WO2024171052A1

WO2024171052A1 - Analytical method

Info

Publication number: WO2024171052A1
Application number: PCT/IB2024/051329
Authority: WO
Inventors: Mila TOPPAZZINI; Claudia Magagnoli; Riccardo DE RICCO; Roberta FRANZINI; Michela DE MARTINO; Jacopo SANTORO; Salvatore Sanna COCCONE; Alessio CORRADO; Dalila IACOPINI
Original assignee: Glaxosmithkline Biologicals Sa; CureVac SE
Priority date: 2023-02-14
Filing date: 2024-02-13
Publication date: 2024-08-22
Also published as: GB202302092D0

Abstract

The present invention relates to liquid chromatography methods (e.g., HPLC) that facilitate high resolution separation of mRNA formulated in lipid nanoparticles (LNPs) from impurities. In aspects, the disclosure relates to a mobile phase with two or more eluents that separates constituents of a mixture into one or more peaks, wherein at least one of the peaks is a well- defined impurity peak that can be quantified to accurately determine the amount of the impurity in the mixture.

Description

ANALYTICAL METHOD

TECHNICAL FIELD

The present invention relates to liquid chromatography methods (e.g., HPLC) that facilitate high resolution separation of RNA formulated in lipid nanoparticles (LNPs) from potency impacting impurities (e.g., mRNA-lipid adduct). In aspects, the disclosure relates to a mobile phase with two or more eluents that separates constituents of a mixture into one or more peaks, wherein at least one of the peaks is a defined impurity peak that can be quantified to accurately determine the amount of the impurity in the mixture.

BACKGROUND

RNA-based vaccines are an established alternative to traditional vaccines. mRNA vaccines may be rapidly designed and deployed due to the flexibility of mRNA sequence design and scalability of the manufacturing process. Additionally, mRNA vaccines have been shown to have a suitable safety and pharmacokinetic profile at least in part due to their design, modification, and rapid biodegradability. mRNA vaccines and therapies may be delivered by lipid nanoparticles (LNPs) that provide a variety of benefits including protection from degradation of the mRNA product by exonucleases and/or endonucleases and promotion of cellular uptake of the LNP and expression of the encapsulated mRNA. Although LNPs are a proven route for mRNA delivery to tissues, degradation may still occur during storage from oxidation, hydrolysis, or transesterification thereby cleaving the mRNA into smaller fragments.

Another type of mRNA degradation has been recently discovered - namely, the formation of lipid-mRNA adducts that are generated during production of the mRNA encapsulated product and/or long-term storage. LNPs that are used to encapsulate RNA (e.g., mRNA) generally comprise a cationic lipid which is an ionizable amino-lipid. Recently, it has been shown that the ionizable amino-lipid might bring or generate impurities (N-oxides) upon oxidation of the tertiary amine group of the lipid (Packer et al., Nature Communications (2021) 12: 6777 (https://doi.org/10.1038/s41467-021-26926-0)). N-oxides can indeed hydrolyze to aldehydes, which might form adducts through covalent addition of reactive lipid species to the nucleobases of mRNA strands. Packer et al. (2021) provide an analytical method to detect these adducts using Reverse Phase-Ion Pair High Performance Liquid Chromatography (RP-IP HPLC) and have shown that these adducts might be associated with a reduction in in vitro protein expression. The lipid-mRNA adduct is referred to herein as the “Late Eluting Peak” (LP or LEP) and might reduce vaccine potency and/or stability according to its amount in the vaccine product. However, in reference to the published analytical method, the elution curve of the LEP is not sharp and well-defined (see, FIG. 1). Therefore, it is difficult to measure accurately the amount of LEP present (e.g., by integration of the LEP peak area under the chromatogram curve). Further, the measurement may be subject to bias by background noise. Accordingly, the published analytical method does not provide a suitable way to accurately measure the amount of LEP present in the sample.

Accordingly, improved analytical techniques are needed to accurately measure the amount of impurities in RNA manufacturing (e.g., for any type of RNA including but not limited to antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs) and single-guide RNAs (sgRNAs)-mediated CRISPR-Cas system). Improved analytical techniques are needed to measure the amount of LEP impurity in mRNA manufacturing, for example, in order to optimize vaccine potency and stability.

SUMMARY

The present invention relates to liquid chromatography methods (e.g., high performance liquid chromatography (HPLC), ion-pair - reversed-phase - ultra-performance liquid chromatography (IP-RP-UPLC), etc.) that facilitate high resolution separation of RNA encapsulated in lipid nanoparticles (LNPs) from impurities (e.g., lipid-mRNA adducts). In aspects, the disclosure relates to a mobile phase with two or more eluents that separates constituents of a mixture into one or more peaks, wherein at least one of the peaks is a defined impurity peak that can be quantified to accurately determine the amount of the impurity present in the mixture. Methods described herein allow for accurate integration of the area under the impurity peak to determine the amount of the impurity present in the mixture (or to determine ratios between components of the mixture) to assess quantitatively the amount of lipid adduct (e.g., to quantify the lipid- mRNA adduct in encapsulated mRNA-based vaccines). In other aspects, the techniques provided herein may be applied to any suitable type of RNA (e.g., mRNA, siRNA, etc.) to determine the amount of lipid adduct impurity present in the mixture. The present techniques apply generally to separation and resolution of chromatographic peaks and measurement of different lipid adduct impurities generated from manufacturing RNA for a variety of applications. Present techniques may be used with any LNP suitable for encapsulating mRNA.

In aspects, the invention may include the development/optimization of a chromatographic method that includes a sample pre-treatment step to reduce time and complexity of sample processing before the sample is injected onto the column. The present method, which does not require traditional lipid extraction, provides significant advantages in terms of reduced sample manipulation and analysis lead time, especially given that extraction often counts for most of the analysis lead time.

In aspects, pre-treating the sample may comprise diluting and/or mixing the sample containing the LNPs with a surfactant, wherein the surfactant is present at a concentration of about 1-10%.

Surfactants may include any suitable surfactant, including but not limited to Brij-35 (polyoxyethylene(23)lauryl ether, 2-(dodecyloxy)ethan-l-ol), Triton X 100 (t- octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenylether), Tween 20, and Sodium Dodecyl Sulfate (SDS). It has been discovered herein that any suitable surfactant may be used for impurity quantification, and that quantification of the LEP does not depend upon complete disruption of the LNPs. Without being bound by any particular theory, it is thought that partial disruption of the LNPs in combination with denaturing conditions of the chromatographic process, allow for quantification of the LEP with a wide variety of different surfactants.

Alternatively, and optionally, pretreating the sample may comprise extracting the RNA and RNA lipid-adduct from the sample using traditional RNA extraction methods, wherein the extract is loaded onto the chromatography column. Thus, the present method may be performed with or without traditional lipid extraction techniques.

In some embodiments, the method includes separating on a chromatography column a lipid nanoparticle (LNP)-formulated mRNA product (e.g., an mRNA product subjected to an LNP encapsulation process) from an impurity comprising: obtaining a sample comprising a mixture of the LNP-formulated mRNA product and the impurity, pre-treating the sample, contacting a stationary phase of a reverse phase (RP) chromatography column with the sample (e.g., by injection) and eluting the components of the sample with a mobile phase that is a mixture of a first eluent and a second eluent. The mRNA product traverses the column with a first retention time and the impurity traverses the column with a second retention time different from the first retention time, wherein the impurity is eluted as a well-defined peak (e.g., single well-defined peak). In aspects, the impurity is a lipid-mRNA adduct. A range of mRNA product concentrations can be analyzed with this analytical method, for example, as low as 1 pg/mL, 2 pg/mL. 3 pg/mL, 4 pg/mL, 5 pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 150 pg/mL, or more, with virtually no limit on the upper range (e.g., limited by the loading capacity of the column) (see, e.g., TIG. 7). In some embodiments. a volume percentage of the first eluent in the mobile phase is about 0% to about 55% or about 0% to about 50% and a volume percentage of the second eluent in the mobile phase is about 45% to about 100% or about 50% to about 100%.

In some embodiments, the first eluent comprises at least two ion pairs in a first solvent or first solvent solution, and the second eluent comprises at least two ion pairs in a second solvent or second solvent solution. Unless the context indicates otherwise, a solvent generally refers to a dissolving medium. A solution may be formed from one or more solvents and one or more solutes.

In some embodiments, the concentration of each of the at least two ion pairs in the first solvent or first solvent solution is about 25 mM-125 mM, about 50 mM-100 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM-100 mM, about 100 mM-125 mM, about 75 mM- 125 mM, about 25 mM-75 mM, or about 50 mM to 150 mM. In some embodiments, the concentration of each of the at least two ion pairs in the second solvent or second solvent solution is about 25 mM-125 mM, about 50 mM-100 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM-100 mM, about 100 mM-125 mM, about 75 mM-125 mM, about 25 mM-75 mM, or about 50 mM to 150 mM.

In some embodiments, the ion pairs of the first eluent are selected from trimethylamine or a salt thereof (e.g., triethylammonium salt), and dibutylamine or a salt thereof (e.g., dibutylammonium salt). In some embodiments, the ion pairs of the second eluent are selected from trimethylamine or a salt thereof (e.g., triethylammonium salt), and dibutylamine or a salt thereof (e.g., dibutylammonium salt).

In some embodiments, the first solvent or first solvent solution comprises one or more solvents selected from the group consisting of: water, alkanols, alkandiols, alkanoic acids, polar aprotic solvents (e.g., acetone, acetonitrile, DMF, DMSO, THF). In some embodiments, the second solvent or second solvent solution comprises one or more solvents selected from the group consisting of: water, alkanols, alkandiols, alkanoic acids, polar aprotic solvents (e.g., acetone, acetonitrile, DMF, DMSO, THF). In aspects, the first solvent or first solvent solution comprises water. In aspects, the second solvent or second solvent solution comprises acetonitrile in water. In other aspects, the triethylammonium salt is triethylammonium acetate and the dibutylammonium salt is dibutylammonium acetate. In still other aspects, the first solvent solution is water, and the second solvent solution is 50% acetonitrile in H2O. In still other aspects, the first eluent comprises or consists of the first solvent or first solvent solution. In still other aspects, the second eluent comprises or consists of the second solvent or second solvent solution.

In some embodiments, for the first eluent, the concentration of triethylammonium acetate is about 25 mM-125 mM, about 50 mM-100 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM-100 mM, about 100 mM-125 mM, about 75 mM-125 mM, about 25 mM-75 mM, or about 50 mM to 150 mM, such as about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, about 106 mM, about 107 mM, about 108 mM, about 109 mM, or about 110 mM. In other embodiments, for the first eluent, the concentration of dibutylammonium acetate is about 0 mM-125 mM, about 25 mM-100 mM, about 0 mM-25 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM to 100 mM, about 0 mM to 50 mM, about 25 mM-75 mM, about 50 mM-100 mM, such as about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, or about 60 mM.

In some embodiments, for the first eluent, the concentration of triethylammonium acetate is about 100 mM (e.g., about 95 mM - 105 mM, about 96 mM - 104 mM, about 97 mM - 103 mM, about 98 mM - 102 mM, about 99 mM - 101 mM, about 100 mM) and the concentration of dibutylammonium acetate is about 50 mM (e.g., about 45 mM - 55 mM, about 46 mM - 54 mM, about 47 mM - 53 mM, about 48 mM - 52 mM, about 49 mM - 51 mM, about 50 mM). In other embodiments, for the first eluent, the concentration of triethylammonium acetate is about 100 mM and the concentration of dibutylammonium acetate is about 50 mM.

In some embodiments, for the second eluent, the concentration of triethylammonium acetate is about 25 mM-125 mM, about 50 mM-100 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM-100 mM, about 100 mM-125 mM, about 75 mM-125 mM, about 25 mM-75 mM, or about 50 mM to 150 mM, such as about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, about 106 mM, about 107 mM, about 108 mM, about 109 mM, or about 110 mM. In other embodiments, for the second eluent, the concentration of dibutylammonium acetate is about 0 mM-125 mM, about 25 mM-100 mM, about 0 mM-25 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM to 100 mM, about 0 mM to 50 mM, about 25 mM-75 mM, about 50 mM-100 mM, such as about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, or about 60 mM.

In some embodiments, for the second eluent, the concentration of triethylammonium acetate is about 100 mM (e.g., about 95 mM - 105 mM, about 96 mM - 104 mM, about 97 mM - 103 mM, about 98 mM - 102 mM, about 99 mM - 101 mM, about 100 mM) and the concentration of dibutylammonium acetate is about 50 mM (e.g., about 45 mM - 55 mM, about 46 mM - 54 mM, about 47 mM - 53 mM, about 48 mM - 52 mM, about 49 mM - 51 mM, about 50 mM). In other embodiments, for the second eluent, the concentration of triethylammonium acetate is about 100 mM and the concentration of dibutylammonium acetate is about 50 mM.

In still other aspects, the first eluent is water, and the second eluent is 50% acetonitrile in H2O (e.g., the first eluent is H2O and the second eluent is FhO/McCN 50:50).

In some embodiments, the volume percentage of the first eluent and the second eluent is not held constant during elution of the mRNA product and the impurity.

In some embodiments, the retention time of the mRNA product and the impurity is between about 1 minute and 5 minutes (e.g., about 1 minute to 4 minutes, about 1 minute to 3 minutes, about 1 minute to 2 minutes). In some aspects, short RNA sequences (e.g., as few as 200-400 bases) may be detected with the present method. In some embodiments, the difference between the retention time of the mRNA product and the impurity is less than about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 90 seconds, about 75 seconds, about 60 seconds, about 45 seconds or about 30 seconds. In some embodiments, the difference between the retention time of the mRNA product and the impurity is greater than 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, or 45 seconds. In some embodiments, the difference between the retention time of the mRNA product and the impurity is about 1 to 45 seconds, about 1 to 15 seconds, about 15 to 30 seconds, about 30 to 45 seconds or about 20 to 40 seconds or longer.

