may be used to compare protein expression from a test recombinant nucleic acid encoding a protein to protein expression from a reference recombinant nucleic acid encoding a protein. The methods may therefore be used to compare two or more different constructs (e.g. constructs that encode the same antigen but which differ in other aspects, such as promoter or regulatory element, codon usage, 3’IITR, 5'UTR, 5’ cap, polyA tail length and / or antigen sequence), to compare two or more different batches or lots of the same construct or to compare the effect of changing different process or formulation parameters. They may also be used to assess the potency of the recombinant nucleic acid over time. Accordingly, in some examples, the samples comprise or are derived from cells treated with nucleic acids (e.g. recombinant nucleic acids). In some examples, the samples comprise cells treated with nucleic acids (e.g. recombinant nucleic acids). In some examples, the cells are treated with an recombinant RNA, for example RNA encapsulated in a lipid nanoparticle or other delivery vehicle. In some examples, the cells are treated with an recombinant DNA, for example DNA encapsulated in a lipid nanoparticle or other delivery vehicle. In some examples, the first sample comprises cells treated with a first RNA. In some examples, the first sample comprises cells treated with a test RNA. In some examples, the second sample comprises cells treated with a second RNA. In some examples, the second sample comprises cells treated with a reference RNA. In some examples, the first sample comprises cells treated with a first RNA-LNP. In some examples, the first sample comprises cells treated with a test RNA-LNP. In some examples, the second sample comprises cells treated with a second RNA-LNP. In some examples, the second sample comprises cells treated with a reference RNA-LNP. Any suitable cells may be used and can be selected by the skilled addressee, for example, based on the LNP and / or RNA being tested. Typically, the cells are eukaryotic cells. In some examples, the cells are HEK293 cells or BHK21 cells. In some examples, the cells are HEK293 cells. In some examples, the cells are BHK21 cells. .

[0235] Labeled amino acids

[0236] In some examples, the methods described herein comprise culturing cells using a media comprising one or more labeled reagents (e.g. labeled amino acids such as isotopically labeled amino acids). As used herein, the term “labeled” refers to attachment of a detectable signal, agent or moiety to a compound. As used herein, the term “detectable signal” refers to a signal that can be detected or measured by a human being or a machine. In one example, a detectable signal can be quantified such that the intensity of the signal is related to (e.g., proportional to) the amount of the compound associated with the signal. Depending on the nature of the signal, a detectable signal may be detected, measured or quantified by a spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. A “detectable signal” may also be referred to as “detectable agent” or “detectable moiety” in this application. In one example, the “detectable signal” is detectable by mass spectrometry.

[0237] In one example, the labeled reagents are isotopically labeled reagents. In one example, the labeled reagents are labeled with a stable isotope. In one example, the labeled reagents are labeled with a heavy isotope. As used herein, a “heavy” isotope is the stable isotope or stable isotopes of an element, which are heavier than the most abundant isotope. In other words, a “heavy isotope” is a stable atom in which there are more neutrons than in the normal isotope of the element, giving it a greater mass. For instance,18O is considered as heavy oxygen, compared to the most abundant16O. Heavy isotopes of nitrogen includes15N (c.f.14N). Heavy isotopes of Carbon include13C and heavy isotopes of hydrogen include2D, also referred to as deuterium. In one example, the labeled reagent may comprise one or more15N,13C and / or2D atoms.

[0238] In one example, the isotopically labeled reagent is an isotopically labeled amino acid. Any suitable isotopically labeled amino acid may be used in the methods described herein. Isotopically labeled amino acids are available from commercial suppliers such as Sigma Aldrich. Any isotopically labeled amino acid that is suitable for use in cell culture may be used. In one example, the isotopically amino acid is one of the amino acids that are naturally incorporated into polypeptides, also called proteinogenic or natural amino acids. For example, arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), glycine (G), proline (P), cysteine (C), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and / or tryptophan (W). In some examples, the isotopically labeled amino acid is an essential amino acid (i.e. not synthesized by cells, such as eukaryotic cells). Essential amino acids include H, I, L, K, M, F, T, W, and V. In some examples, the isotopically labeled amino acid is an essential or a conditionally essential amino acid, for example, H, I, L, K, M, F, T, W, V, R, C, G, Q, P, and Y. In some examples, the isotopically labeled amino acid is arginine (R) and / or lysine (K). In some examples, the isotopically labeled amino acid is arginine (R), leucine (L) and / or lysine (K). Using isotopically labeled lysine as an example, the molecular weight of heavy lysine is higher than the molecular weight of lysine and the difference in molecular weight may be detected by mass spectrometry.

[0239] As would be understood by the person skilled in the art, one or more atoms of the isotopically labeled amino acid may be labeled with a heavy isotope. For example, the isotopically labeled amino acid may comprise one or more13C, one or more2D, one or more15N, and / or one or more18O. In some examples, each C atom is replaced by a heavy C atom (e.g.13C). In some examples, each N atom is replaced by a heavy N atom (e.g.15N). In some examples, each non-exchangeable H atom is replaced by a heavy H atom (e.g.2D). As used herein, “non-exchangeable” H is understood to mean a H atom that may not be replaced by a H atom from a protic solvent due to solvent exchange. In protic solution exchangeable protons such as those in hydroxyl or amine groups exchange protons with the solvent. In one example, isotopically labeled lysine comprises15N-lysine,13C-lysine,2D-lysine,15N,13C-lysine,15N,2D-lysine,13C,2D-lysine or15N,13C,2D-lysine. In one example, isotopically labeled lysine is15N,13C-lysine. In one example, isotopically labeled lysine is2D-lysine. In one example, isotopically labeled arginine comprises15N- arginine,13C- arginine,2D- arginine,15N,13C- arginine,15N,2D- arginine,13C,2D- arginine or15N,13C,2D- arginine. In one example, isotopically labeled arginine is15N,13C- arginine. In one example, isotopically labeled arginine is13C- arginine. In one example, isotopically labeled leucine comprises15N- leucine,13C- leucine,2D- leucine,15N,13C- leucine,15N,2D- leucine,13C,2D- leucine or15N,13C,2D- leucine. In one example, isotopically labeled leucine is15N,13C- leucine. In one example, isotopically labeled leucine is13C- leucine. In one example, isotopically labeled leucine is2D-leucine.

[0240] As would be appreciated by the skilled addressee, not all atoms in the molecule need to be replaced by the heavy isotope. In one example, each carbon and / or nitrogen atom in the molecule are replaced by the heavy isotope. In one example, a subset of the carbon and / or nitrogen atoms in the molecule are replaced by the heavy isotope. In one example, the isotopically labeled lysine is13C6,15N2-lysine. The mass of13C6,15N2- lysine is 8 Daltons greater than regular / light lysine and is referred to as K(+8). In one example, the isotopically labeled lysine is13Ce -lysine (i.e. K(+6)). In one example, the isotopically labeled lysine is15N2-lysine (i.e. K(+2). In one example, the isotopically labeled lysine is 4,4,5,5-2D4-lysine (i.e. K+(4)). In one example, the isotopically labeled lysine is 3,3,4,4,5,5,6,6-2D8-lysine (i.e. K(+8)). In one example, the isotopically labeled arginine is13Ce-arginine (i.e. R(+6)) In one example, the isotopically labeled arginine is13C6,15N4-arginine (i.e. R(+10)). In one example, the labeled leucine is13Ce-leucine (i.e. L(+6)). In one example, the labeled leucine is 5,5,5 -2D3-leucine (i.e. L(+3)).

[0241] Typically the isotopically labeled amino acids have an isotopic purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5%. In some examples, the isotopic purity of the isotopically labeled amino acid is at least 98%. In some examples, the isotopic enrichment of the isotopically labeled amino acid is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% (atom%). In some examples, the isotopic enrichment of the isotopically labeled amino acid is at least 98 atom%.

[0242] Isotopic labeling

[0243] Various methods of isotopic labeling are known in the art. When the peptides are derived from proteins, the proteins in different samples may be differentially isotopically- labeled such that peptides resulting from digestion of the proteins will also be isotopically-labeled. Examples of methods of providing differentially isotopically-labeled proteins and / or peptides include chemically modifying the peptides using chemicals having different isotopes incorporated therein (e.g., acetylation of peptides using acetic anhydride and hexadeutero acetic anhydride such that peptides from one sample include CH3CO- and peptides from another sample include CD3CO-), digesting proteincontaining samples that were prepared using different isotope-enriched cell culture media (e.g., using isotopes of hydrogen (H), carbon (C), nitrogen (N), phosphorous (P), sulfur (S), etc.), and / or by labeling the peptides during digestion of the protein (e.g., enzymatically digesting different protein samples using H216O or H218O such that the peptides in different samples contain either16O or18O (e.g., as described in U.S. Patent Application Publication No. 2002 / 0076817)).

[0244] In some examples, protein-containing samples were prepared using different isotope-enriched cell culture media (e.g., using isotopes of hydrogen (H), carbon (C), nitrogen (N), phosphorous (P), sulfur (S), etc.). The media typically lack one or more standard essential amino acids but are supplemented with a stable, isotopically labeled form of those amino acids. Any suitable cell culture media may be used. The media may comprise any combination of isotopically labeled amino acids. In some examples, the isotopically labeled amino acids comprise arginine and / or lysine. In some examples, the media comprises SILAC medium plus one or more isotopically labeled amino acids, such as lysine and / or arginine. In some examples, the media comprises RPMI SILAC medium plus one or more isotopically labeled amino acids, such as lysine and / or arginine. In some examples, the media comprises RPMI 1640 SILAC medium plus one or more isotopically labeled amino acids, such as lysine and / or arginine.

[0245] In some examples, the media comprises “medium” labeling. In some examples, the media comprises K(+4). In one example, the media comprises 4,4,5,5-D4-lysine. In some examples, the media comprises R(+6). In some examples, the “medium” media comprises K(+4) and / or R(+6). In some examples, the “medium” media comprises K(+4) and R(+6). In one example, the media comprises13Ce-arginine. In some example, the “medium” media comprises 4,4,5,5-D4-lysine and13Ce-arginine. In some examples, the media comprises K(+3). In some examples, the media comprises 5,5,5 - D3-leucine. In some examples, the media comprises K(+3) and R(+6), however any combination may be used provided the molecular weight is different to the other media being used. In some examples, the “medium” media comprises 5,5,5- D3-leucine and13Ce-arginine.

[0246] In some examples, the media comprises “heavy” labeling. In some examples, the media comprises K(+8). In one example, the media comprises13C6,15N2-lysine. In some examples, the media comprises R(+10). In one example, the media comprises13C6,15N4-arginine. In some examples, the “heavy” media comprises K(+8) and R(+10), however any combination may be used provided the molecular weight is different to the other media being used. In one example, the media comprises13C6,15N2-lysine and13C6,15N4-arginine.

[0247] In some examples, the media comprises “super heavy” labeling. In some examples, the media comprises K(+17). In one example, the media comprises13Ce, D9,15N2-lysine. In some examples, the media comprises K(+11 ). In one example, the media comprises D9,15N2-lysine. In some examples, the media comprises R(+17). In one example, the media comprises13Ce, D7,15N4-arginine. In some examples, the media comprises R(+1 1 ). In one example, the media comprises D7,15N4-arginine. In some examples, the media comprises R(+10). In one example, the media comprises13Ce,15N4-arginine. In some examples, the “super heavy” media comprises K(+17) and R(+17), however any combination may be used provided the molecular weight is different to the other media being used. In one example, the media comprises13Ce, D9,15N2-lysine and13Ce, D7,15N4-arginine. In some examples, proline (a non-essential amino acid) is added to the labeled SILAC media to prevent the metabolic conversion of isotopically labeled arginine to isotopically labeled proline in mammalian cell lines with high arginine dehydrogenase activity.

[0248] Depending on the mass difference, isotopically labeled amino acids, media containing the isotopically labeled amino acids or proteins and peptides containing the isotopically labeled amino acids may be classified relative to each other, for example as light (i.e. no isotopic labeling (other than at naturally occurring levels)), medium, heavy, super heavy etc. These terms are relative and are used herein to reflect differential isotopic labeling and not absolute masses. While the above is provided by way of example, the person skilled in the art would understand that any suitable SILAC media may be used provided the isotopically labeled amino acids in the “medium” media can be distinguished by molecular weight from the isotopically labeled amino acids in the “heavy” media.

[0249] Any suitable concentration of labeled amino acid may be used. Typically, the amount of labeled amino acid present in the media is not limiting.

[0250] In some examples, the cells are transferred to and cultured in isotopically labelled media at the same time as dosing. This may allow use of an endogenous reference protein where the labeled reference protein can be used as an indicator of cell number at dosing and / or cell health post dosing.

[0251] In some examples, the cells are transferred to and cultured in isotopically labelled media prior to dosing. In some examples, the cells are seeded in isotopically labelled media at least 1 , 2, 4, 6, 8, 12, 18, 24, 30 or more hrs before dosing. In some examples, the cells are seeded in isotopically labelled media at least 24 hrs before dosing.