In some embodiments, the retention time of the mRNA product is less than the retention time of the impurity, and both the mRNA product and impurity are eluted in less than about 5 minutes. In some embodiments, the peak of the mRNA product does not overlap or does not substantially overlap with the peak of the impurity.

In some embodiments, a step gradient is applied to the mobile phase, comprising the steps of: (i) applying a mixture of about 50% of the first eluent and about 50% of the second eluent;

(ii) applying, after (i), 100% of the second eluent; and (iii) applying, after (ii), a mixture of about 50% of the first eluent and about 50% of the second eluent.

In further aspects, the step gradient comprises:

(i) applying, at an initial time point, a mixture of about 50% of the first eluent and about 50% of the second eluent;

(ii) at a second time point, increasing (e.g., linearly) the percent volume of the second eluent over a time span of about 1.6 to 2.2 minutes (e.g., 1.7 to 2.1 minutes), until reaching 100% volume of the second eluent;

(iii) maintaining, for about 45-75 seconds (e.g., 50-70 seconds), 100% volume of the second eluent;

(iv) at a third time point, decreasing (e.g., linearly) the percent volume of the second eluent over a time span of about less than 25 seconds (e.g., less than 20 seconds, less than 15 seconds) until reaching 50% volume of the second eluent;

(v) maintaining a mixture of 50% of the first eluent and 50% of the second eluent. In further aspects, the step gradient comprises:

(i) applying, at an initial time, a mixture of 50% of the first eluent and 50% of the second eluent;

(ii) applying, at a time of 6 minutes, 100% of the second eluent;

(iii) applying, at a time of 8 minutes, 100% of the second eluent;

(iv) applying, at a time of 10 minutes, a mixture of 50% of the first eluent and 50% of the second eluent.

In further aspects, the step gradient comprises:

(i) applying, at an initial time point, a mixture of about 50-55% (e.g., about 52%) of the first eluent and about 45-50% (e.g., about 48%) of the second eluent;

(ii) at a second time point, increasing (e.g., linearly) the percent volume of the second eluent over a time span of about 3-7 minutes (e.g., about 4-6 minutes, about 5 minutes), until reaching 100% volume of the second eluent;

(iii) maintaining, for about 1.5-2.5 minutes (e.g., about 2 minutes), 100% volume of the second eluent;

(iv) at a third time point, decreasing (e.g., linearly) the percent volume of the second eluent over a time span of about 1-3 minutes (e.g., about 2 minutes) to reach a mixture of about 50-55% volume (e.g., about 52% volume) of the first eluent and about 45-50% (e.g., about 48% volume) of the second eluent;

(v) maintaining a mixture of about 50-55% (e.g., about 52%) of the first eluent and about 45-50% (e.g., about 48%) of the second eluent.

In further aspects, the step gradient comprises:

(i) applying, at an initial time, a mixture of 52% of the first eluent and 48% of the second eluent;

(ii) applying, at a time of 6.6 minutes, a mixture of 47% of the first eluent and 53% of the second eluent;

(iii) applying, at a time of 8.6 minutes, 100% of the second eluent;

(iv) applying, at a time of 9.6 minutes, 100% of the second eluent;

(v) applying, at a time of 9.8 minutes, a mixture of 52% of the first eluent and 48% of the second eluent.

In certain aspects, the initial time may correspond to the injection time of the mixture onto the column In some embodiments, the method comprises detecting the components of the sample using a UV detector with a range/wavelength of 260 nm.

In aspects, the column is an analytical column or a preparative column. In aspects, the column comprises particles (e.g., porous resin particles). In aspects, the particles are hydrophobic or comprise hydrophobic functional groups. In aspects, the particles have a diameter ranging from 2 pm to about 10 pm, about 2 pm to 6 pm, or about 4 jam. In aspects, the particles comprise pores having a diameter ranging from about 100 A to about 3000 A, from about 100 A to about 2000 A, from about 500 A to about 2000A or from about 1000 A to about 2000A.

In aspects, the temperature of the reverse phase column is about 53 °C to about 65 °C, about 55°C to about 63°C, about 57°C to about 61°C, or about 59°C, or about 60°C.

In aspects, the flow rate of the reverse phase column is about 0.2 to 0.5 mL/min, 0.3 to 0.4 mL/min, or about 0.35 mL/min. In aspects, the dimensions of the reverse phase column are about 2.1 x 100 mm. In other aspects, the chromatographic run length is under 20 minutes or under 15 minutes.

In still other aspects, the method further comprises the step of performing a blank subtraction. The present invention may encompass various embodiments, and therefore, it is understood that each of the embodiments of the invention involving any one element or combinations of elements may be included in each aspect or embodiment of the invention. The present invention is not limited to the details of construction or arrangement of components set forth in the description, drawings or examples, and may be practiced or carried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an ultra-performance liquid chromatography (UPLC) method as reported in the art ((see, Packer et al., Nature Communications (2021) 12: 6777

(https://doi.org/10.1038/s41467-021-26926-0)). In this figure, the late eluting peak (LEP) elutes over roughly 5 minutes of a chromatography run, leading to a peak that is difficult to quantify (e.g., difficult to integrate the area under the curve with accuracy). Additionally, the signal is difficult to distinguish, and may be possibly biased by background noise.

FIG. 2A shows an example of an optimized gradient profile for LEP quantification via RP- UPLC chromatography and detection. Chromatography parameters include: UPLC Waters Acquity H-Class with quaternary pumps; Column: DNA-PAC RP 4 pm, 2.1 x 100 mm (Cod 088923); Eluent A: TEAA 100 mM/DBAA 50 mM in H₂0; Eluent B: TEAA 100 mM/DBAA 50 mM in EEO/MeCN 50:50; Column Temperature about 59 °C or 60°C; Autosampler Temperature: 8 °C; Vol inj: generally ranging from about 2,5 pl up to about 7 pl; Wavelength: 260 nm. Additional software parameters may include: Wash solvent: Methanol 10%; Purge Solvent: Water; Pre-injection wash: 0 sec; Post-inject wash: 6 sec; Sample Temperature 4°C; Channel 1 Absorbance: 260 wavelength 4.8 nm resolution; Channel 2 Absorbance: 214 wavelength 4.8 nm resolution. The parameters of the step gradient are shown in Table 1.

FIG. 2B shows a chromatogram according to the parameters of FIG. 2A, with no blank subtraction (full view).

FIG. 2C shows a chromatogram based on FIG. 2B with no blank subtraction (zoom view).

FIG. 2D shows a chromatogram according to the parameters of FIG. 2A with blank subtraction (full view).

FIG. 2E shows a chromatogram based on FIG. 2D with blank subtraction (zoom view).

FIG. 3A shows another example of an optimized gradient profile for LEP quantification via RP-UPLC chromatography and detection. Chromatography parameters include: UPLC Waters Acquity H-Class with quaternary pumps; Column: DNA-PAC RP 4 pm, 2.1 x 100 mm (Cod 088923); Eluent A: TEAA 100 mM/DBAA 50 mM in H2O; Eluent B: TEAA 100 mM/DBAA 50 mM in EEO/MeCN 50:50; Column Temperature about 59 °C or 60°C; Autosampler Temperature: 8 ° C; Vol inj: about 2,5 pl up to about 7 pl; Wavelength: 260 nm. Additional software parameters may include: Wash solvent: Methanol 10%; Purge Solvent: Water; Pre-injection wash: 0 sec; Post-inject wash: 6 sec; Sample Temperature 4 ° C; Channel 1 Absorbance: 260 wavelength, 4.8 nm resolution; Channel 2 Absorbance: 260 wavelength, 4.8 nm resolution. The parameters of the step gradient are shown in Table 2.

FIG. 3B shows a chromatogram according to the parameters of FIG. 3 A (full view). In this example, the solvents have been optimized so that the main peak (the mRNA product) and LEP elute more rapidly.

FIG. 3C shows a chromatogram according to the parameters of FIG. 3B (zoom view).

FIG. 4A shows another example of a chromatogram, showing eluted peaks of mRNA product and LEP.

FIG. 4B shows a zoom view of the chromatogram of FIG. 4A.

FIG. 5 shows an overlay of the chromatograms obtained from samples diluted in different surfactants: 5%Triton (e.g., Triton X-100) (v/v); 5% Brij-35 (p/v); Sodium Dodecyl Sulfate 2,5% (p/v); Tween-20 5% (v/v). Different surfactants showed the same values for the quantification of the impurity, although some surfactants in Dynamic Light Scattering (DLS) analysis did not show complete LNP disruption (see, FIGs. 6A-6C).

FIG. 6A shows an overlay of size distribution by volume of LNP-formulated mRNA product diluted in water, LNP-formulated mRNA product diluted in 5% TritonX-100 (v/v) solution, and the blank solution, Triton X- 100 in water. The different distribution of the LNP-formulated mRNA product with and without the surfactant shows the complete disruption of lipid nanoparticles obtained using TritonX-100.

FIG. 6B shows an overlay of size distribution by volume of LNP-formulated mRNA product diluted in water, LNP-formulated mRNA product diluted in 5% (v/v) Tween 20 solution , and the blank solution, Tween 20 in water. The similar distribution of the LNP-formulated mRNA product with and without the surfactant shows the incomplete disruption of lipid nanoparticles by Tween 20.

FIG. 6C shows an overlay of size distribution by volume of LNP-formulated mRNA product diluted in water, LNP-formulated mRNA product diluted in 5% (p/v) Brij-35 solution , and the blank solution, Brij-35 in water. The similar distribution of the LNP-formulated mRNA product with and without the surfactant shows the incomplete disruption of lipid nanoparticles by Brij-35.

FIG. 7 shows an overlay of chromatograms obtained from samples with different concentrations ofmRNA (10 pg/ml, 20 pg/ml, 40 pg/ml, 80 pg/ml and 100 pg/ml).

FIG. 8 shows results for a range of concentrations of Triton X-100 (e.g., 1.5%-5% (v/v)), where it was determined that 2.5% (v/v) is an optimized concentration of Triton X-100.

FIG. 9A shows a mRNA product peak with a retention time of about 3.10 minutes and a mRNA-lipid adduct peak with a retention time of about 3.50 minutes.

FIG. 9B shows a zoomed in view of FIG. 9A.

DETAILED DESCRIPTION

The inventors have developed a novel chromatography method in order to obtain a single and well-defined LEP peak. The method allows for accurate integration of the area under the impurity peak, also known as a late eluate peak (e.g., LEP) to determine the amount of the impurity in the mixture (or to determine ratios between components of the mixture). The LEP modification results in untranslatable mRNA and may lead to a reduction in protein expression. This method may be broadly applicable to manufacturing protocols, allowing the formation of lipid-mRNA adducts to be monitored and quantified, thereby ensuring that manufacturing standards of LNP -formulated mRNA products or other RNA products are met in terms of quality and purity.

The present method includes the development/optimization of a chromatography process that may be performed without RNA extraction - for example, the sample may be pre-treated with a surfactant prior to chromatography analysis and loaded directly onto the chromatography column (e.g., without a RNA extraction step). Thus, the present method allows analysis of the sample by chromatography, with or without lipid extraction. Different surfactants were explored and suitable surfactants include but are not limited to Triton X-100 (1-5%, 5-10%, 1- 10%), SDS (1-5%, 5-10%, 1-10%), Tween 20 (1-5%, 5-10%, 1-10%) and Brij-35 (1-5%, 5- 10%, 1-10%) (v/v) or (p/v). In some aspects, Triton X-100 may be present in an amount of about 0.5% -2.5%, about 0.5% -2.0%, about 0.5% -1.5%, about 0.5% -1.0%, or about 0.5%, or about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0% of the final percentage in the sample solution (v/v). Notably, considering the denaturing condition of the chromatography, any suitable surfactant may be applicable for impurity quantification (see, FIG. 5), even if incomplete disruption is shown by dynamic light scattering (DLS). Avoiding the lipid extraction step provides a substantial advantage in terms of reduced sample manipulation and analysis lead time, especially given that extraction normally counts for most of the analysis lead time.

Importantly, the inventors discovered an elution gradient comprising at least two different eluents that successfully resolved the LEP into a well-defined peak which could be quantified (e.g., by integration of the area under the curve). There is no mRNA extraction needed and low mRNA concentrations can be analyzed. Additionally, other parameters were optimized and include column temperature, chromatographic run length, and an option to perform blank subtraction.

DEFINITIONS

As used herein, an “ion pair” or “ion pairing agent” refers to a molecule (e.g., a small molecule) that changes the retention time of an analyte as it traverses the stationary phase of a chromatography column (e.g., a RP HPLC column). An ion pair may act as a counter ion to a charged or ionized functional group on an analyte (such as mRNA product(s), impurities (e.g., such as the LEP)) to thereby alter the retention time of the analyte on the column. In aspects, the ion pairs provided herein may be cationic ion pairs that interact with negatively charged functional groups (e.g., mRNA or mRNA/lipid products). In general, an ion pair further includes the small molecule itself and its derivates (e.g., including acetate, phosphate, bicarbonate, chloride, citrate, nitrate, nitrite, oxide, sulfate, etc.). In an aspect, one or more ion pairs provided in the methods herein is a cationic ion pair. Examples of cationic ion pairs include but are not limited to primary amines, secondary amines, tertiary amines, quaternary amines or protonated amines and/or salts thereof, such as triethylammonium acetate (TEAA) and dibutylammonium acetate (DBAA).

As used herein, a “mobile phase” is a polar solution used to carry an analyte (or analytes) through a chromatography column (e.g., a RP HPLC or UPLC column). The mobile phase may comprise water and/or one or more organic solvents. In general, a mobile phase for a IP- RP-HPLC or IP-RP-UPLC column comprises a polar organic solvent, including but not limited to acetone, acetonitrile, alcohols, amides, alkylsulfoxides, dimethylformamide, dimethylsulfoxide (DMSO), esters, ethanol, hexylene glycol, isopropanol, ketones, methanol, methyl acetate, nitrates, propanol, water, or other polar aprotic solvents such as Ci-4 alkanols, Ci-ealkandiols, and C2-4 alkanoic acids. In some embodiments, the mobile phase (e.g., comprising the first eluent and/or the second eluent) comprises acetonitrile and/or water.