[0252] Proteases

[0253] In some examples, the methods described herein may comprise the step of treating the mixture with a protease prior to analysis. Treating the mixture with a protease cleaves the proteins in the mixture producing a mixture of peptides. The resulting peptides may be labeled and / or unlabeled depending on the protease chosen. It is to be appreciated that examples of the method are not limited by the identity of the protease, although certain proteases may provide one or more advantages. Any protease capable of cleaving or digesting the expressed polypeptide to release at least one labeled peptide may be used. It would also be appreciated by the person of skill in the art that the identity of the target protein and / or the reference protein may influence the choice of protease. In addition, the specificity of the protease and the choice of labeled amino acid may also influence the choice of protease. For example, a protease that cleaves N- or C-terminal of an arginine may be preferred when the polypeptide comprises isotopically labeled arginine. Suitable proteases include, but are not limited to, endoproteinase GluC, enterokinase light chain, Factor Xa, furin, chymotrypsin, Lys- C, Lys-N, elastase, Asp-N and endoproteinase ArgC.

[0254] In one example, a suitable protease is trypsin or an enzyme with trypsin-like biochemical activity. Trypsin is often selected when isotopically labeled lysine and arginine are used as trypsin preferentially cleaves peptide bonds at the C-terminal side of lysine and arginine residues (except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis by trypsin). Trypsin can tolerate mild denaturing conditions, such as 0.1% SDS, 1 M urea, and 10% acetonitrile. In one example, a suitable protease is LysC (also referred to as Lysyl endopeptidase) or an enzyme with LysC-like biochemical activity. LysC is often selected when isotopically labeled lysine is used as LysC preferentially cleaves peptide bonds at the C-terminal side of lysine. LysC produces larger peptide fragments (compared to trypsin) resulting in less complex samples. Lys-C can also cleave between K-P. Lys-C retains proteolytic activity under strong protein denaturing conditions such as 8M urea, which can be used to improve digestion of proteolytically resistant proteins. In one example, a suitable protease is LysN or an enzyme with LysN-like biochemical activity. LysN is often selected when isotopically labeled lysine is used as LysN preferentially cleaves peptide bonds at the N-terminal side of lysine. Advantageously, LysN is capable of cleaving methylated lysine. LysN protease is active over a wide range of temperatures (e.g. between 37°C and 50°C, although digestion is possible at both higher and lower temperatures). LysN also remains active under moderate denaturing conditions such as 0.1% SDS, 6M urea or heating to 70°C, which can be used to improve digestion of proteolytically resistant proteins.

[0255] In some examples, two or more proteases may be used in combination, for example trypsin and LysC.

[0256] Any suitable amount of protease may be used and the person skilled in the art would be able to determine the amount of protease required. Suitable proteases may be manufactured using recombinant techniques or are available commercially from suppliers, such as Thermo Fisher Scientific and SigmaAldrich.

[0257] Reference protein

[0258] The methods described herein use a reference protein to help account for variability in the target protein MS signal intensity that may arise due to, for example, the cell count at the time of treatment or transfection with the recombinant nucleic acid. This is relevant for cell based assays as sample variability due to cell count is possible and ideally should be taken into account when determining the potency of a nucleic acid or nucleic acid vaccine. Accordingly, in some examples, the amount of target protein is in the sample is quantitated by comparison to a reference protein. In some examples, the amount of target protein is in the sample is quantitated by normalization using a reference protein. Any suitable reference protein may be used. Suitable reference proteins include proteins that can be used as an indicator of cell number. Reference proteins may be a protein that is expressed from an endogenous nucleic acid or may be a protein expressed from an exogenous nucleic acid.

[0259] In some examples, the reference protein is a protein that is expressed from an endogenous nucleic acid. In some examples, a suitable reference protein typically exhibits constitutive, nonregulated and stable expression, regardless of cell type or experimental design, although in some examples a suitable reference protein exhibits constitutive, nonregulated and stable expression in the cell line being used to prepare the samples. Additionally, the reference proteins should be expressed at a level which is above the limit of detection. In some examples, reference proteins include housekeeping proteins which are often involved in cellular maintenance. Reference proteins include, but are not limited to, ACTB, GAPDH, TUBB, 18S-rRNA, HPRT1 and UBC. However, there is no universally accepted “best reference gene” for normalization. In the methods described herein the inventors have found that beta-actin is a ubiquitously expressed protein that can be utilized, for example, to help control for cell count. Given examples of the methods described herein are “pulsed” methods where cultured cells are transferred to an isotopically labeled media at same time as transfection with the recombinant nucleic acid, it is thought that the amount of isotopically labeled reference protein (e.g. beta-actin protein) is representative of the cell count during and after transfection. Accordingly, it is thought that normalization based on the amount of reference protein (e.g. beta actin) can be utilized to minimize the variability of target protein signal due to cell count. It is further thought that utilization of both “medium” and “heavy” labeled cells (i.e. no unlabeled samples) in the claimed method enables a cell count normalization ratio to be calculated.

[0260] In some examples, the reference protein is a protein that is expressed from an exogenous nucleic acid. Exogenous nucleic acids are any nucleic acid that is added from outside of the cell and includes DNA and RNA. In some examples, the reference protein is a protein that is expressed from a RNA-LNP. This may be referred to as a control RNA-LNP. Any reference protein may be used provide it can be distinguished from the test protein using mass spectrometry. For example, the control RNA-LNP may encode a different strain of the test protein or may encode a different protein. In both cases, the unique sequence of the protein encoded by the control RNA LNP enables the quantification of peptides from the reference protein, avoiding overlap with peptides from the protein being quantified. In some examples, the control RNA-LNP is mixed with the diluted test RNA-LNP at a single dose prior to transfecting the cells with the combined RNA-LNP mixture. This means that the amount of control RNA-LNP added remains constant, while the amount of test RNA-LNP varies to, for example, allow for normalization to the control RNA-LNP. The quantified peptides from the reference protein can be used to normalize the quantified peptides from the test protein (i.e. the protein(s) being quantified), functioning as a control for the LNP dosing process. It has been found that this normalization strategy improves the linearity (R2) across a range of doses and brings the potency slope ratio between two replicates of the same LNP closer to 1 (Example 12). Without wishing to be bound by theory, it is thought that this normalization strategy corrects for inherent well-to-well cell variability and LNP-induced cell toxicity (causing decreased protein expression), particularly at higher LNP doses. In some examples, the control RNA-LNP is the same formulation as the test RNA-LNP, such that only the sequence of the encoding protein differs. In alternative examples, the control RNA-LNP has a different formulation to the test RNA-LNP. For example, the lipid formulation may differ, the construct may differ and / or any salts, buffers or other excipients may differ.

[0261] Reference peptide

[0262] In some examples of the methods described herein, a known amount of a reference peptide may be added to the mixture prior to analysis. The reference peptide may be used as part of the normalization strategy and / or for absolute quantification of the reference protein. The reference peptide is typically a peptide derived from the reference protein (e.g. beta-actin), for example a peptide produced by digestion of the reference protein (e.g. a tryptic peptide from the reference protein). In some examples, the reference peptide is a beta actin peptide. Any suitable peptide derived from beta actin may be used but preferably the peptide comprises an arginine and / or a lysine. In some examples, the beta actin peptide is a tryptic beta actin peptide. In some examples, the beta actin peptide comprises the sequence GYSFTTTAER (SEQ ID NO: 1 ). Other peptides derived from beta actin may be used and can be selected by the person skilled in the art based on the known sequence of beta action and predicted protease cleavage sites (see, e.g. NCBI protein accession number: NP 001092.1 ). In some examples, the reference peptide is isotopically labeled. In some examples, the reference peptide is isotopically labeled such that its molecular weight differs from the naturally occurring (light) variant. In some examples, the reference peptide is isotopically labeled such that its molecular weight differs from the isotopically labeled variants generated by the methods described herein. In some examples, the reference peptide is isotopically labeled such that its molecular weight differs from the naturally occurring (light) variant and from the isotopically labeled variants generated by the methods described herein. In some examples, the reference peptide comprises the sequence GYSFTTTA(+4)ER(+10) (SEQ ID NO: 1 ). As would be understood by the skilled address, the molecular weight of alanine is 4 Daltons greater than the naturally occurring variant (e.g. D4-Alanine) and the molecular weight of arginine is 10 Daltons greater than the naturally occurring variant (e.g.13Ce,15N4-arginine). This means that the above peptide would have a molecular weight of 14 Daltons greater than the light equivalent.

[0263] Any suitable amount of isotopically labeled reference peptide may be used and the person skilled in the art would be able to determine the amount required to produce a signal that is above the detection limit, for example, of the mass spectrometer. Suitable isotopically labeled reference peptides may be prepared using techniques known to the person skilled in the (e.g. chemical synthesis) or obtained from a commercial supplier.

[0264] Standard

[0265] In some examples, the methods described herein comprise determining the absolute amount of the target protein, for example, as a concentration or amount per cell or per sample. In some examples, the methods described herein further comprise the step of combining the second sample with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture; (e) analyzing the second mixture to determine relative amounts of the second variant and third variant of the target protein or peptide thereof; and (f) determining the absolute amount or concentration of the second variant in the second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof. As used herein, the third, unlabeled variant of the target protein may also be referred to as a standard.

[0266] The third, unlabeled variant of the target protein or standard may be prepared by any technique known to the person skilled in the art for preparing proteins. For example production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. Effective culture conditions can be determined by the person skilled in the art include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. The polypeptides of the present invention may be extracted and purified from recombinant cells, such as plant, bacteria or yeast cells, producing said polypeptide by methods known to the person skilled in the art. In one embodiment, the method involves extracting total soluble proteins by homogenizing cells / tissues / plants and isolating the hexa-histidine polypeptide using a Ni-NTA or Talon. Additional purification may be achieved with conventional gel or affinity chromatography.

[0267] Once the third, unlabeled variant of the target protein or standard has been obtained it may be quantitated using any suitable technique known to the person skilled in the art. In some examples, the third, unlabeled variant of the target protein or standard is quantitated by amino acid analysis (also referred to as “AAA”).

[0268] In some examples, the standard may be used to construct a calibration curve for absolute quantification (e.g. copy number per cell) of the target protein. In one example, the second, or other labeled) sample is combined with increasing, known amounts of the standard, the mixture is analyzed, for example, by mass spectrometry and the standard is used to construct a standard curve. In some examples, the standard is digested with a protease (e.g. trypsin) before being combined with the labeled sample. In some examples, the standard is combined with the labeled sample before being digested with a protease (e.g. trypsin). The absolute amount or concentration of the second or other labeled variant in the sample may then be determined using the standard curve.

[0269] In some examples, the standard is used as an internal standard. In some examples, the standard is used for one-point quantification. In some examples, the standard is used for one-point quantification using IDMS. For example, a known amount of the third, unlabeled variant of the target protein (e.g. a recombinant protein) is combined with the first mixture to form a second mixture. The second mixture is then analysed to determine relative amounts of the labeled and unlabeled variants of the target protein or peptide thereof. The absolute amount or concentration of the labeled variants in the first sample may then be determined based on the known amount of the third, unlabeled variant of the target protein or peptide thereof. For example, the concentration of the first variant (e.g. medium labeled variant) can be determined using the following equation:

[0270] C (first variant) =

[0271] Peak area of first variant peptide

[0272] C (third, unlabelled variant) x (

[0273] Peak area of third, unlabelled variant peptide

[0274] For example, the concentration of the second variant (e.g. heavy labeled variant) can be determined using the following equation:

[0275] In the above equations, C(first variant), C(second variant) and C(unlabeled, third variant) are the concentrations for the first variant (e.g. medium labeled variant), second variant (e.g. heavy labeled variant) and third, unlabeled variant (e.g recombinant protein). In some examples, the standard is digested with a protease (e.g. trypsin) before being combined with the first sample. In some examples, the standard is combined with the first sample before being digested with a protease (e.g. trypsin).

[0276] In some examples, quantification of the target protein is performed by stable isotope dilution mass spectrometry (ID-MS). In some examples, quantification of the target protein is performed by stable isotope dilution mass spectrometry (ID-MS) using a light (unlabeled) peptide as the internal standard. In some examples, the light (unlabeled) peptide corresponds to one the peptides produced by digestion (e.g. protease digestion) of the reference protein. In some examples, the light (unlabeled) peptide corresponds to one the peptides produced by digestion (e.g. protease digestion) of the target protein. In some examples, the light (unlabeled) peptide corresponds to one the peptides produced by digestion the target protein using trypsin (e.g. a tryptic peptide).

[0277] Nucleic acid The methods of the present disclosure may be used for quantifying protein expression from a nucleic acid encoding the protein (e.g. a DNA or RNA vaccine). Typically, the nucleic acid is a recombinant nucleic acid which encodes for one or more polypeptides under the control of a promoter and / or other regulatory element.

[0278] The nucleic acid used in the methods of the present disclosure typically comprises a nucleotide sequence encoding a polypeptide of interest (e.g. the target protein in the methods described herein). The nucleotide sequence may encode any polypeptide known to the person skilled in the art, including any naturally or non-naturally occurring or otherwise modified polypeptide. The encoded polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide may have a therapeutic effect when expressed in a cell. In one example, the nucleotide sequence encodes an antigen (e.g., a pathogenic antigen). For example, the antigen can induce an immune response in the subject. Example antigens are defined hereinbelow.

[0279] In some examples, the nucleic acid (e.g. mRNA) may contain one or more intronic sequences capable of being excised from the nucleic acid (e.g. mRNA).

[0280] In some examples, the nucleic acid is RNA. In some examples, the RNA is messenger RNA. As used herein, the term “messenger RNA” (also referred to as mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. The mRNA may or may not be chemically modified. The mRNA of the present disclosure encompasses a non- replicating mRNA (also referred to as conventional mRNA (cRNA)), a self-replicating RNA (sa-mRNA), and any RNA that capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0281] In one example, the mRNA is cRNA. Typically, cRNA comprises, in order from 5’ to 3’: a 5’ cap structure, a 5’-UTR, a nucleotide sequence encoding a polypeptide of interest, a 3’-UTR and a tailing sequence (e.g. a polyadenylation signal or poly-A tail). The cRNA of the present disclosure may further comprise an translation internal ribosome entry site (e.g. Kozak consensus sequence or IRES). In some embodiments, the cRNA may also comprise a chain terminating nucleotide and / or a stem loop. In one example, the cRNA is a monocistronic cRNA. In one example, the cRNA is a multicistronic cRNA.

[0282] In one example, the mRNA is sa-mRNA (also referred to as a replicon or selfamplifying RNA). As used herein, the term “self-replicating RNA” refers to a construct based on an RNA virus that has been engineered to allow expression of heterologous RNA and proteins. Self-replicating RNA can also be referred to as a replicon. Selfreplicating RNA can amplify in host cells leading to expression of the desired gene product in the host cell. In one example, the present disclosure provides a monocistronic self-replicating RNA. In one example, the present disclosure provides a bicistronic selfreplicating RNA. In one example, the present disclosure provides a multi-cistronic selfreplicating RNA. The sa-mRNA of the present disclosure comprises one or more features of a cRNA, however, sa-mRNA further comprises nucleotide sequences encoding non-structural proteins (NSPs) which enables the sa-mRNA to direct its selfreplication. Non-structural proteins include at least one or more genes selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase and other non-structural viral proteins. The skilled person will understand that, in one example, self-replicating RNA can be based on the genomic RNA of RNA viruses. The RNA should be positive (+)-stranded so that it can be directly translated after delivery to a cell without the need for intervening replication steps (e.g., reverse transcription). Translation of the RNA results in the production of non-structural proteins (NSPs) which combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The replicase complex is the component of the sa-mRNA which amplifies the original RNA producing both antisense and sense transcripts, resulting in production of multiple daughter RNAs, and subsequently the encoded polypeptide of interest. For example, In one example, the self-replicating RNA comprises a viral replicase (or viral polymerase).

[0283] For example, the sa-mRNA comprises NSPs derived from (or based on) an alphavirus. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A. AR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus may also include chimeric alphaviruses (e.g., as described by Perri et al, (2003) J. Virol. 77(19): 10394-403) that contain genome sequences from more than one alphavirus. In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, for example, a positive-stranded RNA virus. Suitable positive-stranded RNA viruses suitable for use in the present disclosure will be apparent to the skilled person and include, for example, a picornavirus, a flavivirus, a rubivirus, a pestivirus, a hepacivirus, a calicivirus, or a coronavirus.

[0284] Typically the sa-mRNA also includes a subgenomic (SG) promoter which, when linked to a nucleotide sequence encoding NSPs and / or an polypeptide of interest, drives the expression of the NSPs and / or polypeptide of interest. The present disclosure provides a self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to a SG promoter. SG promoters (also known as ‘junction region’ promoters) suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimal sequence required for initiation of transcription. In one example, the self-replicating RNA comprises the non- structural proteins of the RNA virus, the 5’ and 3’ untranslated regions (UTRs) and the native subgenomic promoter. In another example, the self-replicating RNA comprises a 5'- and a 3'-end UTR of the RNA virus. In one example, the self-replicating RNA comprises a 5’ and 3’ conserved sequence elements (CSE). In one example, the selfreplicating RNA comprises a 5’- and a 3’-end CSE. As would be appreciated by the person skilled in the art, the 5’ and / or 3’ CSE may form part of the 5’ and / or 3’IITR or may form part of other components (e.g. the coding sequence for one or more of the non-structural proteins) of the self-replicating RNA.

[0285] The self-replicating RNA of the present disclosure cannot induce production of infectious viral particles. For example, the self-replicating RNA of the present disclosure does not comprise viral genes encoding structural proteins necessary for production of viral particles.

[0286] In some examples, the nucleic acid is DNA. In some examples, the DNA is plasmid DNA. As used herein, the term “plasmid DNA” (also referred to as mRNA) refers to any extra-chromosomal, independently replicating, circular DNA which encodes a polypeptide of interest and which is capable of being transcribed to produce mRNA which encodes the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Typically the plasmid DNA encodes one or more antigens, or immune response molecules. Such DNA vaccines can include, for example , hantavirus vaccines including those targeting Andes virus, Sin Nombre virus, Hantaan virus, and Puumala virus; South American arenavirus vaccines including those targeting Junin virus, Machup virus, Guanarito virus, and Sabia virus; poxvirus DNA vaccines, alphavirus DNA vaccines, filovirus DNA vaccines, and Zika virus DNA vaccines. The preceding list includes anti- viral DNA vaccines, but DNA vaccines against cancer and other infectious diseases are possible vaccines-of-interest. Although vaccines based on plasmid DNA differ from RNA vaccines in that they must first enter the cell nucleus to undergo transcription to yield the corresponding mRNA, the final step of translation from RNA into the protein antigen in the cytoplasm is the same as for RNA vaccines. Therefore, the methods described herein, which the inventors demonstrate can be used as potency assays for RNA vaccines, can also be used as potency assays for DNA vaccines as both are based measurement of protein antigen expression upon transfection of eukaryotic cells.

[0287] In some examples, the nucleic acid comprises one or more modification(s). Typically, modifications are introduced into a polynucleotide (e.g. mRNA) to increase the translation efficiency and / or stability of the polynucleotide. Suitable modifications to the polynucleotide will be apparent to the skilled person. DNA and / or RNA can be modified in many ways including chemically, structurally, and functionally, by methods known to those of skill in the biotechnological arts. Moreover, mRNA molecules can be modified by the introduction during transcription of alternative nucleotides nucleosides or nucleotides, as described in U.S. Pat. No. 10 8,278,036 (Kariko et al .) ; U.S. Pat. AppL No. 2013 / 0102034 (Schrum); U.S. Pat. AppL No. 2013 / 0115272 (deFougerolles et al) and U.S. Pat. AppL No. 2013 / 0123481 (deFougerolles et aL). In some examples, one or more nucleotide sequence(s) of the nucleic acid are codon optimized. Methods of codon optimization will be apparent to the skilled person and / or described herein. In some example, the nucleic acid (e.g. DNA or RNA) may include one or more alternative components, as known to the person skilled in the art, which impart useful properties including increased stability and / or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. For example, an alternative polynucleotide exhibits reduced degradation in a cell into which the polynucleotide is introduced, relative to a corresponding unaltered polynucleotide. These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and / or viability of contacted cells, as well as possess reduced immunogenicity. The methods described herein may be used to assess the impact of the alterations / modifications on the nucleic acid and protein expression. The methods described herein may be used to assess the impact one or more modifications may have on expression of the polypeptide from the nucleic acid. For example, an increase in the stability of the nucleic acid may by demonstrated by an increase in the quantity of protein measured using the methods described herein relative to the amount of protein produced by unmodified nucleic acid. The nucleic acid may be formulated with a delivery vehicle. Any suitable delivery vehicle may be used. Viral or non-viral delivery vectors may be used. Suitable delivery vehicles include lipid nanoparticles oil-in-water emulsions, cationic nanoemulsions, polymeric carriers, dendrimer carriers, polycation complexes, virus like replicon particles and the like.

[0288] In some examples, the delivery vehicle is a lipid nanoparticle. It will be apparent that the term “lipid nanoparticle” or “LNP” refers to any lipid composition, including, but not limited to, liposomes or vesicles, where an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or multiple; multilamellar) micelle-like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles, wherein solid lipid nanoparticles lack lipid bilayers. Lipid nanoparticles suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. In one example, the lipid nanoparticle comprises a cationic lipid. In one example, the lipid nanoparticle does not comprise a cationic lipid. In one example, the lipid nanoparticle further comprises a PEG-lipid, a sterol structural lipid and / or a neutral lipid. In one example, the lipid nanoparticle comprises a cationic lipid, a PEG-lipid, a sterol structural lipid and / or a neutral lipid. In one example, the lipid nanoparticle comprises 40% cationic lipid, 2% PEG-lipid, 48% sterol structural lipid and 10% neutral lipid, where the % are mol% based on the total amount of lipid.

[0289] In one example, the LNP comprises a PEG-lipid. For example, the PEG-lipid is selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG- DMPE, PEG-DPPC, a PEG-DSPE lipid and combinations thereof.

[0290] In one example, the LNP comprises a structural lipid. For example, the structural lipid is selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol and combinations thereof.

[0291] In one example, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. For example, the neutral lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1 ,2- dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1 ,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE), 1 ,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn- glycero- 3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof.

[0292] In one example, the LNP comprises a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1 ,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy- N,Ndimethyl- 3- aminopropane (DODMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5- bis((9z,12z)- octadeca-9,12,dien-l-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-l- yloxyl)benzyl-4- (dimethylamino)butnoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0293] In one example, the delivery vehicle is a polymeric microparticle. The skilled person will be aware that various polymers can form microparticles to encapsulate or adsorb the nucleic acid. It will be apparent that use of a substantially non-toxic polymer means that particles are safe, and the use of a biodegradable polymer means that the particles can be metabolized after delivery to avoid long-term persistence. Useful polymers are also sterilizable, to assist in the preparation of pharmaceutical grade formulations. Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(a- hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, poly anhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl- pyrrolidinones or polyester-amides, and combinations thereof.

[0294] In one example, the delivery vehicle is an oil-in-water cationic emulsion. Suitable oils for use in an oil-in-water emulsion will be apparent to the skilled person and / or are described herein. For example, the emulsion comprises one or more oils derived, for example, from an animal (e.g., fish) or a vegetable source (e.g., nuts, seeds, grains). The skilled person will recognize that biocompatible and biodegradable oils are preferentially used. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oils, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, corn oil.

[0295] In addition to the oil, the oil-in-water emulsion also comprises a cationic lipid to facilitate formation and stabilization of the emulsion. Suitable cationic lipids will be apparent to the skilled person and / or are described herein. Exemplary cationic lipids include, but are not limited to, limited to: 1 , 2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-Dimethylaminoethane)-carbamoyl] Cholesterol (DC Cholesterol), dimethyldioctadecyl-ammonium (DDA), l,2-Dimyristoyl-3-Trimethyl-

[0296] AmmoniumPropane (DMTAP), dipalmitoyl [C 16:0] trimethyl ammonium propane (DPTAP) and distearoyltrimethylammonium propane (DSTAP).

[0297] In some examples, the oil-in-water emulsion also comprises a non-ionic surfactant and / or a zwitterionic surfactant. The skilled person will be aware of surfactants suitable for use in the present disclosure. Exemplary surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0298] Antigens

[0299] The present inventors have found that the methods described herein are particularly useful for determining the potency of immunogenic compositions comprising RNA-encoding multiple antigens (e.g. multicistronic RNA or a sample comprising two or more RNA encoding different antigens). Antigens suitable for use in the immunogenic compositions described herein will be apparent to the skilled person and, for example, include proteins and peptides derived from any pathogen. For example, the antigen is from a virus, bacteria, a fungus or a protozoan.