As used herein, the term “stationary phase” refers to the immobile support of the column, e.g., comprising a hydrophobic material such as silica or CI-CI 8.

As used herein, the term “messenger RNA (mRNA)” refers to a ribonucleic acid that has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome to synthesize protein encoded by DNA. As disclosed herein, mRNA may be encapsulated by a lipid nanoparticle (LNP) shell for delivery and stability purposes. The mRNAs may be unmodified or modified nucleic acids. In some instances the nucleic acid is a modified nucleic acid.

As referred to herein, a “polynucleotide” or “nucleic acid” sequence is a series of nucleotide bases (also called “nucleotides” as found in DNA and RNA), and refers to any chain of two or more nucleotides, including genomic DNA, cDNA, RNA (unmodified or modified), synthetic polynucleotides, and recombinant polynucleotides, and includes single and/or double stranded molecules (e.g., DNA-DNA, DNA-RNA, RNA-RNA and “protein nucleic acids” (PNA) formed by conjugating nucleobases to an amino acid backbone).

As referred to herein, a “defined” or “well-defined” peak is a peak corresponding to an analyte (e.g., a LEP) eluted as part of a chromatography process, wherein the amount of the analyte can be accurately determined by integrating the area underneath the peak. In some aspects, the well-defined peak is eluted early in the chromatography process, prior to purging the column. In some aspects, the well-defined peak is of a sufficient height above a baseline to allow accurate integration. In some aspects, the well-defined peak is eluted in under 5 minutes, 4 minutes, 3 minutes, in under 2 minutes, in under 90 seconds, in under one minute so that the width of the peak with respect to the time axis is limited, allowing accurate integration of the area under the curve and reduction of background noise. In some aspects, the well-defined peak may have a shoulder. In aspects, the well-defined peak may overlap with other peaks provided that the accuracy in determining the amount of analyte (e.g., LEP) is not substantially affected by the overlap. In other aspects, the well-defined peak does not overlap or does not overlap substantially with other peaks.

As used herein, the term “lipid-mRNA adduct” or “adduct” or “lipid adduct” or “amino-lipid” refers to an RNA molecule which differs from the intended RNA molecule. In particular, an adduct may be a dysfunctional RNA molecule, for example an mRNA molecule whose ability to translate into a protein is impaired or even entirely abolished. As used herein, a “lipid-mRNA adduct” refers to an RNA molecule which is “lipidated” through covalent addition of lipid residues to nucleobases forming part of the RNA. In particular, a lipid adduct may be a dysfunctional lipidated RNA molecule, for example a lipidated mRNA molecule whose ability to translate into a protein is impaired or even entirely abolished, or a lipidated siRNA whose ability to silence translation of a target mRNA is impaired or even entirely abolished. Chromatographic methods may be used for the detection and quantification of RNA lipid adducts, in particular of lipid-mRNA adducts. Suitably, detection and/or quantification of lipid- mRNA adducts in the context of the invention is performed using analytical high performance liquid phase chromatography (HPLC), suitably analytical ion pair reverse phase HPLC (IP-RP- HPLC), for example as described in Packer et al. (2021). In the case of chromatography, quantification of lipid adduct, in particular of lipid-RNA or lipid-mRNA adducts, may be based on comparing the peak area (or “area under the peak”) of the full length functional RNA product and of the late eluting peak (LEP) in a chromatogram. The peak area may be determined by any suitable software which evaluates the signals of the detector system. The process of determining the peak area is also referred to as integration of the area under the peak . Herein, a “surfactant” is a substance which reduces the surface tension, thereby disrupting the LNP shell. According to present examples, the surfactant may be added to a sample containing a LNP-formulated mRNA product, prior to injecting the sample onto a chromatography column, to disrupt the LNP shell. The sample may then be loaded onto a column for quantification of the lipid adduct. In aspects, due to the denaturing conditions of the chromatography process, quantification is possible with a variety of surfactants, and does not require complete disruption of the LNP by the surfactant. Using the present method, pretreatment with a surfactant may be used in lieu of an RNA extraction step.

In aspects, mRNA concentrations ranging from 20 ug/ml, 30 ug/ml, 40 ug/ml or more up to about 100 ug/ml, 110 ug/ml, 120 ug/ml, 150 ug/ml, 200 ug/ml or more are suitable with Triton X-100 pre-treatment.

An LNP-formulated RNA product comprises RNA (e.g., including mRNA) subjected to lipid nanoparticle encapsulation. Thus, such RNA or mRNA product may comprise an impurity (e.g., lipid mRNA adduct). In general, HPLC may be used to separate the RNA or mRNA product from the impurity, allowing quantification of the impurity. In aspects, the LNP- formulated RNA or mRNA product may be pre-treated (e.g., with a surfactant or be subjected to extraction of the RNA and RNA-lipid adduct).

Ionizable amino-lipid

Herein, an “ionizable amino-lipid” is an ionizable lipid that has an amino group. An “ionizable lipid” is a lipid which becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the ionizable amino-lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. Suitably, the ionizable amino-lipid is selected from a tertiary ionizable amino-lipid and a secondary ionizable amino-lipid. More suitably, the ionizable amino-lipid is a tertiary ionizable amino-lipid.

In some embodiments, ionizable amino-lipids as defined herein have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, suitably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise be suitable in the context of the present invention. In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11 (e.g., a pKa of about 5 to about 7).

The ionizable amino-lipid may be cationic. Unless a different meaning is clear from the specific context, the term “cationic” means that the respective structure bears a positive charge, either permanently or not permanently, but in response to certain conditions such as pH. Thus, the term cationic covers both “permanently cationic” and “cationizable”. The term “permanently cationic” means, for example, that the respective compound, or group, or atom, is positively charged at any pH value or hydrogen ion activity of its environment. In some aspects, the positive charge results from the presence of a quaternary nitrogen atom.

The term “cationizable” as used herein means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also in nonaqueous environments where no pH value can be determined, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the respective cationizable group, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of cationizable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. In some embodiments, it is suitable that the cationizable compound or moiety is predominantly neutral at physiological pH values (e.g., about 7.0-7.4) but becomes positively charged at lower pH values. In some embodiments, the range of pKa for the cationizable compound or moiety is about 5 to about 7.

Suitable ionizable amino-lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N- dimethylaminopropane (y-DLenDMA), 98N12-5, l,2-Dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoley oxy-3 -(dimethylamino)acetoxypropane (Dlin-DAC), 1,2-Dilinoley oxy-3 -morpholinopropane (Dlin-MA), l,2-Dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3-dimethylaminopropane (Dlin-S- DMA), 1 -Linoleoyl -2 -linoleyloxy-3 -dimethylaminopropane (Dlin-2-DMAP), 1,2-

Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, Klin-K-DMA, Dlin-K-XTC2-DMA, XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane) HGT4003, l,2-Dilinoleoyl-3 -trimethylaminopropane chloride salt (Dlin-TAP.Cl),

1.2-Dilinoleyloxy-3-(N-methylpiperazino)propane (Dlin-MPZ), or 3-(N,N-Dilinoleylamino)-

1.2-propanediol (DlinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP), 1,2-

Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DM A), 2,2-Dilinoleyl-4- dimethylaminomethyl-[ 1,3] -dioxolane (Dlin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N- dimethyl-2,2-di((9Z, 12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d] [ l,3]dioxol-5- amine, (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 -tetraen- 19-yl-4-(dimethylamino)butanoate (MC3), ALNY- 100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z, 12Z)-octadeca-9, 12- dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5-amine)), l,l’-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (Dlin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin- K-DMA), NC98-5 (4,7, 13-tris(3-oxo-3-(undecylamino)propyl)-N ,N 16-diundecyl-4,7, 10,13- tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,3 lZ)-heptatriaconta-6, 9, 28, 31 -tetraen- 19-yl 4- (dimethylamino) butanoate (Dlin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylbutan-l-amine (MC4 Ether), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); dilinoleyl -methyl -4-dimethylaminobutyrate (Dlin-MC3-DMA); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable ionizable amino-lipids for use in the compositions and methods of the invention include those described in international patent publications W02010053572 (and particularly, CI 2-200 described at paragraph [00225]) and W02012170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1).

In some embodiments, the ionizable amino-lipid is an ionizable aminoalcohol lipidoid.

Ionizable aminoalcohol lipidoids may be prepared by the methods described in U.S. Patent No. 8,450,298, herein incorporated by reference in its entirety.

Suitable ionizable amino-lipids can also be the compounds as disclosed in Tables 1, 2 and 3 and as defined in claims 1-24 ofWO2017075531Al, hereby incorporated by reference.

In another embodiment, suitable ionizable amino-lipids can also be the compounds as disclosed in W02015074085A1 (i.e. ATX-001 to ATX-032 or the compounds as specified in claims 1-26), U.S. Appl. Nos. 61/905,724 and 15/614,499 or U.S. Patent Nos. 9,593,077 and 9,567,296 hereby incorporated by reference in their entirety.

In other embodiments, suitable ionizable amino-lipids can also be the compounds as disclosed in W02017117530A1 (i.e. lipids 13, 14, 15, 16, 17, 18, 19, 20, orthe compounds as specified in the claims), hereby incorporated by reference in its entirety.

In some embodiments, ionizable amino-lipids may also be selected from the lipids disclosed in W02018078053A1 (i.e. lipids derived from formula I, II, and III of W02018078053A1, or lipids as specified in Claims 1 to 12 of W02018078053A1), the disclosure of W02018078053A1 hereby incorporated by reference in its entirety. In that context, lipids disclosed in Table 7 of W02018078053A1 (e.g., lipids derived from formula I- 1 to 1-41) and lipids disclosed in Table 8 of W02018078053A1 (e.g., lipids derived from formula II-l to 11-36) may be suitably used in the context of the invention. Accordingly, formula 1-1 to formula 1-41 and formula II-l to formula 11-36 of W02018078053A1, and the specific disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, ionizable amino-lipids may be derived from formula III of published PCT patent application W02018078053A1. Accordingly, formula III of W02018078053A1, and the specific disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, the ionizable amino-lipid is selected from structures III- 1 to III- 36 of Table 9 of published PCT patent application W02018078053A1. Accordingly, formula III- 1 to III-36 of W02018078053A1, and the specific disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, the ionizable (cationic) lipid has the formula III:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

L¹ or L² is each independently -O(C=O)- or -(C=O)O-;

G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G³ is CI-C24 alkylene, C1-C24 alkenylene, Cs-Cs cycloalkylene, or Cs-Cs cycloalkenylene;

R¹ and R² are each independently, branched or linear, C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R⁵ is H or Ci-Ce alkyl.

In some embodiments, the ionizable (cationic) lipid has the formula III:

L¹ or L² is each independently -O(C=O)- or -(C=O)O-;

G¹ and G² are each independently unsubstituted C1-C12 alkylene;

G³ is C1-C24 alkylene; R¹ and R² are each independently, branched or linear, C6-C24 alkyl;

R³ is OR⁵; and

R⁵ is H.

In some embodiments, the ionizable (cationic) lipid has the formula III and wherein R¹,

R² or both R¹ and R² have one of the following structures:

In some embodiments, R² has the structure:

In some embodiments, the ionizable (cationic) lipid has the formula:

In some embodiments, the ionizable (cationic) lipid has the formula:

In some embodiments, the ionizable (cationic) lipid has the formula III-3 :

The lipid of formula III-3 as suitably used herein has the chemical term ((4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2 -hexyldecanoate), also referred to as ALC- 0315 i.e. CAS Number 2036272-55-4.

In some embodiments, the ionizable (cationic) lipid has the following structure:

Other suitable ionizable amino-lipids are disclosed in W02009086558, W02009127060, W02010048536, W02010054406, W02010088537, W02010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, US20140039032 and WO2017112865. In that context, the disclosures of W02009086558, W02009127060, W02010048536, W02010054406, W02010088537, W02010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541,

US20130225836 and US20140039032 and WO2017112865 specifically relating to ionizable (cationic) lipids suitable for UNPs are incorporated herewith by reference. In other embodiments, the ionizable amino-lipid is a tertiary ionizable amino lipid, suitably selected from:

A preferred lipid in that context may be selected or derived from SM-102 (CAS-Nummer: 2089251-47-6).

The amount of the ionizable amino-lipid may be selected taking the amount of RNA into account (N/P ratio). In this context, the “N/P ratio” is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the ionizable amino-lipid to the phosphate groups (“P”) of the RNA. The N/P ratio may be calculated on the basis that, for example, 1 pg RNA typically contains about 3 nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The “N”-value of the ionizable amino-lipid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and/or cationizable groups. If more than one ionizable amino-lipid is present, the N-value should be calculated on the basis of all ionizable amino-lipids comprised in the lipid nanoparticles.

In one embodiment, the composition has an N/P ratio from about 0.1 to about 20. In one embodiment, the composition has an N/P ratio from about 1 to about 20, suitably from about 2 to about 15, more suitably from about 3 to about 10, even more suitably from about 4 to about 9, most suitably about 6. In one embodiment, the composition has an N/P ratio from about 5 to about 20, more suitably from about 10 to about 18, even more suitably from about 12 to about 16, most suitably about 14.

Lipid nanoparticles

Suitably, the composition according to the invention comprises one or more further lipids which together with the ionizable amino-lipid as defined herein form a delivery system for the RNA. A range of delivery systems have been described which can be used to encapsulate (or complex) RNA in order to protect it and facilitate its delivery to target cells. In a preferred embodiment, the delivery system is a lipid nanoparticle (LNP).

The term “lipid nanoparticle” (or “LNP”) refers to a non-virion particle in which nucleic acid molecules, such as RNA, can be encapsulated. LNPs are not restricted to any particular morphology, and include any morphology generated when an ionizable (or cationic) lipid and optionally one or more further lipids are combined (e.g., in an aqueous environment and/or in the presence of a nucleic acid, such as an RNA). For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP). LNP delivery systems and methods for their preparation are known in the art.