[0300] Viral antigens that can be encoded by the RNA will be apparent to the skilled person and include, for example, proteins and peptides from a Orthomyxoviruses (e.g., Influenza A, B and C), Paramyxoviridae viruses (Pneumoviruses (e.g., Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus), Paramyxovirus types 1 -4 (PIV), Mumps, Sendai viruses, Simian virus 5)), Bovine parainfluenza virus, Nipahvirus, Henipavirus and Newcastle disease virus), Poxviridae (e.g., Variola vera, including but not limited to, Variola major and Variola minor, Metapneumoviruses, such as human metapneumovirus (hMPV) and avian metapneumoviruses (aMPV)), Morbilliviruses (e.g., Measles), Picornaviruses (e.g., Enteroviruses, Rhinoviruses, Heparnavirus, Parechovirus, Cardioviruses and Aphthoviruses), Enteroviruseses (e.g., Poliovirus types 1 , 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus types 1 to 9, 11 to 27 and 29 to 34 and Enterovirus 68 to 71 ), Bunyaviruses (e.g., California encephalitis virus), Phlebovirus (e.g., Rift Valley Fever virus), Nairovirus (e.g., Crimean- Congo hemorrhagic fever virus), Heparnaviruses (e.g., Hepatitis A virus (HAV)), Togaviruses (e.g., Rubivirus, an Alphavirus, or an Arterivirus), Flaviviruses (e.g., Tick- borne encephalitis (TBE) virus, Dengue (types 1 , 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus), Pestiviruses (e.g., Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV)), Hepadnaviruses (e.g., Hepatitis B virus, Hepatitis C virus), Rhabdoviruses (e.g., Lyssavirus (Rabies virus) and Vesiculovirus (VSV)), Caliciviridae (e.g., Norwalk virus, and Norwalk-like Viruses (e.g., Hawaii Virus and Snow Mountain Virus); Coronaviruses (e.g., severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV)), Retroviruses (e.g., Oncovirus, a Lentivirus or a Spumavirus), Reoviruses (e.g., Orthoreo virus, a Rotavirus, an Orbivirus, or a Coltivirus), Parvoviruses (e.g., Parvovirus B 19), Delta hepatitis virus (HDV), Hepatitis E virus (HEV), Human Herpesviruses (e.g., Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8)), Papovaviruses (e.g., Papillomaviruses and Polyomaviruses), Adenoviruess and Arenaviruses.

[0301] In one example, the antigen / s of the present disclosure is a viral antigen from a respiratory virus. Respiratory viral antigens that can be encoded by the RNA will be apparent to the skilled person and include, for example, proteins and peptides from a Orthomyxoviruses (e.g., Influenza A, B and C), Paramyxoviridae viruses (Pneumoviruses (e.g., Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus), Paramyxoviruses (PIV), and Metapneumovirus such as human metapneumovirus (hMPV) and avian metapneumoviruses (aMPV)), Picornaviruses (e.g., Rhinoviruses) and Coronaviruses (e.g., severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV)).

[0302] In one example, the antigen / s of the present disclosure is a viral antigen from an influenza virus. Influenza viruses are enveloped RNA viruses, belonging to the family Orthomyxoviridae. Three genera of this family, influenza virus A, B and C, cause influenza in humans. Influenza virus A and B are further classified, based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA).

[0303] Suitable influenza viral antigens include haemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 , M2, NB and BM2), a heterotrimeric RNA-dependent RNA polymerase (made up of one polymerase acidic subunit (PA), and two polymerase basic subunits (PB1 and PB2)), nucleoprotein (NP), and two non-structural proteins (NS1 and NS2; NS2 is also known as nuclear export protein (NEP)), and pro-apoptotic peptide (PB1 -F2).

[0304] At the time of filing there are 18 described HA (H1 -H18) and 11 described NA (N1 -N11) subtypes (e.g. “strains”) of influenza A viruses. There are two antigenically and genetically distinct lineages of influenza B viruses: the Victoria lineage (“BA / ictoria”) and the Yamagata lineage (“B / Yamagata).

[0305] Non limiting examples of influenza A subtypes include: H1 N1 , H1 N2, H1 N3, H1 N4, H1 N5, H1 N6, H1 N7, HINS, H1 N9, HINIO, HINII, H2NI, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8, H2N9, H2N10, H2NII, H3NI, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8, H3N9, H3N10, H3NII, H4NI, H4N2, H4N3, H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H4N10, H4NII, H5N1 H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H5N10, H5NII, H6NI, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N9, H6N10, H6NII, H7NI, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, N7N8, H7N9, H7N10, H7NII, HSNI, H8N2, H8N3, H8N4, H8N5, H8N6, H8N7, H8N8, H8N9, H8N10, H8NII, H9NI, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H9N10, H9NII, HIODNI, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, HIONS, H10N9, HIONIO, HIONII, HIINI, H1 1 N2, H1 1 N3, H11 N4, H1 1 N5, H1 1 N6, H1 1 N7, HUNS, H11 N9, HIINIO, HIINII, H12NI, H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H12N10, H12NII, H13NI, H13N2, H13N3, H13N4, H13N5, H13N6, H13N7, H13N8, H13N9, H13N10, H13NII, H14NI, H14N2, H14N3, H14N4, H14N5, H14N6, H14N7, H14N8, H14N9, H14N10, H14NII, H15NI, H15N2, H15N3, H15N4, H15N5, H15N6, H15N7, H15N8, H15N9,

[0306] H15N10, H15NII, H16NI, H16N2, H16N3, H16N4, H16N5, H16N6, H16N7, H16N8,

[0307] H16N9, H16N10, H16NII, H17NI, H17N2, H17N3, H17N4, H17N5, H17N6, H17N7,

[0308] H17N8, H17N9, H17N10, H17NII, H18NI, H18N2, H18N3, H18N4, H18N5, H18N6,

[0309] H18N7, H18N8, H18N9, H18N10, and H18NII. Influenza virus strains for use in seasonal vaccines change from season to season. Authorities such as the World Health Organisation (WHO) and Centre for Disease Control (CDC) publish a list of the predominant circulating influenza strains each year and further publish recommended influenza strains for inclusion in influenza vaccines, or reference strains to guide selection of the strains for inclusion in influenza vaccines. Accordingly, the first, second, and / or fourth influenza strain may be recommended for inclusion in an immunogenic composition by a public health authority (e.g., the WHO).

[0310] As used herein, the term “seasonal influenza virus strain” refers to a strain of influenza virus to which a subject population is exposed to on a seasonal basis. The immunogenic composition described herein may be suitable for protecting against seasonal virus strains that are presently being spread or are endemic within a human population. In addition, the immunogenic composition described herein may be suitable for protecting against emerging seasonal virus strains. Nucleotide sequences of all known influenza strains and antigens can be readily determined via online databases, such as the Influenza Virus Resource

[0311] (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / ).

[0312] Non-limiting examples of influenza A strains include: influenza A / Califomia / 07 / 2009 (HINI); influenza A / Michigan / 45 / 2015 / (HINI); influenza A / Netherlands / 602 / 2009 (HINI); influenza A / Vietnam / 1194 / 2004 (H5NI); influenza A / Vietnam / 1203 / 2004 (H5NI); influenza A / Hong Kong / 4801 / 2014 (H3N2); A / Hong Kong / 2671 / 2019 / (H3N2); influenza A / Panama / 2007 / 1999 (H3N2); influenza

[0313] A / Kansas / 14 / 2017 / (H3N2); influenza A / South Australia / 34 / 2019 / (H3N2); influenza A / Tasmania / 503 / 2020 / (H3N2); influenza A / Cambodia / e08263601 / 2020 / (H3N2); influenza A / Darwin / 6 / 2021 / (H3N2);. influenza A / Perth / 20 / 2020 / (H3N2).

[0314] Non-limiting examples of influenza B strains include B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus; B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0315] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two seasonal influenza virus strains. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from three seasonal influenza virus strains. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from four seasonal influenza virus strains. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from three influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from four influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza A viruses and one influenza B virus. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza A viruses and two influenza B viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1 N1 virus, an influenza A H3N2 virus, and an influenza B virus. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1 N1 virus, an influenza A H3N2 virus, an influenza B / Victoria virus, and an influenza B / Yamagata virus.

[0316] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1 N1 strain selected from: A / Califomia / 07 / 2009 (HINI); influenza A / Michigan / 45 / 2015 / (HINI); influenza A / Netherlands / 602 / 2009 (HINI); and A / Sydney / 5 / 2021 (H1 N1 ).

[0317] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H3N2 strain selected from: influenza A / Hong Kong / 4801 / 2014 (H3N2); A / Hong

[0318] Kong / 2671 / 2019 / (H3N2); influenza A / Panama / 2007 / 1999 (H3N2); influenza

[0319] A / Kansas / 14 / 2017 / (H3N2); influenza A / South Australia / 34 / 2019 / (H3N2); influenza A / Tasmania / 503 / 2020 / (H3N2); influenza A / Cambodia / e08263601 / 2020 / (H3N2); influenza A / Darwin / 6 / 2021 / (H3N2); influenza A / Perth / 20 / 2020 / (H3N2); influenza A / turkey / Turkey / 1 / 2005 and / or influenza A / chicken / Ohio / 494832 / 2007).

[0320] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage) and / or B / Singapore / INFTT-16-0610 / 2016 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from two influenza B strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Victoria strain and an influenza B / Yamagata strain. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Victoria strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Yamagata strain selected from: B / Phuket / 3073 / 2013 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an A / Sydney / 5 / 2021 (H1 N1 ); an A / Darwin / 6 / 2021 (H3N2); a B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus; and a B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0321] In one example, the antigen / s of the present disclosure is a viral antigen from a coronavirus, for example an alphacoronavirus, a betacoronavirus, a gammacoronavirus and / or a deltacoronavirus.

[0322] In one example, the antigen / s of the present disclosure is a viral antigen from a an alphacoronavirus. Examples of alphacoronaviruses include Alphacoronavirus 1 , Human coronavirus 229E (HCoV 229E), Human coronavirus NL63 (HCoV NL63), Miniopterus bat coronavirus 1 , Miniopterus bat coronavirus HKU8, Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2 and Scotophilus bat coronavirus 512.

[0323] In one example, the antigen / s of the present disclosure is a viral antigen from a a gammacoronavirus. Examples of gammacoronaviruses include Avian coronavirus and Beluga whale coronavirus SW1 .

[0324] In one example the antigen / s of the present disclosure is a viral antigen from a deltacoronavirus. Examples of deltacoronaviruses include Bulbul coronavirus HKU11 and Porcine coronavirus HKU15

[0325] In one example, the antigen / s of the present disclosure is a viral antigen from a a betacoronavirus. Examples of betacoronaviruses include Betacoronavirus 1 (Bovine Coronavirus, Human coronavirus OC43), Hedgehog coronavirus 1 , Human coronavirus HKU1 (HCoV HKU1 ), Middle East respiratory syndrome-related coronavirus (MERS- CoV), Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS- CoV-2) and Tylonycteris bat coronavirus HKU4.

[0326] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from a betacoronavirus selected from the group consisting of Middle East respiratory syndrome-related coronavirus (MERS-CoV) and Severe acute respiratory syndrome-related coronavirus (SARS-CoV or SARS-CoV-2). For example, the viral antigen is from MERS-CoV. In another example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from SARS-CoV. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from SARS-CoV-2.

[0327] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein of a coronavirus. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein of SARS-CoV-2. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020.

[0328] The present disclosure contemplates that the viral antigen can be a modified S protein. In an example, a modified S protein comprises a mutation in the receptor binding domain. For example, the mutation is selected from the group consisting of S438F, N439K, N440K, L441 I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471 O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, P479L, P479L, P479S, N481 D, N481 H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491 R, Q493L, S494P, Y495N, T500N, N501 S and Y505H, Y508H. In an example, a modified S protein comprises a mutation in the receptor binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483A and E484D.

[0329] In an example, a modified S protein comprises a mutation selected from the group consisting of P337S, F338L, F338C, G339D, E340K, V341 I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417R, K417N, 1418V, Y421 S, Y423C, Y423F, Y423S, D427Y, R509K, V510L, V511 E, V512L, L518I, H519O, A520S, A520V, P521 R, P521 S, A522P, A522S and D614G.

[0330] In one example, the antigen / s of the present disclosure is a viral antigen from a respiratory syncytial virus strain. In an example, respiratory syncytial virus strain is a RSV-A. In an example, respiratory syncytial virus strain is a RSV-B.

[0331] In one example, the antigen is an attachment glycoprotein (G), the fusion protein (F), or the small hydrophobic protein (SH). In one example, the RNA encodes the G protein from an RSV-A strain. In one example, the RNA encodes the G protein from an RSV-B strain.

[0332] In one example, the antigen / s of the present disclosure is a viral antigen from a parainfluenza virus (HPIV) strain. In one example, the parainfluenza virus strain is HPIV- 1 , HPIV-2, HPIV-3, or HPIV-4. In one example, HPIV-4 is HPIV-4A or HPIV-4B. In one example, the antigen is the nucleocapsid protein (NP), the phosphoprotein (P), the fusion glycoprotein (F), the matrix protein (M), the hemagglutinin-neuraminidase (HN) glycoprotein, or the RNA polymerase (L). For example, the RNA encodes one or more of the NP, P, F, M, HN, or L protein from HPIV-1 , HPIV-2, HPIV-3, or HPIV-4.