Suitably, at least about 80%, 85%, 90%, 95% of lipid-based carriers, suitably the LNPs, have a spherical morphology.

LNPs are typically suitable for intramuscular, intradermal and/or intravenous administration.

In some embodiments, the ionizable amino-lipid is present in the LNP in an amount from about 20 mol% to about 70 mol% (based upon 100% total moles of lipid in the LNP). Suitably, the ionizable amino-lipid is present in the LNP in an amount from about 30 mol% to about 65 mol%. In one embodiment, the ionizable amino-lipid is present in the LNP in an amount from about 40 mol% to about 60 mol%, such as about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol%, respectively. In one embodiment, the ionizable amino-lipid is present in the LNP in an amount from about 47 mol% to about 48 mol%, such as about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mol%, respectively, wherein 47.4 mol% is particularly suitable.

The composition according to the invention can comprise two or more (different) ionizable amino-lipids as defined herein. Ionizable amino-lipids may be selected to contribute to different advantageous properties. For example, ionizable amino-lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the LNP (or liposomes, nanoliposomes, lipoplexes). In particular, the ionizable amino-lipids can be chosen so that the properties of the mixed-LNP are more desirable than the properties of a single-LNP of individual lipids. If more than one ionizable amino-lipid are present, the percentages above apply to the total amount of ionizable amino-lipids.

In addition to the ionizable amino-lipid, the composition according to the invention typically comprises one or more additional lipids selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., PEGylated lipid) which together with the ionizable amino-lipid form an LNP.

Suitably, the composition according to the invention comprises a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” (or “PEG-modified lipid” or “PEG lipid”) refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

A polymer conjugated lipid as defined herein (e.g., a PEGylated lipid), may serve as an aggregation reducing lipid.

Suitably, the LNP comprises a PEGylated lipid. Suitable PEGylated lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative PEGylated lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the PEGylated lipid is N-[(methoxy polyethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3 -amine (PEG-c- DMA). In some embodiments, the PEGylated lipid is PEG-2000-DMG. In one embodiment, the PEGylated lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-O- (co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate.

In some embodiments, the PEGylated lipid comprises PEG-DMG or PEG-cDMA.

In embodiments, the PEGylated lipid is suitably derived from formula (IV) of published

PCT patent application W02018078053A1. Accordingly, PEGylated lipids derived from formula (IV) of published PCT patent application W02018078053A1, and the respective disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, the PEGylated lipid has the formula IV :

wherein R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In one embodiment, R⁸ and R⁹ are saturated alkyl chains.

In some embodiments, the RNA, suitably mRNA, is complexed with one or more lipids thereby forming LNPs, wherein the LNP comprises a polymer conjugated lipid, suitably a PEGylated lipid, wherein the PEGylated lipid is suitably derived from formula (Iva) of published PCT patent application W02018078053A1. Accordingly, PEGylated lipid derived from formula (Iva) of published PCT patent application W02018078053A1, and the respective disclosure relating thereto, is herewith incorporated by reference.

In some embodiments, the PEGylated lipid is of formula (Iva):

wherein n has a mean value ranging from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In an embodiment n is about 49. In another embodiment n is about 45. In further embodiments, the PEGylated lipid is of formula (Iva) wherein n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2000g/mol to about 3000 g/mol or about 2300g/mol to about 2700g/mol, suitably about 2500g/mol.

In some embodiments, the PEGylated lipid has the formula Iva:

wherein n has a mean value ranging from 30 to 60, suitably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most suitably wherein n has a mean value of 49 or 45; or wherein n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2500g/mol.

The lipid of formula Iva as suitably used herein has the chemical term 2 [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.

Further examples of PEGylated lipids suitable in that context are provided in US20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEGylated lipid, based on the total moles of lipid in the LNP.

In further embodiments, LNPs comprise from about 0.1% to about 20% of the PEGylated lipid on a molar basis, for example, about 0.5 to about 15%, about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2,5%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the LNP). In embodiments, LNPs comprise from about 1.0% to about 2.0% of the PEGylated lipid on a molar basis, for example, about 1.2 to about 1.9%, about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about 1.8%, about 1.5 to about 1.8%, about 1.6 to about 1.8%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about

1.9%, most suitably 1.7% (based on 100% total moles of lipids in the LNP). In various embodiments, the molar ratio of the ionizable amino-lipid to the PEGylated lipid ranges from about 100 : 1 to about 25: 1.

In some embodiments, the LNP comprises a PEGylated lipid at around 0.5 to 10 molar %, optionally 0.5 to 5 molar % or 0.5 to 3 molar %.

Suitably, the composition according to the invention comprises one or more stabilizing lipids (or helper lipids), which stabilize the formation of particles during their formulation or during the manufacturing process (e.g., neutral lipid and/or one or more steroid or steroid analogue).

Suitable stabilizing lipids (or helper lipids) include neutral lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

In some embodiments, the composition according to the invention comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), pahnitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- Icarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidylethanolamine (DSPE), 16- O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), sphingomyelin (SM) or mixtures thereof. In various embodiments, the molar ratio of the ionizable amino-lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

Suitably, the neutral lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Suitably, the molar ratio of the ionizable amino-lipid to DSPC may be in the range from about 2: 1 to about 8: 1.

Suitable stabilizing lipids (or helper lipids) also include steroids or steroid analogues.

In some embodiments, the steroid is a sterol, suitably cholesterol.

Suitably, the molar ratio of the ionizable amino-lipid to cholesterol may be in the range from about 2: 1 to about 1 : 1. In some embodiments, the cholesterol may be PEGylated.

The sterol can be about 10mol% to about 60mol% or about 25mol% to about 55mol% or about 25mol% to about 40mol% of the lipid particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60mol% of the total lipid present in the lipid particle. In another embodiment, the LNPs include from about 5% to about 50% on a molar basis of the sterol, for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Suitably, in addition to the RNA, an ionizable amino-acid, the composition according to the invention comprises a PEGylated lipid, a neutral lipid and a sterol. Suitably, the composition according to the invention comprises RNA, an ionizable amino-acid, a PEGylated lipid, a neutral lipid and a sterol. More suitably, the composition according to the invention comprises mRNA, an ionizable amino-acid, a PEGylated lipid, a neutral lipid and a sterol.

In one embodiment, the composition comprises:

(i) at least one ionizable amino-lipid as defined herein, suitably lipid of formula III-3;

(ii) at least one neutral lipid as defined herein, suitably l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC); (iii) at least one sterol as defined herein, suitably cholesterol; and

(iv) at least one a PEGylated lipid as defined herein (e.g., PEG-DMG or PEG-cDMA), suitably a PEGylated lipid that is or is derived from formula Iva.

In one embodiment, the ionizable amino-lipid has formula III-3, the neutral lipid is DSPC, the steroid is cholesterol and the PEGylated lipid which has formula Iva.

Suitably, lipids (i) to (iv) form LNPs and are present in a molar ratio of about 20-60% ionizable amino-lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEGylated lipid. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 50: 10:38.5: 1.5. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.5: 10.0:40.8: 1.7. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.4: 10.0:40.9: 1.7.

In one embodiment, the composition according to the invention comprises the ionizable amino-lipid of formula III-3, DSPC, cholesterol and the PEGylated lipid of formula Iva in a molar ratio of about 47.4: 10.0:40.9: 1.7.

Other useful LNP compositions are described in the following references: W02012/006376; WO2012/030901; WO2012/031046; WO2012/031043; WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825; WO2014/136086; W02015/095340; WO2015/095346; W02016/037053; W02017/070620 which are incorporated herein by reference.

Suitably, the LNPs have a mean diameter of from about 50nm to about 200nm, from about 60nm to about 200nm, from about 70nm to about 200nm, from about 80nm to about 200nm, from about 90nm to about 200nm, from about 90nm to about 190nm, from about 90nm to about 180nm, from about 90nm to about 170nm, from about 90nm to about 160nm, from about 90nm to about 150nm, from about 90nm to about 140nm, from about 90nm to about 130nm, from about 90nm to about 120nm, from about 90nm to about lOOnm, from about 70nm to about 90nm, from about 80nm to about 90nm, from about 70nm to about 80nm, or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm,

95nm, lOOnm, 105nm, HOnm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 160nm, 170nm, 180nm, 190nm, or 200nm and are substantially non-toxic. As used herein, the mean diameter may be represented by the z-average size as determined by dynamic light scattering as commonly known in the art.

Suitably, the LNPs have a polydispersity index (Pdl) of 0.4 or less, suitably of 0.3 or less. Typically, the Pdl is determined by dynamic light scattering.

Suitably, at least 50%, more suitably at least 60%, 70% 80%, 85%, 90% or 95% of the RNA is encapsulated in the LNP. In this context, “encapsulated RNA” is understood as RNA (suitably mRNA) that is complexed with the lipids forming the LNP and/or that is contained within the interior space of the LNP. The proportion of encapsulated RNA can typically be determined using a RiboGreen assay.

Suitably, the composition contains less than about 30%, suitably less than 20%, 15%, 10% or 5% non-encapsulated RNA (or free RNA). In this context, the term “free RNA” or “non-encapsulated RNA” is understood as RNA (suitably mRNA) that is not encapsulated in the LNPs as defined herein. In a therapeutic composition, free RNA, may represent a contamination or an impurity.

RNA

The method of the invention comprises separating a RNA molecule (e.g., a mRNA product) from an impurity (a lipid adduct) on a chromatography column, for example, generated by electrophilic degradants and impurities originating in an ionizable liquid. This modification may make the mRNA untranslatable and lead to a loss of protein expression (see, Packer et al., Nat. Comm. 12, 6777 (2021)).

The term “RNA” is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosinemonophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogs thereof, which are connected to each other along a so-called backbone. The backbone is typically formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence.

Suitably, the RNA molecule is selected from an antisense RNA, such as an antisense oligonucleotides (ASOs), a small interfering RNA (siRNA), a microRNA (miRNAs), a messenger RNA (mRNA) and an RNA forming part of a single-guide RNA (sgRNA)-mediated CRISPR-Cas system.

Suitably, the amount of RNA, suitably mRNA, in the composition according to the invention ranges from about 0.1 to about 1000 pg, for example from about 1 to about 500 pg, especially from about 2 to about 500 pg, in particular from about 10 to about 250 pg.

Suitably, the RNA molecule has a length of at least 200, more suitably at least 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides, 2 kB, 3kB, or 4 kB, and any range inclusive therein.

In a preferred embodiment, the RNA molecule is a coding RNA.

In a preferred embodiment, the RNA molecule is a messenger RNA (mRNA).

An mRNA is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by ribosomes in the process of producing a protein. The mRNA may be selected from non-replicating mRNA and self-replicating mRNA (or which may also be referred to as self-amplifying mRNA or SAM). A non-replicating mRNA typically encodes a protein of interest and contains 5 ' and/or 3 ' untranslated regions (UTRs), a 5 ’ cap and a poly(A) tail. A self-replicating mRNA also encodes a viral replication machinery that enables intracellular mRNA amplification. In one embodiment, the RNA molecule is a non-replicating mRNA.

In one embodiment, the RNA molecule is a self-replicating mRNA. The RNA, suitably mRNA, may be prepared in many ways, for example, by chemical synthesis in whole or in part, by digesting longer nucleic acids using nucleases (e.g., restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), from genomic or cDNA libraries, etc. In particular, the mRNA may be prepared enzymatically using a DNA template.

The RNA, suitably mRNA, may be an artificial nucleic acid.

The term “artificial nucleic acid” as used herein is intended to refer to a nucleic acid that does not occur naturally. In other words, an artificial nucleic acid may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecules may be non-natural due to its individual sequence (e.g., G/C content modified coding sequence, UTRs) and/or due to other modifications, for example, structural modifications of nucleotides. Typically, artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, an artificial nucleic acid is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type or reference sequence/the naturally occurring sequence by at least one nucleotide (via e.g., codon modification as further specified below). The term “artificial nucleic acid” is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules. Accordingly, it may relate to a plurality of essentially identical nucleic acid molecules.

In some embodiments, the mRNA may be a modified and/or stabilized mRNA, suitably a modified and/or stabilized artificial mRNA.

According to some embodiments, the mRNA may thus be provided as a “stabilized” artificial nucleic acid or stabilized coding nucleic acid that is to say a nucleic acid showing improved resistance to in vivo degradation and/or a nucleic acid showing improved stability in vivo, and/or a nucleic acid showing improved translation efficiency in vivo. In the following, specific suitable modifications/adaptations in this context are described which are suitable to stabilize the nucleic acid.

The mRNA may be codon optimized. In some embodiments, the mRNA comprises at least one codon modified coding sequence. In some embodiments, the coding sequence of the mRNA is a codon modified coding sequence. Suitably, the amino acid sequence encoded by the codon modified coding sequence is not modified compared to the amino acid sequence encoded by the corresponding wild type or reference coding sequence. Suitably, the mRNA may be codon optimized for expression in human cells.

By “codon optimized” it is intended that modification with respect to codon usage may increase translation efficacy and/or half-life of the nucleic acid. The term “codon modified coding sequence” relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference coding sequence. Suitably, a codon modified coding sequence in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translation efficiency in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably (cf. Table 1 of W02020002525) to optimize/modify the coding sequence for in vivo applications as outlined herein.

In some embodiments, the mRNA may be modified, wherein the C content of the at least one coding sequence is increased, suitably maximized, compared to the C content of the corresponding wild type or reference coding sequence (herein referred to as “C maximized coding sequence”). The amino acid sequence encoded by the C maximized coding sequence of the mRNA is suitably not modified compared to the amino acid sequence encoded by the respective wild type or reference coding sequence. The generation of a C maximized nucleic acid sequences may suitably be carried out using a modification method according to WO2015/062738. In this context, the disclosure of WO2015/062738 is included herewith by reference.

In some embodiments, the mRNA may be modified, wherein the codons in the at least one coding sequence may be adapted to human codon usage (herein referred to as “human codon usage adapted coding sequence”). Codons encoding the same amino acid occur at different frequencies in humans. Accordingly, the coding sequence of the mRNA is suitably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage. For example, in the case of the amino acid Ala, the wild type or reference coding sequence is suitably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (see e.g., Table 1 of W02020002525). Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the RNA to obtain sequences adapted to human codon usage.

In embodiments, the mRNA may be modified, wherein the codon adaptation index (CAI) may be increased or suitably maximised in the at least one coding sequence (herein referred to as “CAI maximized coding sequence”). In some embodiments, all codons of the wild type or reference nucleic acid sequence that are relatively rare in, for example, a human are exchanged for a respective codon that is frequent in the, for example, a human, wherein the frequent codon encodes the same amino acid as the relatively rare codon. Suitably, the most frequent codons are used for each amino acid of the encoded protein (see Table 1 of W02020002525, most frequent human codons are marked with asterisks). Suitably, the mRNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. In some embodiments, the codon adaptation index (CAI) of the at least one coding sequence is 1 (CAI=1). For example, in the case of the amino acid Ala, the wild type or reference coding sequence may be adapted in a way that the most frequent human codon “GCC” is always used for the amino acid. Accordingly, such a procedure (as exemplified for Ala) may be applied for each amino acid encoded by the coding sequence of the mRNA to obtain CAI maximized coding sequences.

In some embodiments, the mRNA may be modified, wherein the G/C content of the at least one coding sequence may be modified (or optimized) compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as “G/C content modified_coding sequence”). In this context, the terms “G/C optimization” or “G/C content modification” relate to a nucleic acid that comprises a modified, suitably an increased number of guanosine and/or cytosine nucleotides as compared to the corresponding wild type or reference coding sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides. Suitably, nucleic acid sequences having an increased G /C content are more stable or show a better expression than sequences having an increased A/U. The amino acid sequence encoded by the G/C content modified coding sequence of the mRNA is suitably not modified as compared to the amino acid sequence encoded by the respective wild type or reference sequence. In some embodiments, the G/C content of the coding sequence of the nucleic acid is increased by at least 10%, 20%, 30%, suitably by at least 40% compared to the G/C content of the coding sequence of the corresponding wild type or reference nucleic acid sequence. The generation of a G/C content optimized mRNA sequence may be carried out using a method according to W02002/098443. In this context, the disclosure of W02002/098443 is included in its full scope in the present invention. In some embodiments, the mRNA may be modified by altering the number of A and/or U nucleotides in the nucleic acid sequence with respect to the number of A and/or U nucleotides in the original nucleic acid sequence (e.g., the wild type or reference sequence). In some embodiments, such an A/U alteration is performed to modify the retention time of the individual nucleic acids in a composition, to (i) allow co-purification using a HPLC method, and/or to allow analysis of the obtained nucleic acid composition. Such a method is described in detail in published PCT application WO2019092153A1. Claims 1 to 70 of WO2019092153A1 herewith incorporated by reference.

In some embodiments, the modified RNA sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified (or optimized) sequence, A/U alteration, or any combination thereof.

In some embodiments, the RNA sequence has a G/C content of at least about 45%, 50%, 55%, or 60%. In particular embodiments, the RNA sequence has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

Suitably, when transfected into mammalian host cells, the mRNA comprising a modified sequence has a stability of between 12-18 hours, or greater than 18 hours (e.g., 24, 36, 48, 60, 72, or greater than 72 hours) and are capable of being expressed by the mammalian host cell (e.g., a muscle cell).

Suitably, when transfected into mammalian host cells, the mRNA comprising a modified RNA sequence is translated into protein, wherein the amount of protein is at least comparable to, or suitably at least 10% more than, or at least 20% more than, or at least 30% more than, or at least 40% more than, or at least 50% more than, or at least 100% more than, or at least 200% or more than the amount of protein obtained by a naturally occurring or wild type or reference coding sequence transfected into mammalian host cells.

In some embodiments, the mRNA comprises at least one poly(N) sequence, for example, at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or combinations thereof.

In one embodiment, the mRNA comprises at least one poly(A) sequence. Suitably, a poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life.

The terms “poly(A) sequence”, “poly(A) tail” or “3’-poly(A) tail” as used herein will be recognized and understood by the person of ordinary skill in the art, and are, for example, intended to be a sequence of adenosine nucleotides, typically located at the 3 ’-end of a linear RNA (or in a circular RNA), of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric, for example, a poly(A) sequence of 100 adenosine nucleotides has essentially the length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, for example, a poly(A) sequence of 100 adenosine nucleotides may have a length of more than 100 nucleotides comprising 100 adenosine nucleotides and in addition the at least one nucleotide - or a stretch of nucleotides different from an adenosine nucleotide.

The poly(A) sequence may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. In some embodiments, the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.

In some embodiments, the mRNA comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides. In some embodiments, the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In other embodiments, the poly(A) sequence comprises about 100 adenosine nucleotides (A 100). In other embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides.

In further embodiments, the mRNAs used herein comprise at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, suitably by 10 non-adenosine nucleotides (A30- N10-A70).

The poly(A) sequence as defined herein may be located directly at the 3 ’ terminus of the mRNA. In some embodiments, the 3’-terminal nucleotide (that is the last 3’-terminal nucleotide in the polynucleotide chain) is the 3 ’-terminal A nucleotide of the at least one poly(A) sequence. The term “directly located at the 3’ terminus” is to be understood as being located exactly at the 3 ’ terminus - in other words, the 3 ’ terminus of the nucleic acid consists of a poly (A) sequence terminating with an A nucleotide.

In one embodiment, the mRNA comprises a poly(A) sequence of at least 70 adenosine nucleotides, suitably consecutive at least 70 adenosine nucleotides, wherein the 3 ’-terminal nucleotide is an adenosine nucleotide.

In embodiments, the poly(A) sequence of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly(A) sequence is obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template. In other embodiments, poly(A) sequences are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively, by using immobilized poly(A)polymerases, for example, using a methods and means as described in WO2016174271.

In one embodiment, the mRNA comprises at least one poly(C) sequence.

The term “poly(C) sequence” as used herein is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In an embodiment, the poly(C) sequence comprises about 30 cytosine nucleotides.

In some embodiments, the mRNA comprises a poly(A) tail sequence, preferably comprising 30 to 200 adenosine nucleotides and/or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.

In some embodiments, the mRNA comprises at least one histone stem-loop (hSL) or histone stem loop structure.

The term “histone stem-loop” (e.g., abbreviated as “hSL” in the sequence listing) is intended to refer to nucleic acid sequences that form a stem-loop secondary structure predominantly found in histone mRNAs.

Histone stem-loop sequences/structures may suitably be selected from histone stemloop sequences as disclosed in W02012019780, the disclosure relating to histone stem-loop sequences/histone stem -loop structures incorporated herewith by reference. A histone stemloop sequence that may be used may be derived from formulae (I) or (II) of W02012019780. According to a further embodiment, the mRNA comprises at least one histone stem-loop sequence derived from at least one of the specific formulae (ia) or (Ila) of the patent application W02012019780.

In other embodiments, the mRNA does not comprise a hsL as defined herein.

The mRNA may be modified by the addition of a 5 ’-cap structure, which suitably stabilizes the RNA and/or enhances expression of the encoded antigen and/or reduces the stimulation of the innate immune system (after administration to a subject).

The term “5 ’-cap structure” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5 ’-end of an RNA (e.g., an mRNA).

For example, the 5' end of the mRNA may be capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine -7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the mRNA molecule may be further modified by a 2'-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy.

In some embodiments, the 5 ’-cap structure is connected via a 5 ’-5 ’-triphosphate linkage to the RNA.

5 ’-cap structures which may be suitable are capO (methylation of the first nucleobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1 -methyl -guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8- oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In some embodiments, the mRNA comprises a 5’ cap, preferably m7G, capO, capl, cap2, a modified capO or a modified capl structure, suitably a 5 ’-capl structure. A 5 ’-cap (such as capO or capl) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co-transcriptional capping) using cap analogues.

The term “cap analogue” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of a nucleic acid molecule, particularly of an RNA molecule, when incorporated at the 5 ’-end of the nucleic acid molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5 ’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3 ’-direction by a templatedependent polymerase, particularly, by template-dependent RNA polymerase. Examples of cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g., ARCA; m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG and their tetraphosphate derivatives). Further cap analogues have been described previously (W02008016473, WO2008157688, WO2009149253, WO2011015347, and WO2013059475). Further suitable cap analogues in that context are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017/053297, WO2017066782, WO2018075827 and WO2017066797 wherein the disclosures referring to cap analogues are incorporated herewith by reference.

In some embodiments, a modified cap 1 structure is generated using tri -nucleotide cap analogue as disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827 and WO2017066797. In particular, any cap structures derivable from the structure disclosed in claim 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate a modified capl structure. Further, any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate a modified capl structure.

In some embodiments, the mRNA comprises a capl structure.

In some embodiments, the 5 ’-cap structure may be added co-transcriptionally using trinucleotide cap analogue as defined herein, suitably in an RNA in vitro transcription reaction as defined herein.

In some embodiments, the capl structure of the mRNA is formed using co- transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG. A suitable capl analogues in that context is m7G(5’)ppp(5’)(2’OMeA)pG.

In other embodiments, the capl structure of the mRNA is formed using co- transcriptional capping using tri -nucleotide cap analogue 3’0Me- m7G(5’)ppp(5’)(2’OMeA)pG.

In other embodiments, a capO structure of the mRNAs used herein is formed using co- transcriptional capping using cap analogue 3’0Me-m7G(5’)ppp(5’)G.

In other embodiments, the 5 ’-cap structure is formed via enzymatic capping using capping enzymes (e.g., vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases) to generate capO or capl or cap2 structures. The 5 ’-cap structure (capO or capl) may be added using immobilized capping enzymes and/or cap-dependent 2’-0 methyltransferases using methods and means disclosed in WO2016193226.

For determining the presence/absence of a capO or a capl structure, a capping assays as described in published PCT application WO2015101416, in particular, as described in claims 27 to 46 of published PCT application W02015101416 can be used. Other capping assays that may be used to determine the presence/absence of a capO or a cap 1 structure of an RNA are described in PCT/EP2018/08667, or published PCT applications WO2014152673 and WO2014152659.

In some embodiments, the mRNA comprises an m7G(5’)ppp(5’)(2’OMeA) cap structure. In such embodiments, the mRNAs comprise a 5’-terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide of m7GpppN, in that case, a 2’0 methylated Adenosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises such a capl structure as determined using a capping assay.

In other embodiments, the mRNAs used herein comprise an m7G(5’)ppp(5’)(2’OMeG) cap structure. In such embodiments, the mRNAs comprise a 5’-terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide, in that case, a 2’0 methylated guanosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the coding RNA (species) comprises such a capl structure as determined using a capping assay.

Accordingly, the first nucleotide of the mRNA sequence, that is, the nucleotide downstream of the m7G(5’)ppp structure, may be a 2’0 methylated guanosine or a 2’0 methylated adenosine.

Suitably, the mRNAs used herein comprise a ribosome binding site, also referred to as Kozak sequence. In some embodiments, the A/U (A/T) content in the environment of the ribosome binding site of the mRNAs used herein may be increased compared to the A/U (A/T) content in the environment of the ribosome binding site of its respective wild type or reference nucleic acid. This modification (an increased A/U (A/T) content around the ribosome binding site) increases the efficiency of ribosome binding to the mRNA. An effective binding of the ribosomes to the ribosome binding site in turn has the effect of an efficient translation of the mRNA

In some embodiments, the mRNA comprises at least one heterologous untranslated region (UTR), for example, a 5’ UTR and/or a 3’ UTR.

The term “untranslated region” or “UTR” or “UTR element” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a part of a nucleic acid molecule typically located 5’ or 3’ of a coding sequence. An UTR is not translated into protein. An UTR may be part of a nucleic acid (e.g., a DNA or an RNA). An UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites, miRNA binding sites, promotor elements, etc.

In some embodiments, the mRNA comprises a protein-coding region (“coding sequence” or “cds”), and 5 ’-UTR and/or 3 ’-UTR. Notably, UTRs may harbor regulatory sequence elements that determine nucleic acid (e.g., RNA turnover, stability, and localization). Moreover, UTRs may harbor sequence elements that enhance translation. In medical application of nucleic acid sequences (including DNA and RNA), translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy. Certain combinations of 3 ’-UTRs and/or 5 ’-UTRs may enhance the expression of operably linked coding sequences encoding peptides or proteins of the invention. Nucleic acid molecules harboring the UTR combinations advantageously enable rapid and transient expression of antigenic peptides or proteins after administration to a subject, suitably after intramuscular administration. Accordingly, the mRNA comprising certain combinations of 3 ’-UTRs and/or 5 ’-UTRs as provided herein is particularly suitable for administration as a vaccine or therapeutic, in particular, suitable for administration into the muscle, the dermis, or the epidermis of a subject.

In some embodiments, the mRNA comprises at least one heterologous 5 ’-UTR and/or at least one heterologous 3 ’-UTR. The heterologous 5 ’-UTRs or 3 ’-UTRs may be derived from naturally occurring genes or may be synthetically engineered. In embodiments, the mRNA comprises at least one coding sequence as defined herein operably linked to at least one

(heterologous) 3’-UTR and/or at least one (heterologous) 5 ’-UTR.

In some embodiments, the mRNA comprises at least one heterologous 3 ’-UTR.

The term “3 ’-untranslated region” or “3’-UTR” or “3’-UTR element” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a part of a nucleic acid molecule located 3’ (i.e. downstream) of a coding sequence and which is not translated into protein. A 3 ’-UTR may be part of a nucleic acid (e.g., a DNA or an RNA), located between a coding sequence and an (optional) terminal poly(A) sequence. A 3 ’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites, miRNA binding sites, etc.

In some embodiments, the mRNA comprises a 3 ’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).

In some embodiments, a 3 ’-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.