[0333] In one example, the antigen / s of the present disclosure is a viral antigen from a human metapneumovirus (HMPV) strain. In one example, the human metapneumovirus virus strain is HMPV A1 , HMPV A2a, HMPV A2b, HMPV B1 , or HMPV B2. In one example, the antigen is the nucleocapsid protein (N), the phosphoprotein (P), the fusion

[0334] (F), the matrix protein (M), the M2 protein, the SH protein, the attachment glycoprotein

[0335] (G), or the RNA polymerase (L). For example, RNA encodes one or more of the N, P, M, F, M2, SH, G, or L protein from HMPV A1 , HMPV A2a, HMPV A2b, HMPV B1 , or HMPV B2.

[0336] In one example, the antigen / s of the present disclosure is a viral antigen from an Epstein-Barr virus (EBV) strain. In one example, the EBV strain is EBV-1 or EBV-2. EBV-1 and EBV-2 can further be categorised according to the EBV nuclear antigen (EBNA-1 ). In one example, the EBV strain has nuclear antigen EBNA-2, EBNA-3A / 3, EBNA-3B / 4, or EBNA-3C / 6.

[0337] In one example, the antigen is EBNA-1 . For example, RNA encodes one or more of the EBNA-1 protein from EBV-1 or EBV-2.

[0338] In one example, the nucleic acid (e.g. RNA) comprises a nucleotide sequence that encodes an antigen from a virus. In one example, the nucleic acid comprises a nucleotide sequence that encodes one or more antigens from a respiratory virus, for example, influenza virus, coronavirus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV) or human parainfluenza virus (PIV). In one example, the nucleic acid comprises a nucleotide sequence that encodes one or more antigens from a Epstein-Barr virus (EBV).

[0339] In examples wherein the virus is influenza, the nucleic acid may have a coding region encoding at least one antigenic peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1 ), matrix protein 2 (M2), non-structural protein 1 (NS1 ), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1 , PB1 -F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant thereof. In one example, the coding region encodes at least one antigenic peptide or protein derived from hemagglutinin (HA) and / or neuraminidase (NA) of an influenza virus or a fragment or variant thereof. The HA and / or NA may, independently, be derived from an influenza A virus or an influenza B virus or a fragment of either.

[0340] In examples wherein the virus is coronavirus, the nucleic acid may have a coding region encoding at least one antigenic peptide or protein derived from Spike (S) protein and / or nucleocapsid (N) protein. In one example, the antigen is from SARS-CoV-2. The S and / or N may, independently, be derived from an variant of SARS-CoV-2 (e.g. the original strain, alpha, delta, omicron) or a fragment of either.

[0341] In examples wherein the virus is RSV, the nucleic acid may have a coding region encoding at least one antigenic peptide or protein derived from the fusion (F) protein and / or glycoprotein (G) protein.

[0342] In examples wherein the virus is PIV, the nucleic acid may have a coding region encoding at least one antigenic peptide or protein derived from fusion (F) protein and / or hemagglutinin-neuraminidase (HN) protein. In one example, the antigen is from PIV3.

[0343] In examples wherein the virus is HMPV, the nucleic acid may have a coding region encoding at least one antigenic peptide or protein derived from the fusion (F) protein and / or glycoprotein (G) protein.

[0344] Protein extraction

[0345] The present inventors have also developed an in-well cell lysis method that is suitable for use in the methods described herein, for example in the high-throughput methods. The method substantially reduces the amount of time required to perform the methods described herein, for example, relative to conventional cell lysis methods which use an enzyme such as trypsin or TrypLE to detach cells, followed by centrifugation and cell resuspension in a lysis buffer.

[0346] Accordingly, the present application also provides an in-well method for extracting proteins from adhered cells, the method comprising incubating the adhered cells with lysis buffer in the absence of an exogenous protease. In some examples, the lysis buffer comprises a surfactant. Any suitable surfactant may be used. In some examples, the surfactant is an anionic surfactant, In some examples, the surfactant is RapiGest surfactant (also known as sodium 3-[(2-methyl-2-undecyl-1 ,3-dioxolan-4- yl)methoxy]-1 -propanesulfonate). In some examples, the surfactant is as described in US 8,580,533. For example, the surfactant may represented by represented by formula (I): wherein p is 0, 1 or 2;

[0347] R is alkyl;

[0348] Ri and R2 are each, independently, hydrogen or methyl; and

[0349] R3is selected from -OSO3_, -R4OSO3_, -R4OR5SO3_, and -OR5SO3_, wherein R4and R5 are each, independently, lower alkyl.

[0350] For example, the surfactant may be represented by formula II: wherein Re is alkyl; and

[0351] R7 is selected from -OSO3_, -R4OSO3_, -R4OR5SO3_, and -ORsSOs-, wherein R4 and R5 are each, independently, lower alkyl.

[0352] In some examples, the surfactant has the following chemical structure:

[0353] In some examples, the surfactant has the following chemical structure:

[0354] In some examples, the lysis buffer further comprises a nuclease, for example a DNase. Any suitable nuclease may be used. The nuclease is present in a sufficient amount to degrade or partially degrade the released cellular DNA such that the viscosity of the sample is reduced. The wells may form part of a 6-well, 12-well, 24- well, 48-well, 96-well or 384-well plate. In some examples, the treated cell mixture is mixed, for example at 1000 rpm for 10 min using a plate shaker to lyse the cells. The cell lysate may then be transferred from the plate. In some examples, the corresponding heavy and medium labeled lysates are mixed at equal volumes. In some examples the method does not comprise adding a protease to detach the cells from the plate. In some examples, the method does not comprise adding a trypsin-like protease to detach the cells from the plate. In some examples, the method does not comprise adding TrypLE enzyme to detach the cells.

[0355] In some examples, the method further comprises removing the insoluble material. Any suitable technique may be used. However, the present inventors have found that centrifuging the sample without a chloroform extraction step is sufficient to separate the insoluble material. The supernatant can then be used for further analysis. Accordingly, in some examples, the method further comprises a centrifugation step. In some examples, the method does not comprise a chloroform extraction step.

[0356] Embodiments of this in-well lysis strategy provide an alternative to a cell harvesting strategy, which uses TrypLE enzyme to detach cells, followed by centrifugation and cell resuspension in a suitable lysis buffer (e.g. Rapigest). Removing the centrifugation and cell resuspension steps greatly reduces the assay time and / or minimizes the potential for variability with a simplified process.

[0357] Uses

[0358] The methods described herein are useful for quality control during manufacture of a nucleic acid and for characterization of a nucleic acid as an active pharmaceutical ingredient (API) in final therapeutic products, including vaccines. In some examples, the methods and uses described herein are useful for determining the potency of a nucleic acid vaccine. In some examples, the methods and uses described herein are useful for determining the potency of a RNA-LNP. For example, the methods and uses described herein may be used to compare a test RNA-LNP (e.g. from a new batch, new process, new formulation etc.) to a reference standard RNA-LNP. The methods and uses herein may also be used to assess the impact of modifications to the nucleic acid, changes to the formulation, changes to the process and the like on the potency of the nucleic acid. The methods and uses herein may also be used to quantify the amount of protein produced by the nucleic acid. The methods an uses herein may also be used to monitor the potency of a recombinant nucleic acid, e.g. an RNA-LNP, over time.

[0359] The methods and uses described herein may be applied to any vaccine antigen. This can be achieved by adjusting the MRM MS parameters to monitor peptides of interest from the target protein of interest. The methods and uses described herein are particularly advantageous for analysis of multivalent vaccine drug products as it is possible to adjust the MS parameters to include peptide targets from several antigens simultaneously. The methods and uses described herein may be applied to various cell lines. The Examples described herein demonstrated that the methods can be used across several different cell lines but the person skilled in the art would appreciate that these methods can be readily applied to other cell lines. The methods described herein are also capable of being used for either relative or absolute quantification of antigens. For example, the method may be used to quantify the average amount of each antigen per cell.

[0360] The present application also provides a process for releasing or accepting a batch of a pharmaceutical composition comprising a recombinant nucleic acid encoding a target protein, comprising

[0361] (i) determining the relative in vitro potency of a test sample of the recombinant nucleic acid from the batch according to the methods described herein; and

[0362] (ii) releasing further pharmaceutical compositions from the batch for in vivo use if the results of step (i) indicate an acceptable relative in vitro potency value.

[0363] The present application also provides a process for releasing or accepting a batch of a batch of a pharmaceutical composition comprising an RNA encapsulated in an LNP, comprising

[0364] (i) determining the relative in vitro potency of a test sample of the pharmaceutical composition from the batch according to the methods described herein; and

[0365] (ii) releasing further pharmaceutical compositions from the batch for in vivo use if the results of step (i) indicate an acceptable relative in vitro potency value.

[0366] Kit

[0367] The present application further provides kits comprising various reagents and materials useful for carrying out the methods described herein. The quantitative methods described herein may be performed by diagnostic laboratories, experimental laboratories, or commercial laboratories and others. The disclosed kits can be used in these and other different settings.

[0368] As used herein, the term “kit” refers to any delivery system for delivering materials. Such delivery systems may include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme (for example, a protease) for use in the methods described herein, while a second container may contain one or more labeled reagents (such as, isotopically labeled lysine and / or isotopically labeled arginine). Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0369] For example, materials and reagents for quantifying protein expression may be assembled together in a kit. In one example, a kit comprises labeled reagents (e.g. isotopically labeled amino acids) and instructions for using the kit according to the methods described herein. In one example, the kits may further comprise proteases and instructions for using the same. The kits may also further comprise minimal media to which labeled or unlabeled amino acids can be added prior to use and instructions for using the same. The kit may further comprise a peptide for use as a standard and instructions for use. In one example, the kit comprises a protease, isotopically labeled lysine and isotopically labeled arginine. In one example, the kit comprises a protease, isotopically labeled arginine, isotopically labeled lysine and an isotopically labeled peptide. The kit may also comprise a suitable standard as defined herein. Kits or other articles of manufacture as described herein may include one or more containers to hold various reagents. Suitable containers include, for example, bottles, vials, ampules and the like. The container may be formed from a variety of materials such as glass or plastic.

[0370] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0371] EXAMPLES

[0372] Example 1 - Synthesis of RNA

[0373] The synthesis of RNA was conducted under RNAse-free conditions. All tubes, vials, pipette tips, pipettes, buffers, etc. were required to be nuclease-free. The following constructs were prepared:

[0374] • sa-mRNA expressing H5 and N1 antigen and NSP1 -4. • sa-mRNA expressing H1 and N1 antigen and NSP1 -4.

[0375] • sa-mRNA expressing H3 and N2 antigen and NSP1 -4.

[0376] • sa-mRNA expressing influenza B / Victoria HA and NA antigens and NSP1 -4.

[0377] • sa-mRNA expressing influenza B / Yamagata HA and NA antigens and NSP1 -4.

[0378] • sa-mRNA expressing SARS-CoV2 spike antigen and NSP1 -4.

[0379] • sa-mRNA expressing SARS-CoV2 spike and nucleocapsid antigens and NSP1 - 4.

[0380] DNA templates encoding the self-replicating RNAs were produced in competent Escherichia coli cells that were transformed with a DNA plasmid. Individual bacterial colonies were isolated and the resultant plasmid DNA amplified in E. coli cultures. Following fermentation, the plasmid DNA was isolated and linearized by restriction digest. Restriction enzymes were then removed using phenol / chloroform extraction and ethanol precipitation. mRNA was made by in vitro transcription from the linearized DNA template using a T7 RNA polymerase. Subsequently, the DNA template was removed by DNase digestion. Enzymatic capping using VCE was performed to add CapO and provide functional mRNA. The resultant mRNA was purified and resuspended in nuclease-free water.

[0381] Example 2- Preparation of LNP

[0382] An RNA-containing lipid nanoparticle (RNA-LNP) composition was prepared using an ionizable cationic lipid, additional helper lipids and the mRNA produced as described in Example 1 .

[0383] LKY750, 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1 ,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG2k) were combined in a 40:10:48:2 molar ratio in ethanol at a concentration of 3.2 mM. A solution of mRNA in 50 mM citrate buffer at pH 6 was prepared at 0.025 mg / mL. The lipid solution in ethanol was then rapidly mixed with the mRNA in citrate buffer using a staggered herringbone micromixer such as a NanoAssemblr benchtop instrument (Precision Nanosystems). The total flow rate (TFR) was 12 mL / min and the flow rate ratio (FRR) was 2:1. This mixing ratio resulted in an 8:1 ratio of ionizable cationic lipids to RNA phosphate groups (N:P ratio) and a lipid to RNA mass ratio of 37:1. The mixed solution was diluted 10- fold into 50 mM citrate buffer at pH 6 and subjected to tangential flow filtration (TFF) using a 300k molecular weight cut-off membrane (mPES) until concentrated to the original volume.

[0384] Subsequently, the citrate buffer was replaced with a buffer containing 20 mM T ris buffer at pH 7.5, 80 mM sodium chloride, and 3% sucrose using diafiltration with a 10- fold volume of the new buffer. The LNP solution was concentrated to a volume of between 5-10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1 °C / min using a Corning® CoolCell® LX Cell Freezing Container until the samples reach -80°C. Samples will be stored at -80°C until needed for further assays. Samples may be filtered with an anion exchanger before or after storage at -80°C.

[0385] LNPs containing mRNA were analyzed to determine RNA concentration using the Ribogreen assay.

[0386] Example 3- Quantification of protein expression

[0387] This example demonstrates the quantification of protein expressed from the LNP of Example 2.

[0388] The media for use in this Example were prepared as follows:

[0389] • Light media: DMEM (ThermoFisher), 10% FBS, 1 % Pen / Strep, 1 % L-glutamine.

[0390] • Medium media: Dialyzed FBS (10 kDa MWCO, ThermoFisher), Pen / Strep and R(+6) and K(+4) were combined with R- and K-free RPM1 1640 media for SILAC.

[0391] • Heavy media: Dialyzed FBS (10 kDa MWCO, ThermoFisher), Pen / Strep and R(+10) and (+8)K were combined with R- and K-free RPMI 1640 media for SILAC.

[0392] LNPs containing mRNA were diluted to a concentration of 5 ng / pL using serum free media. The diluted LNP was diluted further to the required concentration using either medium or heavy labeled media.

[0393] BHK21 cells were initially grown in light medium. To determine the potency of the LNP formulation, the BHK21 were treated in a 6-well plate. Each well was seeded with 0.5x106cells in 2 mL of light medium 24 h before treatment and incubated in a growth chamber in 5% CO2 at 37 °C. The cells were grown to cell confluency of about 80%. The growth medium was changed to medium or heavy medium containing the desired amount of LNP (±ApoE) and the cells were incubated in a growth chamber in 5% CO2 at 37 °C for a further 24 h. Untreated BHK21 cells were used as a control.

[0394] Cells were harvested by aspirating the media from each well of the 6-well plate. TrypLE (400 pL) was added to each well and the plate incubated at 37 °C for approximately 5 min. The detached cells were dislodged from the plates by gentle pipetting of the TrypLE solution. The cells dislodged from the wells of plates incubated with the medium media were transferred to individual 1 .5 mL tubes. The 400 uL of cells dislodged from the wells of plates incubated with the heavy media were spiked into each tube containing medium cells. The spiked samples were centrifuged (500rcf, 4 °C) to pellet the cells. The supernatant was removed and the cells resuspended in PBS (200 uL). The resuspended cells were centrifuged (500rcf, 4 °C) to pellet the cells and the supernatant removed.

[0395] The cells were lysed by resuspending the cell pellet in a solution of RapiGest (25 pL; 0.5% v / v in 50 mM Tris pH 8.5). The resuspended cells were incubated at 100 °C for 5 minutes in a heat block. 75 pL of Tris buffer, pH 8.5 (150 mM) was then added to the samples.

[0396] 10 pL of sequencing grade Trypsin (0.5 mg / mL) was added to each sample and the sample was vortexed to combine and quick spin. The samples were then incubated at 37 °C for 2 hrs. The trypsin digest was stopped by adding 10 pL of 0.45 M HCI to each sample and vortexing. The samples were then incubated for a further 20 min at room temperature before being vortexed again.

[0397] 100 pL chloroform was added to each sample and the samples vortexed. The samples were centrifuged at 21 ,000 g for 10 min at RT before the supernatant loaded into glass LC vials for analysis.

[0398] After trypsin digestion, multiple reaction monitoring (MRM) for each light, medium, and heavy HA and NA peptide as well as a beta-actin peptide was performed. Briefly, analysis of the trypsin digested samples was conducted with a Vanquish UPLC (Thermo) and a TSQ Altis triple quadrupole mass spectrometer (Thermo) using a Hypersil gold reverse phase column C18. Mobile phases of 0.1% formic acid in water and 0.1 % formic acid in acetonitrile were used for separation at a flow rate of 0.35 mL / min and a column temperature of 50°C. 25 pL of sample was injected with a runtime of 30 min. The parameters used for LC-MS analysis are provided in Tables 1 , 2 and 3. MRM peptide settings are provided in Figure 6.

[0399] Table 1 : General Liquid Chromatography Parameters Table 3: Mass Spectrometer Settings Mass spectra were obtained in the negative ion mode, using MS / MS multiple reaction monitoring. The mass spectrometer running parameters used are provided in Table 3. Spectra were first analyzed using Skyline software (available online at httpsv' / skyline.ms / projec home / software / Skyline / begin.view) or TraceFinder software (ThermoFisher).

[0400] The peak areas for each peptide being monitored were calculated from the MS spectrum. A number of different beta-actin normalization strategies were used. The first, termed the “simple direct normalization,” uses the ratio of “heavy” to “medium beta actin signal as a normalization factor. In this strategy, the cell count normalization factor for each sample is calculated by dividing the heavy beta actin (from the GYS peptide) peak area by the medium heavy beta actin (from the GYS peptide) peak area. For the individual peptide representing the target protein, the medium peptide is divided by the peak area for heavy peptide peak area for each sample. The relative amount (or cell count normalized ratio) of the target protein is then determined by multiplying this peptide ratio by the cell count normalization factor for each sample. In other words, the cell count normalized ratio is for each sample is determined using equation (1 ) as shown below: cell count normalized ratio =

[0401] Medium peptide peak area Heavy beta actin peak area - * - Heavy peptide peak area Medium beta actin peak area

[0402] The second method used either an indirect light (IL) or total (IT) beta-actin normalization factor. In this strategy, as shown in Figure 2, a ratio (R) of light beta-actin to heavy beta-actin in a control sample of heavy cells is determined empirically by equation (I) below.

[0403] (I) R = LH_C / LH-C

[0404] The heavy cells are also spiked into the targeted medium (M) cells as an internal standard. The light beta-actin from the heavy cells (LH) after spiking can then be calculated using the R from equation (I) and measured heavy beta-actin (HMH) in the mixture as shown in equation (II) below.

[0405] (II) LH=R*HMH The light beta-actin from the medium cells (LM) is then calculated by subtracting the total measured light beta actin signal (LMH) with the light beta-actin from the heavy cells (LH) using equation (III) below.

[0406] (Ill) LM = LMH - LH

[0407] A normalization factor (NF) can then be calculated using either the “light beta-actin” or “total beta-actin” method using the equations (IVa) or (IVb) below, respectively:

[0408] (IVa) NFIL= LH / LM

[0409] (IVb) NFIT=(LH + HH) / (LM+ MM)

[0410] The medium to heavy relative protein abundance in the MH samples can be calculated and normalized using NF:

[0411] The normalization methods appeared to be successful in minimizing variability observed in the raw results ratio following beta-actin normalization (Figure 5). The results show a clear dose-dependent response upon normalization using both strategies. Due to its simplicity, the simple normalization method was chosen for later experiments, however both can be used.

[0412] This example demonstrates that the “pulsed” approach adopted herein which utilizes both “medium” and “heavy” isotopically labeled media immediately following dosing (i.e. concurrently with transfection) allows for a targeted analysis of antigens to assess LNP potency. The methods described herein allow for cell count normalization strategy, for example based on beta-actin based normalization, where beta-actin is generated with isotopic labeling only following LNP dosing. This allows for the beta-actin MS signal to be representative of cell count at the time of dosing, enabling a cell count normalization strategy based on the amount of “heavy” and “medium” beta actin in each sample. Preliminary results show a clear dose-dependent response following normalization (Figure 7).

[0413] Example 4: Comparing test LNP to reference LNP The methods described herein, which for example use “medium” and “heavy” isotopically labeled media, allow a direct comparison between two samples or between a test sample and a reference sample. For example, cells in “medium” media may be dosed with a test LNP, while cells in “heavy” media may be dosed with a reference LNP. The standard “heavy” cells can then be spiked into the sample “medium” cells before lysis and digestion, allowing for simultaneous processing of the reference and test LNP dosed cells. In this way dosed “heavy” cells can be utilized both as an internal standard for normalization and as a direct sample comparison.

[0414] In the present example, the potency of a new LNP lot was tested against the potency of an older LNP reference standard. Following the methods provided in Examples 1 -3, cells in “medium” media were dosed with the new LNP sample and cells in “heavy” media were dosed with the previous LNP and the beta-actin normalized Medium / Heavy ratio was calculated for HA peptides and NA peptides. The beta-actin normalized Medium / Heavy ratio for different antigen peptides was approximately 0.5 (Table 4). This indicates that the antigen MS signals for the new LNP sample were roughly half that of the old LNP sample, meaning the potency of the new lot is roughly half that of the old lot.

[0415] Table 4. Beta-actin normalized Medium / Heavy ratio can be used to directly compare two different LNP lots.

[0416] Example 5: Direct quantification of antigens

[0417] The methods described herein allow for the direct quantification of the expressed antigens, for example using isotope dilution mass spectrometry (IDMS). In this example, transfected cells that are cultured in heavy SILAC medium can be utilized as an internal standard. These heavy cells are spiked into the target sample (cultured in medium media), as well as into light peptide / protein standards of known concentrations. In the present example, samples cultured in medium SILAC media were spiked with cells cultured in heavy SILAC media. The same mixture of heavy cells (post tryptic digest and clean up) was spiked into a light peptide serial dilution to generate a standard curve for quantification. The heavy cells were used as an internal standard so that the medium cell samples can be quantified using the standard curve.

[0418] Preliminary experiments of cells dosed with LNP at various amounts shows that peptides from the target antigen (HA B / Yamagata) can be directly quantified. As shown in Figure 8, two peptides generated from the same target protein (GVL and SYF) show close agreement in their quantification.

[0419] Example 6: Quantification of antigens using recombinant protein

[0420] This example demonstrates the quantification of protein expressed from the LNP of Example 2 using a number of different quantification methods. The methods described herein produce isotopically labeled target antigens, which enables the use of light peptides or recombinant proteins that match the targeted proteins to be directly added to the sample for simultaneous sample processing and quantification. In the present example, five methods of quantification were tested, which each yielded similar results. With the first method, a standard curve was generated using heavy cells that had been dosed with LNP and then spiked with light peptides of varying concentrations. The heavy cells were then spiked into the target sample (e.g. medium cells) as an internal standard for quantification. The second method generated a standard curve using recombinant proteins of a single concentration spiked into manufactured heavy peptides of varying concentrations. The recombinant proteins were then added to the target sample (e.g. medium cells and / or heavy cells) as an internal standard for quantification. Method three used single point quantification with recombinant proteins, where the light recombinant proteins of a known concentration were spiked into the sample and underwent sample processing along with the target medium and heavy labeled antigens. The relative peak areas and the concentration of the recombinant protein were then used to calculate the corresponding medium and heavy peptide concentrations according to the following equations: . Method four used a standard curve generated using manufactured light peptides of one concentration (which are also spiked into the sample (e.g. heavy and / or medium cells) as an internal standard) and manufactured heavy peptides of varying concentrations. Method five used single point quantification with manufactured light peptides of a known amount spiked into the sample. The following methods were used to prepare the samples for quantification.

[0421] The cells (treated and untreated) were cultured and transfected as described in Example 3. Cells were harvested by aspirating the media from each well of the 6-well plate. TrypLE (400 pL) was added to each well and the plate incubated at 37 °C for approximately 5 min. The detached cells were dislodged from the plates by gentle pipetting of the TrypLE solution. The cells dislodged from the wells of plates incubated with the medium media were transferred to individual 1 .5 mL tubes. The 400 uL of cells dislodged from the wells of plates incubated with the heavy media were spiked into each tube containing medium cells. The spiked samples were centrifuged (500rcf, 4 °C) to pellet the cells. The supernatant was removed and the cells resuspended in PBS (200 uL). The resuspended cells were centrifuged (500rcf, 4 °C) to pellet the cells and the supernatant removed.

[0422] The cells were lysed by resuspending the cell pellet in a solution of RapiGest (25 pL; 0.5% v / v in 50 mM Tris pH 8.5). Where required, 50 pL of the corresponding recombinant proteins of a known concentration (10 ug / mL) were added to the cells. The resuspended cells were incubated at 100 °C for 5 minutes in a heat block. 25 pL of Tris buffer, pH 8.5 (150 mM) was then added to the samples. If the corresponding recombinant protein was not added, 75 pL of Tris buffer, pH 8.5 (150 mM) was added to the samples instead of 25 pL.

[0423] 10 pL of sequencing grade Trypsin (0.5 mg / mL) was added to each sample and the sample was vortexed to combine and quick spin. The samples were then incubated at 37 °C for 2 hrs. The trypsin digest was stopped by adding 10 pL of 0.45 M HCI to each sample and vortexing. The samples were then incubated for a further 20 min at room temperature before being vortexed again.

[0424] 100 pL chloroform was added to each sample and the samples vortexed. The samples were centrifuged at 21 ,000 g for 10 min at RT before the supernatant loaded into glass LC vials for analysis.