In embodiments, the mRNA comprises at least one heterologous 3 ’-UTR, wherein the at least one heterologous 3 ’-UTR comprises a nucleic acid sequence is derived or selected from a 3 ’-UTR of a gene selected from PSMB3, AUB7, alpha-globin (referred to as “muag”). CASP1, C0X6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or variant of any one of these genes.

In some embodiments, the mRNA comprises a 3’ UTR comprising or consisting of a nucleic acid sequence derived from a 3 ’-UTR of a gene selected from PSMB3, AUB7, CASP1, C0X6B 1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes. Nucleic acid sequences in that context can be derived from published PCT application

W02019077001A1, in particular, claim 9 of W02019077001A1. The corresponding 3’-UTR sequences of claim 9 ofW02019077001Al are herewith incorporated by reference.

In some embodiments, the mRNA comprises a 3’-UTR as described in WO2016107877, the disclosure of WO2016107877 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 3’-UTR as described in W02017036580, the disclosure of W02017036580 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 152-204 of W02017036580, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 3’-UTR as described in WO2016022914, the disclosure of WO2016022914 relating to 3’-UTR sequences herewith incorporated by reference. Particularly suitable 3’-UTRs are nucleic acid sequences according to SEQ ID NOs: 20-36 of WO2016022914, or fragments or variants of these sequences.

In some embodiments, the mRNA comprises at least one heterologous 5’-UTR.

The terms “5 ’-untranslated region” or “5’-UTR” or “5’-UTR element” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a part of a nucleic acid molecule located 5’ (i.e. “upstream”) of a coding sequence and which is not translated into protein. A 5’-UTR may be part of a nucleic acid located 5’ of the coding sequence. Typically, a 5’-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence. A 5’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites, miRNA binding sites etc. The 5’-UTR may be post- transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5’- cap structure (e.g., for mRNA as defined herein). In some embodiments, the mRNA comprises a 5’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).

In some embodiments, a 5’-UTR comprises one or more of a binding site for proteins that affect an RNA stability or RNA location in a cell, or one or more miRNA or binding sites for miRNAs.

In embodiments, the mRNA comprises at least one heterologous 5 ’-UTR, wherein the at least one heterologous 5 ’-UTR comprises a nucleic acid sequence is derived or selected from a 5 -UTR of gene selected from HSD17B4, RPL32, ASAHI, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.

Nucleic acid sequences in that context can be selected from published PCT application W02019077001A1, in particular, claim 9 of W02019077001A1. The corresponding 5’-UTR sequences of claim 9 ofW02019077001Al are herewith incorporated by reference (e.g., SEQ ID NOs: 1-20 of W02019077001A1, or fragments or variants thereof).

In some embodiments, the mRNA comprises a 5 ’-UTR as described in W02013143700, the disclosure of WO2013143700 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WO2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WO2016107877, the disclosure of WO2016107877 relating to 5 ’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in W02017036580, the disclosure of W02017036580 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 of W02017036580, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WO2016022914, the disclosure of WO2016022914 relating to 5’ -UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 of WO2016022914, or fragments or variants of these sequences.

In some embodiments, the mRNA comprises an heterologous 5 ’-UTR that comprises or consists of a nucleic acid sequence derived from a 5 ’-UTR from HSD17B4 and at least one heterologous 3’-UTR comprises or consists of a nucleic acid sequence derived from a 3’-UTR ofPSMB3.

In one embodiment, the mRNA comprises from 5 ’ to 3 ’ : i) 5'-capl structure; ii) 5 ’-UTR derived from a 5 ’-UTR of a HSD17B4 gene; iii) the coding sequence; iv) 3 ’-UTR derived from a 3 ’-UTR of a PSMB3 gene; v) optionally, a histone stem -loop sequence; and vi) poly(A) sequence comprising about 100 A nucleotides, wherein the 3’ terminal nucleotide of said RNA is an adenosine.

Chemical Modifications

In some embodiments, the RNA, suitably mRNA, comprises a coding sequence that consists only of G, C, A and U nucleotides and therefore does not comprise modified nucleotides (except of the 5’ terminal cap structure (capO, capl, cap2)).

In some embodiments, the RNA, suitably mRNA, is a modified RNA, suitably mRNA, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. A modified RNA, suitably mRNA, may comprise one or more nucleotide analogs or modified nucleotides (nucleotide analogues/modifications, for example, backbone modifications, sugar modifications or base modifications). As used herein, "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)) and/or one or more chemical modifications in or on the phosphates of the backbone. A nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, modified ribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate. The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, see the following references: US Patent Numbers 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.

A backbone modification as described herein is a modification, in which phosphates of the backbone of the nucleotides of the RNA, suitably the mRNA, are chemically modified. A sugar modification as described herein is a chemical modification of the sugar of the nucleotides of the RNA, suitably mRNA. Furthermore, a base modification as described herein is a chemical modification of the base moiety of the nucleotides of the RNA, suitably mRNA. In this context, nucleotide analogues or modifications are suitably selected from nucleotide analogues which are applicable for transcription and/or translation.

In some embodiments, the RNA, suitably the mRNA, comprises at least one chemical modification.

Modified nucleobases (chemical modifications) which can be incorporated into modified nucleosides and nucleotides and be present in the RNA, suitably mRNA, molecules include: m5C (5 -methylcytidine), m5U (5 -methyluridine), m6A (N6-methyladenosine), s2U (2 -thiouridine), Um (2'-0-methyluridine), mlA (1 -methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1- methylinosine); m'lm (1 ,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2’-0- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2-O-methylcytidine); k2C (lysidine); mlG (1 -methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'- O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O- dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxy queuosine); galQ (galtactosyl-queuosine); manQ (mannosyl- queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5 -hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5 -methoxy carbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O- methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5- aminomethyl-2-thiouridine); mnm5U (5 -methylaminomethyluridine); mnm5s2U (5- methylaminomethyl-2 -thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5 -carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L-O-methyl uridine); cmnm5s2U (5 -carboxymethylaminomethyl -2- thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4- methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5 -hydroxymethylcytidine); m3U (3 -methyluridine); cm5U (5 -carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); m62Am (N6,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m5D (5- methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); mlGm (1 ,2'-0- dimethylguanosine); m'Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S- taurinomethyl-2-thiouridine)); iniG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5 -aminouracil, 5-(Ci-Ce)-alkyluracil, 5- methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5-(hydroxymethyl)uracil, 5- chlorouracil, 5 -fluorouracil, 5 -bromouracil, 5 -hydroxy cytosine, 5-(Ci-C6)-alkylcytosine, 5- methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5 -chlorocytosine, 5- fluorocytosine, 5 -bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7- deaza-7-substituted guanine, 7-deaza-7-(C2-Ce)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6- chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.

In some embodiments, the nucleotide analogue s/modifications which may be incorporated into a modified RNA, suitably mRNA, are selected from 2-amino-6- chloropurineriboside-5 ’-triphosphate, 2 -Aminopurine-riboside-5 ’-triphosphate; 2- aminoadenosine-5 ’ -triphosphate, 2 ’ -Amino-2 ’ -deoxy cytidine -triphosphate, 2-thiocytidine-5 ’ - triphosphate, 2-thiouridine-5 ’-triphosphate, 2’-Fluorothymidine-5 ’-triphosphate, 2’-O- Methyl-inosine-5 ’-triphosphate 4-thiouridine-5 ’-triphosphate, 5 -aminoallylcytidine -5 ’- triphosphate, 5 -aminoallyluridine-5’ -triphosphate, 5 -bromocytidine-5 ’ -triphosphate, 5- bromouridine-5 ’-triphosphate, 5 -Bromo-2’ -deoxy cytidine-5 ’-triphosphate, 5-Bromo-2’- deoxyuridine-5 ’-triphosphate, 5-iodocytidine-5 ’-triphosphate, 5 -Iodo-2’ -deoxy cytidine-5 ’- triphosphate, 5 -iodouridine-5 ’ -triphosphate, 5-Iodo-2’-deoxyuridine-5’-triphosphate, 5- methylcytidine-5 ’ -triphosphate, 5-methyluridine-5 ’ -triphosphate, 5 -Propynyl-2 ’ - deoxycytidine -5 ’-triphosphate, 5-Propynyl-2’-deoxyuridine-5 ’-triphosphate, 6-azacytidine-5 ’- triphosphate, 6-azauridine-5 ’-triphosphate, 6-chloropurineriboside-5 ’-triphosphate, 7- deazaadenosine-5 ’-triphosphate, 7 -deazaguanosine-5 ’-triphosphate, 8-azaadenosine-5 ’- triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazole-riboside-5’-triphosphate, Nl- methyladenosine-5’ -triphosphate, Nl-methylguanosine-5’ -triphosphate, N6-methyladenosine- 5 ’-triphosphate, O6-methylguanosine-5 ’-triphosphate, pseudouridine-5 ’-triphosphate, or puromycin-5 ’-triphosphate, xanthosine-5 ’-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5 -methylcytidine -5 ’-triphosphate, 7 -deazaguanosine-5 ’-triphosphate, 5- bromocytidine-5 ’-triphosphate, and pseudouridine-5 ’-triphosphate, pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5 -carboxymethyl -uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl -pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5 -taurinomethyl -2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl -pseudouridine, 4-thio-l -methyl - pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5 -aza-cytidine, pseudoisocytidine, 3- methyl -cytidine, N4-acetylcytidine, 5 -formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5 -methyl -cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5 -methyl -cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy- 1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl -guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5’-O-(l-thiophosphate)-adenosine, 5’-O- ( 1 -thiophosphate)-cytidine, 5 ’ -0-( 1 -thiophosphate)-guanosine, 5 ’ -0-( 1 -thiophosphate)- uridine, 5’-O-(l-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio- cytidine, Pseudo-iso-cytidine, 5 -aminoallyl -uridine, 5-iodo-uridine, Nl-methyl-pseudouridine, 5,6-dihydrouridine, alpha -thio-uridine, 4-thio-uridine, 6-aza-uridine, 5 -hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha -thio-guanosine, 6- methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, Nl-methyl- adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6- Chloro-purine, N6-methyl-adenosine, alpha -thio-adenosine, 8-azido-adenosine, 7-deaza- adenosine.

In some embodiments, the chemical modification is selected from pseudouridine, Nl- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5 -methylcytosine, 5 -methyluridine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2- thio-5 -aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio- 1 -methyl -pseudouridine, 4-thio-pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5 -methoxyuridine and 2'-O- methyl uridine.

Particularly suitable in that context are pseudouridine (y), N1 -methylpseudouridine (m h|/). 5 -methylcytosine, and 5 -methoxyuridine, more suitably pseudouridine (\|/) and Nl- methylpseudouridine (m h|i). still more suitably N1 -methylpseudouridine (m I \|/).

In some embodiments, essentially all, for example, essentially 100% of the uracil in the coding sequence of the RNA, suitably mRNA, have a chemical modification, suitably a chemical modification is in the 5 -position of the uracil.

In some embodiments, the RNA, suitably mRNA, comprises a chemical modification being a uridine modification, preferably wherein 100% of the uridine positions in the mRNA are modified. Incorporating modified nucleotides such as pseudouridine (y), Nl- methylpseudouridine (m h|i). 5-methylcytosine, and/or 5 -methoxyuridine into the coding sequence of the RNA, suitably mRNA, used herein may be advantageous as unwanted innate immune responses (upon administration of the coding mRNA or the vaccine) may be adjusted or reduced (if required).

In some embodiments, the coding sequence of the RNA, suitably mRNA, comprises at least one modified nucleotide selected from pseudouridine (v) and N1 -methylpseudouridine (ml\|/), suitably wherein all uracil nucleotides are replaced by pseudouridine (v) nucleotides and/or N1 -methylpseudouridine (ml\|/) nucleotides, optionally wherein all uracil nucleotides are replaced by pseudouridine ( ) nucleotides and/or N1 -methylpseudouridine (ml ) nucleotides.

In some embodiments, the RNA, suitably mRNA, does not comprise Nl- methylpseudouridine (m I ) substituted positions. In further embodiments, the RNAs, suitably mRNAs, used herein do not comprise pseudouridine (y), N1 -methylpseudouridine (m h|i). 5- methylcytosine, and 5 -methoxyuridine substituted position.

In some embodiments, the chemical modification is N1 -methylpseudouridine and/or pseudouridine. In some embodiments, the chemical modification is N1 -methylpseudouridine.

In the context of nucleic acid-based vaccine or therapeutic production, it may be required to provide GMP-grade nucleic acid (e.g., a GMP grade RNA or DNA). GMP-grade RNA or DNA may be produced using a manufacturing process approved by regulatory authorities. Accordingly, in some embodiments, RNA production is performed under current good manufacturing practice (GMP), implementing various quality control steps on DNA and RNA level, suitably according to WO2016180430. In embodiments, the RNA, suitably mRNA of the invention is a GMP-grade RNA. RNA synthesis

In some embodiments, the RNA, suitably mRNA, may be prepared using any method known in the art, including chemical synthesis such as solid phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

Suitably, the RNA, suitably mRNA, used herein is in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “zfi vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system (in vitro). RNA may be obtained by DNA- dependent in vitro transcription of an appropriate DNA template, which may be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases. In an embodiment of the present invention the DNA template is linearized with a suitable restriction enzyme, before it is subjected to RNA in vitro transcription. Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein; optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g., T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgC12, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, and/or polyamines such as spermidine at optimal concentrations, for example, a buffer system comprising TRIS-Citrate as disclosed in W02017109161.

In embodiments, the nucleotide mixture used in RNA in vitro transcription may additionally comprise modified nucleotides as defined herein. In that context, suitable modified nucleotides may in particular be selected from pseudouridine (y), N1 -methylpseudouridine (m 1 q/)_ 5 -methylcytosine, and 5 -methoxyuridine. In embodiments, uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) by pseudouridine (y) and/or N1 -methylpseudouridine (m 1 \|/) to obtain a modified RNA.

In other embodiments, the nucleotide mixture used in RNA in vitro transcription does not comprise modified nucleotides as defined herein. In embodiments, the nucleotide mixture used in RNA in vitro transcription only comprises G, C, A and U nucleotides, and, optionally, a cap analog as defined herein.