[0425] 5 pL of a known amount of super heavy beta actin peptide is spiked into the sample following loading into glass LC vials. Optionally, 10 pL of a known amount unlabeled peptide may also be spiked into the sample following loading into glass LC vials. Unlabeled peptides are only added if the unlabeled recombinant protein is not added earlier.

[0426] After trypsin digestion, multiple reaction monitoring (MRM) for each light, medium, and heavy HA and NA peptide as well as a beta-actin peptide was performed. Briefly, analysis of the trypsin digested samples was conducted with a Vanquish UPLC (Thermo) and a TSQ Altis triple quadrupole mass spectrometer (Thermo) using a Hypersil gold reverse phase column Cis. Mobile phases of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile were used for separation at a flow rate of 0.35 mL / min and a column temperature of 50°C. 25 pL of sample was injected with a runtime of 30 min. Typical LC and MS parameters are provided in Tables 1 , 2 and 3. MRM peptide settings are provided in Figure 6. Mass spectra were obtained in the negative ion mode, using MS / MS multiple reaction monitoring. Spectra were first analyzed using Skyline software (available online at https: / / 'skyline.ms / projecVhome / 'software / Skyline / begin.view) or TraceFinder software (ThermoFisher). Then, peak area of target peptides reported with TraceFinder (or Skyline) were exported as excel xlsx (or text csv) file, and analyzed to normalize the data, compute relative abundance or absolute concentrations of proteins / peptides, statistically analyze the data, and visualize the results. Figure 9 shows the quantification of expressed antigen using a number of the methods of quantification.

[0427] Example 8: Analysis of multivalent drug product

[0428] To demonstrate the potential of the methods described herein for analysis of multivalent drug products, a quadrivalent influenza LNP mixture was prepared. Peptides from each of the four antigen subtypes were monitored using MS from the same sample (labeled QIV for quadrivalent LNP). Cells dosed with one type of LNP containing a single subtype were also measured separately for comparison. The peptides that were monitored are provided in Table 5.

[0429] Table 5: Seasonal Influenza peptides for MS analysis

[0430] Preliminary results show that each of the four HA protein subtypes were able to be measured when the cells were treated with monovalent LNPs and were also be observed when the cells were treated with the quadrivalent influenza LNP mixture (Figure 10). In the former case, no signal was present for the HA subtypes not expressed by the treatment LNP. This experiment demonstrates that the methods described herein have a high degree of specificity towards each drug product within a multivalent mixture, highlighting the potential of these methods for analysis of multivalent drug products.

[0431] Example 9: Analysis of Combo drug products

[0432] To further demonstrate the multiplexing potential of the methods herein described for the analysis of combined multivalent drug products, cells were dosed with LNP using either Covid LNP #1 , Covid LNP #2, Influenza Seasonal Quadrivalent (QIV), or a mixture of both the QIV and Covid LNPs. Preliminary results show that each of the HA and NA subtypes, as well as S and NP proteins, were able to be measured with cells that are treated with the respective LNPs, and also when the cells were treated with a combination of drug products (Figure 11 ). No signal was present for antigens in samples that were not exposed to one of the dosed LNPs. This example demonstrates the high degree of specificity towards a combined multivalent drug product. The methods described herein can distinguish between samples that were dosed with either Covid or Influenza LNPs, as well a mixture of the two, measuring each unique antigen peptide across different LNPs within a single sample, highlighting the multiplexing capability of these methods.

[0433] Example 10: Use of beta actin as an indictor of cell health following dosing.

[0434] The present example demonstrates that isotopically labeled beta actin peptides can be used as indicators of cell heath following LNP dosing. Medium and heavy labeled cells were prepared, dosed with LNP and analyzed as described in Example 3 (0.5M and 1 M cell seeding) The concentration of light, medium and heavy labeled beta actin was determined and plotted against LNP dosing concentration. As shown in Figure 12, the medium labeled beta actin concentration (blue) decreases proportionally with an increasing dose of LNP. This could be indicative of poor cell health or cell death upon the addition of high doses of LNP. Light beta actin (green) levels are consistent across samples, indicating an even cell seeding density. Heavy beta actin (red) peptide levels are stable across different samples, as all cells cultured in heavy SILAC media were all dosed with the same amount of LNP.

[0435] Example 11: Characterization of a sa-mRNA quadrivalent influenza vaccine.

[0436] The present example describes characterization of an sa-mRNA vaccines using an integrated mass spectrometry assay (IDMS). As demonstrated herein, the assay can be used to evaluate vaccine potency, stability, and / or performance across different cell lines, RNA backbones, and formulations. The methods described herein enables quantification of protein expression levels, providing valuable insights into vaccine efficacy and design optimization.

[0437] LNP were prepared as described in Example 2. Quadrivalent influenza vaccines were prepared by encapsulating 4 seasonal influenza sa-mRNA bi-cistronic constructs in LNPs. The 4 seasonal influenza strains were influenza A H1 N1 , influenza A H3N2, influenza B / Yamagata (B / Yam), and influenza B / Victoria (B / Vic). Four vaccine drug products were prepared, denoted DP-1 to DP-4. Two formulations (different lipid composition) were prepared, denoted DS-A and DS-B.

[0438] LNPs containing RNA were diluted to a concentration of 5 ng / pL Opti-MEM. The diluted LNP was diluted further to the required concentration using Opti-MEM. The highest concentration tested was 0.1 pg / ml. Typically, the following four concentrations were used 0.05, 0.025, 0.0125 and 0.00625 pg / ml.

[0439] BHK21 cells were initially grown in DMEM (EMEM) + 10% FBS + 1% Pen / strep + 1% Glutamax. The cells were harvested, washed and resuspended in RT Opti-MEM at a cell count of 1 X106cells per in 250 pL. The resuspended cells were kept at RT prior to combining with the LNP.

[0440] To determine the potency of the LNP formulation, the BHK21 were treated in a 6-well plate. Each well contained 1.7 mL pre-warmed DMEM with 4% FBS. The appropriate amount of LNP solution was added to each well (total volume LNP added was 200 pL). 100 pL cell suspension was then added to each well and the cells incubated in a growth chamber in 5% CC^ at 37 °C for 16-19 hours. Untreated BHK21 cells were used as a control.

[0441] To determine the potency of the naked RNA, the BHK21 were treated in a 6-well plate. RNA is transferred into the cells using electroporation (120v, 25ms and 1 pulse). As electroporation displayed a lower transfection ability compared to the LNP, the amounts used for potency curve was 200ng, 100ng, 50ng and 25ng.

[0442] Cells were harvested by aspirating the media from each well of the 6-well plate. Each well was washed with 1 mL DPBS buffer. 200 pL 6 M guanidine and 200 pL RAPA buffer were added to each well and the sample sonicated to lyse the cells. 1 mL -20 °C acetone was added to precipitate proteins. The precipitate was pelleted by centrifugation (5 ~10 mins, 4 °C, 14,000 rpm). The pellet was resuspended in 90 pl ABC 0.1 M + (lodoacetamide 4pM) and incubated for 30mins. DTT was then added to a final concentration 4 pM and the sample further incubated for 30mins. 3 mL trypsin was then added and the sample incubated at 37 °C. The trypsin digest was stopped by adding 1 pL formic acid to each sample and vortexing. The samples were then centrifuged to pellet insoluble material and the supernatant collected. The supernatant was 0.22 pm filtered before 19 pL of the filtered supernatant was transferred to a glass vial and 1 pL heavy labeled peptide mixture was added. The sample was analyzed by IDMS. Table 3 provides a peptides being monitored. in this example. Figure 13 illustrates example calibration curves for a subset of selected peptides.

[0443] Table 6: Peptides used for analysis for each antigen

[0444] Optimization of trypsinization is illustrated in Figure 14. The correlation curve between cell number and GAPDH peptide intensity is illustrated in Figure 15.

[0445] The methods described in this example can be used to assess the potency of both naked RNA and LNP-formulated RNA vaccines. In this example, potency is defined as the amount of protein translated from one million cells per nanogram of RNA provided. Figure 16 illustrates use of the exemplified method for determining the potency of an sa-mRNA expressing H5. Two peptides were monitored using MS (IQIIPK and LVLATGLR). The method described in this example may be used to quantify antigens expressed from monovalent RNA constructs encoding H1 N1 or H3N2 (Figure 17) or monovalent RNA constructs encoding Byam HA, Bvic HA, NA (Figure 18).

[0446] The methods used in this example can be used to investigate performance across cell lines (e.g., BHK-21 , HEK-293, C2C12) and RNA backbones: The methods can also be used to study the stability of sa-mRNA vaccines over time, under various storage conditions, and in different formulations. Stability is evaluated by monitoring changes in protein expression levels using the exemplified method. The methods described in this example may also be used to elucidate the influence of modifications introduced in the untranslated regions (UTR) and / or signal adjuvant genes (SAG) on RNA translation and vaccine immunogenicity. This may allow for the identification of optimal vaccine candidates with broad applicability and / or enhanced efficacy. The method described in this example may be used to quantify the potency of formulations having different lipid compositions (Figure 19 and 20). The method described in this example may be used to quantify antigens with NS1 modified H5 RNAs (Figure 21 ). The method described in this example may be used to quantify antigens with 3’ and 5’ UTR modified H1 RNAs (Figure 22). The method described in this example may be used to quantify H1 N1 antigens for RNA constructs in HEK293 or C2C12 cells (Figure 23).

[0447] The methods described in this example can be used to compare H1 / N1 antigens for monovalent and quadrivalent RNA constructs (Figure 24), H3 / N2 antigens for monovalent and quadrivalent RNA constructs (Figure 25) and Bvic / Byam HA / NA antigens for monovalent and quadrivalent RNA constructs (Figure 26).

[0448] Figure 27 provides the results of ELISA, ELLA and pseudovirus MN assays for RNA vaccines. ELISA, ELLA and pseudovirus MN assays were performed using methods known to the person skilled in the art. The methods used in this example can be used to study the stability of sa-mRNA quadrivalent influenza vaccines over time, under various storage conditions, and in different formulations. Stability is evaluated by monitoring changes in protein expression levels using the exemplified method. The methods described in this example can be used to perform stability tests (Figure 28). Figure 29 provides a comparison on the method described in this example and serology results (FACS, Cytation).

[0449] Example 12: Normalization using a control RNA-LNP.

[0450] The present example describes normalization using a control RNA-LNP. The test and control RNA-LNP were prepared as described in Example 2. In this example, the control RNA-LNP encoded influenza HA (H2) and NA (N3) and the test RNA-LNP encoded influenza H1 , H3, HB / Victoria, N1 , N2, and NB / Victoria. In the present example, H2N3 LNP was selected as the control RNA-LNP to avoid N1 signal overlap that would be present if using H5N1 RNA-LNP as a control.

[0451] The test RNA-LNP was thawed on ice and mixed by pipetting to ensure homogeneity. The test RNA-LNP was serially diluted with a starting concentration of 40,000 ng / mL using light media. The final concentration range was between 40,000 ng / mL and 356 ng / mL

[0452] The control RNA-LNP was thawed on ice and mixed by pipetting to ensure homogeneity. The control RNA-LNP was diluted to a concentration of 250 ng / mL using light media.

[0453] Equal volumes (1 :1 ) of each serially diluted test LNP:RNA and control RNA:RNA were combined and the samples mixed well by pipetting. 100 pL of the combined LNP:RNA mixture was then added to the cells plated in a 6 well plate as described in Example 3. The plate was tipped and the RNA-LNP mixture as added directly to the media in each well. The plate was gently rocked using a front-to-back and side-to-side motion (not circular) several times to mix. The dosed plates were placed in a 37 °C, 5% CO2 incubator humidified incubator and incubated at 37 °C for 20-24hrs. The cells were lysed and samples prepared using the in well cell lysis and sample preparation method described in Example 13. The resulting samples were analyzed using MS and the data is shown in Table 7. The inventors found that normalization using a control RNA-LNP improves linearity and repeatability.

[0454] Table 7: Normalization using a control RNA-LNP.

[0455] The present inventors have also used H2N3 RNA LNP (A / chicken / Ohio / 494832 / 2007) as a control for covid LNP constructs, which express spike protein rather than hemagglutinin and neuraminidase of H2N3. H5N1 (A / turkey / Turkey / 1 / 2005) and B / Ya (B / Singapore / INFTT-16-0610 / 2016) LNP constructs have also been used successfully as control RNA-LNPs. This demonstrates that the identity of the control RNA-LNP is not important, it sufficient that the protein expressed has a different MS spectra such that it avoids MS signal overlap.

[0456] Example 13: In-well cell lysis and sample preparation.

[0457] The present example describes the in-well cell lysis method and sample preparation for MS analysis. As demonstrated herein, the in-well cell lysis method can be used in place of a method which uses a protease to detach adhered cells from the plate. The methods described herein provide a simplified process which helps reduce the total time for the method and minimize the potential for variability.