In some embodiments, the nucleotide mixture (i.e. the fraction of each nucleotide in the mixture) used for RNA in vitro transcription reactions may be optimized for the given RNA sequence, suitably as described in WO2015188933.

In this context, the in vitro transcription has been performed in the presence of a sequence optimized nucleotide mixture and optionally a cap analog.

Suitably, the method may be used to quantify impurities in a composition that is a therapeutic composition. In one embodiment, the therapeutic composition is an immunogenic composition, suitably a vaccine composition.

The therapeutic composition according to the invention may be administered via various suitable routes, including parenteral, such as intramuscular, intradermal, intranasal, or subcutaneous administration. Suitably, the therapeutic composition is suitable for intramuscular administration to a subject.

Subjects to which administration of the therapeutic composition is contemplated include, but are not limited to mammals, including humans and/or other primates, as well as commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys. Suitably, the therapeutic composition is for administration to human subjects.

Suitably, the therapeutic composition further comprises at least one pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein suitably includes the liquid or non-liquid basis of the composition for administration. If the composition is provided in liquid form, the carrier may be water (e.g., pyrogen-free water); isotonic saline or buffered (aqueous) solutions, such as phosphate, citrate etc. buffered solutions. Water or suitably a buffer, more suitably an aqueous buffer, may be used, containing a sodium salt, suitably at least 50mM of a sodium salt, a calcium salt, suitably at least 0.0 ImM of a calcium salt, and optionally a potassium salt, suitably at least 3mM of a potassium salt. According to some embodiments, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides (e.g., chlorides, iodides, or bromides), in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Examples of sodium salts include NaCl, Nal, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of the optional potassium salts include KC1, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include CaC12, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2.

Pharmaceutically acceptable carriers or excipients must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated. Compounds which may be used as pharmaceutically acceptable carriers or excipients may be sugars, such as, for example, lactose, glucose, trehalose, mannose, and sucrose; starches, such as, for example, com starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, com oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

Methods of separation

The present disclosure relates to high performance liquid chromatography methods (e.g., HPLC methods) for analyzing mixtures comprising LNP-formulated mRNA products (and also including RNA, RNA variants, RNA fragments, siRNA, etc.) and impurities (e.g., lipid-mRNA adducts). In some aspects, the disclosure relates to improved HPLC methods for quantification of an impurity from manufacturing lipid nanoparticle (LNP)-formulated mRNA products or other RNA products. In some aspects, lipid nanoparticle (LNP)-formulated mRNA product is a monovalent product comprising mRNA that encodes a single type of protein. In other aspects, the lipid nanoparticle (LNP) -formulated mRNA product is a multivalent product comprising two or more mRNAs that encode different proteins. For example, a first mRNA may encode an influenza antigen and a second mRNA may encode a SARS-COV-2 antigen. In aspects, the size of the first mRNA may range anywhere from about IkB to about 4 kB to about 1.5 kB to about 2 kB, and the size of the second mRNA may be about 3kB to about 5 kB. In aspects, the mRNA has a total concentration of about 0.1%, 0.5%, 1% 2%, 3%, 4%, and 5% or more.

In aspects, a method of separating a lipid nanoparticle (LNP)-formulated mRNA product on a chromatography column from an impurity is provided, comprising the steps of: (i) obtaining a sample comprising a mixture of the LNP-formulated mRNA product and the impurity; (ii) pretreating the sample; (iii) contacting a stationary phase of a reverse phase (RP) chromatography column with the sample; (iii) eluting the components of the sample with a mobile phase that is a mixture of a first eluent and a second eluent, such that the mRNA of the LNP-formulated mRNA product traverses the column with a first retention time and the impurity traverses the column with a second retention time different from the first retention time, wherein the impurity is eluted as a single defined or well-defined peak. Here, techniques are provided to avoid traditional RNA sample extraction, for example, by adding a surfactant to the sample during a pre-treatment, prior to loading the sample onto the chromatography column. In aspects, the LEP may be quantified for a range of mRNA lengths (e.g., 1 to 4 kB). In other aspects, the LEP may be quantified for a range of mRNA lengths of at least 200, more suitably at least 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides.

The methods provided herein involve the separation and analysis of samples (e.g., mixtures of analytes) using chromatography comprising one or more LNP-formulated mRNA products and one or more impurities, for example, an LNP encapsulated mRNA and an RNA lipid adduct. An LNP-encapsulated mRNA is typically composed of a lipid shell encapsulating a mRNA core. Due to various environment conditions or impurities arising during manufacturing processes, impurities may be present in a sample of the product. In preferred embodiments, the sample comprises greater than 60%, 70%, 80% or 90% of the LNP-encapsulated mRNA product. In still further embodiments, the sample comprises 95% or greater of the LNP- encapsuled mRNA product with 5% or less of impurities.

Aspects of the present subject matter may be applied to analyze impurities in multivalent mRNA formulations. Multivalent formulations may comprise two, three, four, five, six, seven, eight or more types of mRNA, each encoding a different antigen from one or more different organisms, bacteria, or viruses. Thus, the multivalent formulation may comprise a first mRNA, a second mRNA, a third mRNA, etc. Alternatively, a monovalent formulation may comprise a single type of mRNA encoding one antigen.

Lor example, in one aspect, one or more mRNAs may be derived from one or more strains of influenza and/or one or more mRNAs may be derived from one or more strains/variants of SARS-COV-2. In some aspects, the length of the mRNA may range anywhere from about IkB to about 4 kB. In other aspects, the length of the first mRNA may be about 1.5 kB to about 2 kB and the length of the second mRNA may be about 3kB to about 5 kB.

According to present embodiments, extraction of mRNA (e.g., for multivalent formulations) is not required. Instead, a detergent/surfactant may be added to the sample allowing the sample to be directly analyzed by IP-RP-HPLC (e.g., bypassing RNA extraction, for instance, using phenol/chloroform) .

Present embodiments are applicable to any suitable range of mRNA concentrations, for example, from about 1 ug/ml to about 20 ug/ml of mRNA. The present method is suitable to test the same quality attribute of different products (i.e. different monovalent and/or multivalent formulations) without changing the operative conditions. Accordingly, the following examples are representative of any suitable monovalent or multivalent formulation.

The percent (%) of LEP present is determined based on the proportion between the Total Area of the mRNA (sum of the area under the mRNA product peak and the mRNA-lipid adduct peak) and the LEP Area (the area of the mRNA-lipid adduct peak). In multivalent formulations, the percentage of the mRNA-lipid adduct is the cumulative value from the contributions of the single constructs.

The term “purified RNA (or mRNA)” as used herein is understood as RNA which has a higher purity after certain purification steps (e.g., HPLC, TFF, Oligo d(T) purification, precipitation steps) than the starting material (e.g., in vitro transcribed RNA). Typical impurities that are essentially not present in purified RNA include peptides or proteins (e.g., enzymes derived from DNA dependent RNA in vitro transcription such as RNA polymerases, RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double stranded RNA fragments, abortive sequences etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogue), template DNA fragments, buffer components (HEPES, TRIS, MgCh) etc. Other potential impurities that may be derived from, for example, fermentation procedures comprise bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents etc.). Accordingly, it is desirable in this regard for the “degree of RNA purity” to be as close as possible to 100%. It is also desirable for the degree of RNA purity that the amount of full-length RNA transcripts is as close as possible to 100%. Accordingly, “purified RNA (or mRNA)” as used herein has a degree of purity of more than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or more. The degree of purity may for example be determined by an analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of the peak for the target RNA product and the total area of all peaks representing the by-products. Alternatively, the degree of purity may for example be determined by an analytical agarose gel electrophoresis or capillary gel electrophoresis.

In some embodiments, the RNA is purified using RP-HPLC, suitably using Reversed-Phase High pressure liquid chromatography (RP-HPLC) with a macroporous styrene/divinylbenzene column (e.g., particle size 30pm, pore size 4000 A) and additionally using a filter cassette with a cellulose based membrane with a molecular weight cutoff of about lOOkDa. The RNA may in particular be purified using PUREMESSENGER (CureVac, Tubingen, Germany; RP-HPLC according to W02008077592) and/or tangential flow filtration (as described in WO2016193206) and/or oligo d(T) purification (see W02016180430).

In some embodiments, the RNA, suitably mRNA, is purified by RP-HPLC and/or TFF to remove double -stranded RNA, non-capped RNA and/or RNA fragments. In embodiments, the RNA, suitably mRNA, comprises about 5%, 10%, or 20% less double stranded RNA side products as an RNA, suitably mRNA, that has not been purified with RP- HPLC and/or TFF.

In some embodiments, the RP-HPLC and/or TFF purified RNA, suitably mRNA, comprises about 5%, 10%, or 20% less double stranded RNA side products as an RNA, suitably mRNA, that has been purified with Oligo dT purification, precipitation, filtration and/or AEX.

In some embodiments, the encapsulated mRNA (in LNPs) may contain lipid adduct impurities. The present techniques allow the quantification of such impurity by resolving the peaks on a chromatography column.

HPLC apparatus

Typically, an HPLC apparatus comprises a reservoir containing a mobile phase, a sample input port for injection of the sample (e.g., containing one or more analytes), a chromatography column (e.g., reverse phase) containing the stationary phase, and a detection apparatus, output port, and fraction collection apparatus. HPLC apparatus and methods for HPLC detection of RNA (e.g., including mRNA, LNP encapsulated RNA molecules are generally described, for example in Packer et al. 2021.

Any suitable reverse phase HPLC column (e.g., comprising a hydrophobic stationary phase) may be used in the methods provided herein. In general, an “HPLC column” is a solid structure or support (e.g., glass tube) that contains a medium (e.g., a stationary phase) through which the mobile phase and sample (e.g., a sample containing a mixture of analytes, such as LNP- encapsulated mRNA and impurities/byproducts) are eluted. Without wishing to be bound by any particular theory, the composition and chemical properties of the stationary phase determine the retention time of the analytes. In some embodiments, the stationary phase is nonpolar (e.g., reverse phase), which may include non-polar resins, silica (e.g., alkylated and nonalkylated silica), polystyrenes (e.g., alkylated and non-alkylated polystyrenes), polystyrene divinylbenzenes, etc. In some embodiments, the stationary phase comprises particles, for example, porous particles which may be hydrophobic or comprise hydrophobic functional groups (e.g., such as polystyrene divinylbenzene).

The particle size (e.g., as measured by the diameter of the particle) of the stationary phase of a reverse phase HPLC can vary. In some embodiments, the particle size may range from about 1 pm to about 100 pm (e.g., any value between 1 and 100, inclusive) in diameter. In other embodiments, the particle size ranges from about 2 pm to about 10 pm, about 2 pm to about 6 pm, or about 4 pm in diameter.

The pore size of particles (e.g., as measured by the diameter of the pore) can also vary. In some embodiments, the particles comprise pores having a diameter of about 100 A to about 10,000 A. In some embodiments, the particles comprise pores having a diameter of about 100 A to about 5000 A, about 100 A to about 1000 A, about 500 A to about 2000 A, or about 1000 A to about 2000 A. In some embodiments, the stationary phase may comprise polystyrene divinylbenzene, for example, as used in DNA Pac-RP columns.

In aspects, the temperature of the HPLC column (e.g., the stationary phase within the column) can also vary. In some embodiments, the column has a temperature from about 25 °C to about 99 °C. (e.g., any temperature between 25 °C and 99 °C. In other embodiments, the column has a temperature from about 45 °C to about 80 °C, from about 50 °C to about 75 °C, from about 55 °C to about 65 °C (or any temperature in between these ranges). In some embodiments, the column has a temperature from about 56 °C , about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, or about 64 °C). In some embodiments, the column has a temperature of about 60 °C.

In some embodiments, HPLC methods as described herein further comprise quantifying the amount of the product and/or impurity. Any detection apparatus or modality suitable for HPLC may be used, including but not limited to: absorbance detectors (e.g., UV/VIS detectors), fluorescence detectors, electrochemical detectors, and mass spectrometric detectors.

Mobile phase

The “mobile phase” is a solution (e.g., aqueous, organic/polar solvent, or mixture) used to carry an analyte (or analytes) through the stationary phase of an HPLC column. In some aspects, the mobile phase may comprise water and/or one or more organic solvents. The concentration of organic solvent in the mobile phase may vary. In some embodiments, the volume percentage (v/v) of an organic solvent in a mobile phase varies from 0% (absent) to about 100%. In other embodiments, the volume percentage of organic solvent in a mobile phase is between about 15% and about 75% (v/v), about 30% and about 60% (v/v), or about 45% to about 55% (v/v). A volume percentage (v/v) may refer to, for a solution, the ratio of a volume of a solute as compared to the volume of the solution as a whole. In still other embodiments, the concentration of organic solvent in a mobile phase is about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, or about 50% to about 60% (v/v).

In some embodiments, a mobile phase for use in HPLC methods as described herein comprises multiple (e.g., 2, 3, 4, 5, or more) eluents. In some embodiments, as described herein, the mobile phase comprises two eluents (e.g., a first eluent A, and a second eluent B). In some embodiments, an eluent comprises one or more organic solvents (e.g., a polar solvent, such as acetonitrile) and one or more ion pairing agents, and optionally, water.

The mobile phase may comprise concentrations of two or more eluents. For example, the mobile phase may comprise two eluents (e.g., a first eluent and a second eluent).

In aspects, the volume percentage (v/v) of the first eluent may be about 0%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% (v/v).

In aspects, the volume percentage (v/v) of the second eluent may be about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% (v/v).

The pH of the mobile phase (e.g., the pH of each solvent or solvent solution) may vary. In some embodiments, the pH of the mobile phase is between about pH 5.0 and pH 9.5 (e.g., about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5). In some embodiments, the pH of the mobile phase is between about pH 6.5 and pH 8.5 (e.g., about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.3, or about 8.5). In some embodiments, the pH of the mobile phase is about 7.0.