[0458] LNP were prepared as described in Example 2. Influenza vaccines were prepared by encapsulating seasonal influenza sa-mRNA bi-cistronic constructs in LNPs. Cells were treated with LNP as described in the earlier examples using medium and heavy media.

[0459] Cells were harvested by aspirating the media from each well of the 6-well plate. 100 pL of Lysis buffer (0.1% Rapigest and 1 pL nuclease (Universal Nuclease, ThermoFisher 250 U / pL) per 1 mL Rapigest) was added to the middle of each well. The plate was shaken in an Incu-mixer at room temp (Temp set to off) at 1000 rpm for 10 minutes. The lysate was allowed to settle to the bottom of the well and 90 pL of the medium lysate and 90 pL 0 the heavy lysate was combined in an 1 .5 mL tube. The combined lysate was then spiked with recombinant HA / NA. Briefly, the stock rHA / rNA mixture (10 ng / pL) was thawed at RT and the thawed mixture vortexed to mix. 40 pL of the rHA / rNA protein mixture was added to each sample and the sample mixed. The samples were heated at 100 °C for 5 min with a second hot block placed on the lids of the 1 .5 mL tubes. The heated samples were fast cooled in a room temperature metal block for a minimum of 5 min at RT for fast cooling.

[0460] The samples were then digested using trypsin. Briefly, 14 pL of sequencing grade trypsin was added to each sample and the sample mixed by vortexing. The sample tubes were placed in a thermomixer that has been set to 37°C, 600 rpm, and incubated for 2:00 hours. The samples in the thermomixer were covered with a ThermoTop prior to mixing. After the incubation period, the samples were removed from the thermomixer and cooled at RT for at least 5 min. 5 pL 2N HCI was added to each sample and the sample mixed by vortexing. The HCI treated samples were incubated at room temperature for a minimum of 20 min. The samples were then centrifuged (13,000 rpm, 10 min, room temperature). 180 pL of the supernatant was transferred into glass LC vials and the supernatant was analysed by MS. An schematic of methods described herein incorporatying the in-well cell lysis step is provided in Figure 30.

[0461] Example 14: High-throughput assay in a multi-well plate format

[0462] The present example demonstrates that the method described herein can be used in a 96-well plate format. This was unexpected due to the reduced number of cells that can be plated per well (0.01 x106cells / well vs 0.25x106cells / well in a 6 well plate).

[0463] RNA-LNP were prepared as described above. The wells in a 96-well plate were pre-seeded a cell seeding density of 0.01 x 106cells / well in either medium or heavy labelled SILAC media. The test RNA-LNP was diluted in light SILAC media in a fresh 96-well plate. Equal volumes of control RNA-LNP (at a single concentration) was directly added each well of the test RNA-LNP plate. 20 pL of the RNA-LNP mixture was added to each of the pre-seeded wells and the dosed plates were placed in a 37 °C, 5% CO2 incubator humidified incubator and incubated at 37 °C for 20-24hrs.

[0464] The media was aspirated and the cells were then lysed in the wells by adding 50 pL of 0.05% Rapigest plus nuclease in 50 mM Tris buffer, pH 8.5 to each well of the 96-well plate. 1 uL of 250 U / pL universal nuclease (ThermoFisher) was added per mL of 0.05% Rapigest. The plate was incubated at room temperature in an Incu-mixer at 1000 rpm for 10min. All of the heavy cell lysate was transferred into the well containing the corresponding medium cell lysate. 10pL of recombinant protein standard (stock concentration = 10 ng / pL) was added to each well. The 96-well plate was then placed directly on a heat block and incubated at 100 °C for 5min. 5pL of sequencing grade trypsin was added to each well and the mixture incubated at 37°C without shaking for 2 hrs. 5pL of 1 M HCI was then added to each well and the mixture incubated for at least 60 min at 4 °C to facilitate the precipitation of degraded RapiGest. The entire sample was transferred to a 96 well Filter plate (0.2 pm) and the sample filtered into a 96 well collection plate by centrifugation or a positive pressure-96 processor. The samples were then directly injected into LC-MS directly from the 96-well collection plate and analysed as described herein.

[0465] The present inventors have demonstrated that performing the method in a 96- well plate format can be used to quantify protein expressed from a test RNA-LNP. As shown in Table 8, the dose response curves from two different Covid LNP peptides have a high linearity when normalized to the H2 internal dosing control. This suggests that the low number of LNP-dosed cells in a 96-well plate format is sufficient to produce a robust MS signal from trypically digested target peptides to determine potency, highlighting the high throughput potential of the methods described herein.

[0466] Table 8: High throughput assay in 96-well plate format

Claims

CLAIMS:

1. A method for quantification of a target protein in a sample, the method comprising:(a) combining(i) a first sample of the target protein to be quantified, wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled, and(ii) a second sample comprising a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in the second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, to form a first mixture(b) analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; and(c) normalizing the amount of the target protein relative to the reference protein; and(d) quantifying the amount of target protein in the first sample.

2. The method of claim 1 , wherein step (b) comprises subjecting the first mixture to mass spectrometry to form a spectra comprising one or more sets of matched peptide peaks and comparing relative intensities or peak areas of matched peptide peaks in the spectra to determine the relative amount of the first variant.

3. The method of claim 1 , wherein normalizing comprises multiplying the relative amount of the first variant by a normalization factor, wherein the normalization factor is determined by dividing the relative amount of the second variant of the reference protein by the relative amount of the first variant of the reference protein.

4. The method of claim 1 , wherein the mixture is treated with a protease to prior to analysis.

5. The method of claim 4, wherein the protease is selected from the group consisting of trypsin, endoproteinase GluC, enterokinase light chain, Factor Xa, furin, chymotrypsin, Lys-C, Lys-N, elastase, Asp-N and endoproteinase ArgC.

6. The method of claim 1 , wherein a known amount of isotopical ly labeled reference peptide is added to the first mixture prior to analysis.

7. The method of claim 6, wherein the reference peptide is a beta actin peptide.

8. The method of claim 7, wherein the beta actin peptide comprises the sequence GYSFTTTAER (SEQ ID NO: 1).

9. The method of claim 7, wherein the beta actin peptide comprises the sequence GYSFTTTA(+4)ER(10) (SEQ ID NO: 1).

10. The method of claim 6, wherein the absolute amount of reference protein in the mixture is determined by comparison to the known amount of the reference peptide.11 . The method of claim 1 , wherein the isotopically labeled amino acids are labeled with one or more isotopes selected from2D,15N and13C.

12. The method of claim 1 , wherein the isotopically labeled amino acids are selected from the group consisting of isoleucine, glutamic acid, aspartic acid, glycine, arginine, lysine and combinations thereof.

13. The method of claim 1 , wherein the isotopically labeled amino acids are selected from the group consisting of arginine, lysine and combinations thereof.

14. The method of claim 1 , wherein the first sample is isotopically labeled with lysine (K(+4)) and arginine (R(+6)) and the second sample is isotopically labeled with heavy lysine (K(+8)) and heavy arginine (R(+10)).

15. The method claim 1 , wherein the reference protein is beta actin.

16. The method claim 1 , further comprising the step of purifying the target protein and the reference protein prior to analysis.

17. The method of claim 1 , further comprising the steps of:(d) combining the first and / or second sample with a known amount of a third, unlabeled variant of the target protein or a peptide thereof to form a second mixture;(e) analyzing the second mixture to determine relative amounts of the first and / or second variant and third variant of the target protein or peptide thereof;(f) determining the absolute amount or concentration of the first and / or second variant in the second sample based on the known amount of the third, unlabeled variant of the target protein or peptide thereof.

18. The method of claim 17, wherein the second mixture is treated with a protease before analysis.

19. The method of claim 17, wherein the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled peptide.

20. The method of claim 17, wherein the third, unlabeled variant of the target protein or a peptide thereof is an unlabeled recombinant protein.

21. The method of claim 17, wherein a standard curve is used for determining the absolute amount or concentration of the second variant in the second sample.

22. The method of claim 21 , wherein the standard curve is produced by combining the second sample with a serial dilution of a known amount of the third, unlabeled variant of the target protein or a peptide thereof23. The method of claim 17, wherein one-point quantification is used for determining the absolute amount or concentration of the second variant in the second sample.

24. The method of claim 1 , wherein the first sample comprises cells treated with a RNA-LNP.

25. The method of claim 1 , wherein the second sample comprises cells treated with a RNA-LNP.

26. The method of claim 1 , wherein the first and second samples comprise cells treated with different amounts of RNA-LNP, different batches of RNA-LNP or different RNA-LNP.

27. The method of claim 1 , wherein the method is employed to determine the potency of a RNA-LNP.

28. The method of claim 1 , wherein the target protein is selected from the group consisting of a recombinant protein, a biotherapeutic protein, an antibody, a fusion protein, and a glycosylated protein.

29. The method of claim 1 , wherein the target protein is recombinant protein produced from a nucleic acid vaccine.

30. The method of claim 1 , wherein the reference protein is an endogenous reference protein or an exogenous reference protein.31 . A method for normalizing an amount of a target protein in a sample based on cell count, the method comprising:(a) combining(i) a sample comprising a first variant of a reference protein, wherein at least one amino acid in the first variant is isotopically labeled; and(ii) a sample comprising a second variant of a reference protein, wherein the least one amino acid is differentially isotopically labeled such that the first variant and second variant have a different molecular weight; to form a mixture;(b) analyzing the mixture to determine relative amounts of the first variant and second variant; and(c) dividing the amount of the second variant by the amount of first variant to determine a normalization factor, wherein the normalization factor is used for normalizing an amount of a target protein in a sample based on cell count.

32. The method of claim 31 , wherein the reference protein is an endogenous reference protein or an exogenous reference protein.

33. A method for determining the relative in vitro potency of a recombinant nucleic acid encoding a target protein, the method comprising: transfecting a first population of cells with a test sample of the recombinant nucleic in media comprising an isotopically labeled amino acid to form a first sample,wherein the first sample comprises a first variant of the target protein and a first variant of a reference protein, wherein at least one amino acid in each first variant is isotopically labeled; transfecting a second population of cells with a reference sample of the recombinant nucleic acid in media comprising an isotopically labeled amino acid to form a second sample, wherein the second sample comprises a second variant of the target protein and a second variant of the reference protein, wherein at least one amino acid in each second variant is differentially isotopically labeled such that the first variant and second variant have a different molecular weight, combining the first sample and the second sample to form a first mixture; analyzing the first mixture to determine relative amounts of the first variant and second variant of the target protein and reference protein; normalizing the amount of the target protein relative to the reference protein in the first sample and the second sample; and comparing the amount of expression of the target protein for the first sample with the amount of expression of the target protein for the second sample to determine the relative in vitro potency of the composition.

34. The method of claim 33, wherein analyzing comprises subjecting the first mixture to mass spectrometry to form a spectra comprising one or more sets of matched peptide peaks and comparing relative intensities or peak areas of matched peptide peaks in the spectra to determine the relative amount of the first variant.

35. The method of claim 33, wherein normalizing comprises multiplying the relative amount of the first variant by a normalization factor, wherein the normalization factor is determined by dividing the relative amount of the second variant of the reference protein by the relative amount of the first variant of the reference protein.

36. The method of claim 33, wherein the mixture is treated with a protease to prior to analysis.

37. The method of claim 36, wherein the protease is selected from the group consisting of trypsin, endoproteinase GluC, enterokinase light chain, Factor Xa, furin, chymotrypsin, Lys-C, Lys-N, elastase, Asp-N and endoproteinase ArgC.

38. The method of claim 33, wherein the isotopically labeled amino acids are labeled with one or more isotopes selected from2D,15N and13C.

39. The method of claim 33, wherein the isotopically labeled amino acids are selected from the group consisting of arginine, lysine and combinations thereof.

40. The method of claim 33, wherein the first sample is isotopically labeled with lysine (K(+4)) and arginine (R(+6)) and the second sample is isotopically labeled with heavy lysine (K(+8)) and heavy arginine (R(+10)).

41. The method of claim 33, wherein the reference protein is an endogenous reference protein or an exogenous reference protein.

42. The method of claim 33, wherein the reference protein is beta actin.

43. The method of claim 33, wherein the first and the second population of cells are cultured in unlabeled media for about 18 to 30 hours prior to transfection, or about 24 hours prior to transfection.

44. The method of claim 33, wherein the first and second population of cells are transfected for at least about 12 hours, at least about 18 hours, or about 24 hours.

45. A method comprising the steps of obtaining a population of cells that has been treated with a self-amplifying mRNA encoding one or more target proteins; and quantifying the amount of the one or more target proteins expressed from the RNA.

46. A method comprising the steps of obtaining a population of cells that has been treated with a self-amplifying mRNA encoding one or more target proteins; and quantifying the potency of the self-amplifying mRNA encoding one or more target proteins.

47. An isotopically labeled peptide comprising the sequence GYSFTTTA(+4)ER(+10) (SEQ ID NO: 1).

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