In some embodiments, the pH of the first eluent is between about pH 5.0 and pH 9.5 (e.g., about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5). In some embodiments, the pH of the first eluent is between about pH 6.5 and pH 8.5 (e.g., about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.3, or about 8.5). In some embodiments, the pH of the first eluent is about 7.0.

In some embodiments, the pH of the second eluent is between about pH 5.0 and pH 9.5 (e.g., about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5). In some embodiments, the pH of the second eluent is between about pH 6.5 and pH 8.5 (e.g., about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.3, or about 8.5). In some embodiments, the pH of the second eluent is about 7.0.

Ion Pairs

In RP HPLC, retention of analytes on an HPLC column may be controlled by adjusting concentrations of an ion pairing agent (ion pair), which affect the electrostatic interactions between analytes of a sample (e.g., mRNA, LEP) and the stationary phase of the reverse phase chromatography column.

An “ion pairing agent” or an “ion pair” acts as a counter ion to a charged functional group on an analyte (e.g., ionized or ionizable functional group). By adjusting the concentrations of the ion pairs, the retention time of the analyte traversing the stationary phase of an HPLC column may be modulated. In aspects, an ion pairing agent may be a cationic ion pairing agent that interacts with negatively charged functional groups and may include any suitable counter-ion (e.g., acetate, bicarbonate, chloride, citrate, nitrate, nitrite, oxide, phosphate, sulfate, etc.). Examples of cationic ion pairing agents include but are not limited to dibutylammonium acetate (DBAA), hexylammonium acetate (HAA), tetrabutylammonium phosphate (TBAP), and triethylammonium acetate (TEAA), as well as select protonated or quaternary amines (including e.g., primary, secondary and tertiary amines) and/or salts thereof.

In general, ion pairing agents may promote separation of an individual analyte on the basis of size, or on the basis of composition and ion pairs may comprise ion pairing agents including dibutylammonium salts, hexylammonium salts, tetrabutylammonium salts and triethylammonium salts.

The concentration of one or more ion pairing agents in a mobile phase (e.g., a solvent solution) may vary over time (e.g., a part of a step gradient). In aspects, the concentration of each of the one or more ion pairs (e.g., each ion pairing agent) in a solvent solution (e.g., a first eluent and/or a second eluent) may range from about 10 mM to about 500 mM (e.g., about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140mM, about 150 mM, about 200 mM, about 500 mM), inclusive. In some embodiments, the concentration of an ion pairing agent in a mobile phase (e.g., a first eluent and/or a second eluent) may range from about 10 mM-100 mM, about 20 mM-90 mM, about 30 mM-80 mM, about 40 mM-70 mM, about 40 mM-60 mM, about 45 mM-55 mM or about 10 mM-25 mM, about 25 mM-50 mM, about 50 mM-75 mM, about 75 mM-100 mM or about 25mM-75 mM. In some embodiments, the concentration of an ion pairing agent in a mobile phase (e.g., a first eluent and/or a second eluent) may range from about 50 mM-150 mM, about 60 mM-140 mM, about 70 mM-130 mM, about 80 mM-120 mM, about 90 mM-

110 mM, about 95 mM-105 mM or about 50 mM-75 mM, about 75 mM-100 mM, about 100 mM-125 mM, about 125 mM-150 mM, or about 75 mM-125 mM.

Examples of ion pairing agent concentrations include but are not limited to about 100 mM TEAA/50 mM DBAA, about 75 mM TEAA/50 mM DBAA, about 50 mM TEAA/50 mM DBAA, about 25 mM TEAA/50 mM DBAA, about 75 mM TEAA/25 mM DBAA, about 25 mM TEAA/75 mM DBAA. Examples of ion pairing agent concentrations include but are not limited to about 100 mM TEAA/50 mM DBAA, about 95 mM TEAA/50 mM DBAA, about 90 mM TEAA/50 mM DBAA, about 85 mM TEAA/50 mM DBAA, about 80 mM TEAA/50 mM DBAA, about 75 mM TEAA/50 mM DBAA. Examples of ion pairing agent concentrations include but are not limited to about 100 mM TEAA/50 mM DBAA, about 100 mM TEAA/45 mM DBAA, about 100 mM TEAA/40 mM DBAA, about 100 mM TEAA/35 mM DBAA, about 100 mM TEAA/25 mM DBAA, about 100 mM TEAA/55 mM DBAA, about 100 mM TEAA/60 mM DBAA, about 100 mM TEAA/65 mM DBAA, about 100 mM TEAA/70 mM DBAA, or about 100 mM TEAA/75 mM DBAA. Examples ofion pairing agent concentrations include but are not limited to about 75-125 mM TEAA and 40-60 mM DBAA. In aspects, a “mobile phase” comprises an aqueous solution including water and/or one or more organic solvents along with ion pairs which carry analytes through an HPLC column. In aspects, a mobile phase for a reverse phase column comprises a polar organic solvent, including but not limited to alcohols, alkylsulfoxides, amides, esters, ketones, and nitrates. In some embodiments, a mobile phase comprises one or more organic solvents selected from the group consisting of acetone, acetonitrile, alcohols, amides, alkylsulfoxides, dimethylformamide, dimethylsulfoxide (DMSO), esters, ethanol, hexylene glycol, isopropanol, ketones, methanol, methyl acetate, nitrates, propanol, water or other polar aprotic solvents such as Ci-4 alkanols, Ci-e alkandiols, and C2-4 alkanoic acids. In some embodiments, the mobile phase comprises acetonitrile and water.

Elution Gradient

In some aspects, the mobile phase may comprise different elution gradients, which are manipulated by increasing, keeping constant, or decreasing the percent volume of an organic solvent, allowing for high resolution separations of mRNAs and impurities. In an aspect, the method comprises the step of passing the mobile phase through the stationary phase of the column, wherein the mobile phase is a mixture a first eluent and a second eluent, and wherein the first eluent and the second eluent independently comprise one or more ion-pairs selected from the group consisting of: trimethylamine or a salt thereof (e.g., triethylammonium salt) and dibutylamine or a salt thereof (e.g., dibutylammonium salt). In aspects, the triethylammonium salt is triethylammonium acetate (TEAA) and the dibutylammonium salt is dibutylammonium acetate (DBAA). The first and second eluents separate the analytes of the mixture, with the mRNA product traversing the column with a retention time that is different than the one or more impurities of the mixture.

In some embodiments, the ratio of the first eluent to the second eluent is held constant (e.g., isocratic) during elution of the mRNA product. However, the relative ratio of the first eluent to the second eluent can vary throughout the elution step. For example, in some embodiments, the ratio of the first eluent is increased relative to the second eluent during the elution step. In some embodiments, the ratio of the first eluent is decreased relative to the second eluent during the elution step.

For example, in some embodiments, the ratio of triethylammonium (e.g., TEAA) to dibutylammonium (e.g., DBAA) ranges from about 1: 100 to about 100: 1, about 1:75 to about 75: 1, about 1:50 to about 50: 1, about 1:25 to about 25: 1, 5: 1 to about 1:5, about 3: 1 to about

1:3, about 2: 1 to about 1:2, or about 1: 1 during elution.

In aspects, the sample, comprising the ionizable amino-lipid (and optionally other lipids to form an LNP) and the RNA (suitably mRNA), may be treated with a surfactant, such as Triton (e.g., Triton X-100), SDS, Tween or Brij-35 (e.g., about 0.75% to about 1.75%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) to disrupt LNPs. Therefore, present techniques may be used to quantify impurities in a sample that has been treated with surfactant. In some aspects, a concentration of Triton ranging from 1.5% to 5% (v/v) may be suitable, a concentration of about 2 to 3% (v/v) Triton may be suitable, or a concentration of about 2.5% (v/v) Triton may be suitable to dissociate the ionizable amino-lipid and to release the RNA. The pre-treated sample may be subjected to the analytical method of the invention.

In general, at least 10 pl/mL or more of mRNA may be detected and evaluated in standard conditions (e.g., mRNA concentration of 50 pg/ml and with an injection volume of 7 pl). The LOQ resulted to be 1% for samples with a concentration of mRNA of 50 pg/ml and with an injection volume of 7 pl.

The injection volume of the sample onto the stationary phase of the reverse phase chromatography column is about 2.5 pl to 25 pl, about 2.5 pl to 20 pl, about 2.5 pl to 15 pl, about 2.5 pl to 10 pl, about 2.5 pl to 5 pl, or about 5 pl to 10 pl, about 10 pl to 15 pl, about 15 pl to 20 pl, about 20 pl to 25 pl, about 25 pl to 30 pl, or about 2.5 pl, about 5 pl, about 7 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl.

The following clauses may be used to illustrate various aspects of the invention:

1. A method of separating a lipid nanoparticle (LNP)-formulated mRNA product from an impurity comprising: obtaining a sample comprising a mixture of the LNP-formulated mRNA product and the impurity; pre-treating the sample; contacting a stationary phase of a reverse phase (RP) chromatography column with the sample; eluting the sample with a mobile phase that is a mixture of a first eluent and a second eluent, such that the mRNA product traverses the column with a first retention time and the impurity traverses the column with a second retention time different from the first retention time, wherein the impurity is eluted as a single well-defined peak.

2. The method of clause 1, wherein pre-treating the sample comprises:

(i) diluting or mixing the sample with a surfactant (e.g., Triton, SDS, Tween 20, Brij-35), wherein the surfactant is present at a concentration of 1-10% (about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%), or

(ii) extracting the mRNA product and the impurity (e.g., a lipid-mRNA adduct) from the sample, before contacting the column with the sample.

3. The method of clause 2, wherein pre-treating the sample comprises: diluting or mixing the sample with Brij-35, wherein Brij-35 is present at a concentration of 1-10%.

4. The method of clause 2, wherein pre-treating the sample comprises: diluting or mixing the sample with Triton XI 00, wherein Triton XI 00 is present at a concentration of 1-10%.

5. The method of any of clauses 2 to 4, further comprising: after pre -treating the sample, contacting the column with the sample without extracting the RNA product and impurity (e.g., lipid-mRNA adduct) from the sample. The method of any one of the preceding clauses, wherein the impurity is a lipid- mRNA adduct. 6. The method of any one of the preceding clauses, wherein a volume percentage of the first eluent in the mobile phase is 0% to 55% (v/v) and a volume percentage of the second eluent in the mobile phase is 45% to 100% (v/v).

7. The method of any one of the preceding clauses, wherein the first eluent comprises at least two ion pairs in a first solvent or first solvent solution, and wherein the second eluent comprises at least two ion pairs in a second solvent or second solvent solution.

8. The method of clause 7, wherein a concentration of each of the at least two ion pairs in the first solvent or first solvent solution is 25 mM-125 mM, 50 mM-100 mM, 25 mM-50 mM, 50 mM-75 mM, 75 mM-100 mM, 100 mM-125 mM, 75 mM-125 mM, 25 mM-75 mM, or 50 mM - 150 mM.

9. The method of clause 7 or clause 8, wherein a concentration of each of the at least two ion pairs in the second solvent or second solvent solution is 25 mM-125 mM, 50 mM- 100 mM, 25 mM-50 mM, 50 mM-75 mM, 75 mM-100 mM, 100 mM-125 mM, 75 mM-125 mM, 25 mM-75 mM, or 50 mM - 150 mM.

10. The method of any one of clauses 7 to 9, wherein the ion pairs of the first eluent are a triethylammonium salt and a dibutylammonium salt and the ion pairs of the second eluent are a triethylammonium salt and a dibutylammonium salt.

11. The method of any one of clauses 7 to 10, wherein the first solvent or first solvent solution and the second solvent or second solvent solution comprises one or more solvents selected from the group consisting of: water, alkanols, alkandiols, alkanoic acids, polar aprotic solvents (e.g., acetone, acetonitrile, DMF, DMSO, THF).

12. The method of clause 11, wherein the first solvent or first solvent solution comprises water, and wherein the second solvent or second solvent solution comprises acetonitrile in water (e.g., 50% acetonitrile in H2O).

13. The method of any one of clauses 10 to 12, wherein the triethylammonium salt is triethylammonium acetate, the dibutylammonium salt is dibutylammonium acetate, the first solvent solution is water, and the second solvent solution is TbO/acetonitrile

50:50.

14. The method of any one of clauses 10 to 13, wherein, for the first eluent, the concentration of triethylammonium acetate is about 100 mM (e.g., 95 mM - 105 mM, 96 mM - 104 mM, 97 mM - 103 mM, 98 mM - 102 mM, 99 mM - 101 mM, 100 mM) and the concentration of dibutylammonium acetate is about 50 mM (e.g., 45 mM - 55 mM, 46 mM - 54 mM, 47 mM - 53 mM, 48 mM - 52 mM, 49 mM - 51 mM, 50 mM); and for the second eluent, the concentration of triethylammonium acetate is about 100 mM (e.g., 95 mM - 105 mM, 96 mM - 104 mM, 97 mM - 103 mM, 98 mM - 102 mM, 99 mM - 101 mM, 100 mM) and the concentration of dibutylammonium acetate is about 50 mM (e.g., 45 mM - 55 mM, 46 mM - 54 mM, 47 mM - 53 mM, 48 mM - 52 mM, 49 mM - 51 mM, 50 mM).

15. The method of clause 14, wherein, for the first eluent, a concentration of triethylammonium acetate is 100 mM and a concentration of dibutylammonium acetate is 50 mM, and wherein, for the second eluent, a concentration of triethylammonium acetate is 100 mM and a concentration of dibutylammonium acetate is 50 mM.

16. The method of any preceding clause, wherein the first eluent comprises 100 mM triethylammonium acetate and 50 mM dibutylammonium acetate in water.

17. The method of any preceding clause, wherein the second eluent comprises 100 mM triethylammonium acetate and lOOmM dibutylammonium acetate (DBAA) in 50% water and 50% acetonitrile.

18. The method of any one of the preceding clauses, wherein the volume percentage of the first eluent and the volume percentage of the second eluent is not held constant during elution of the mRNA product and the impurity. The method of any one of the preceding clauses, wherein the retention time of the mRNA product is less than the retention time of the impurity, and wherein both the mRNA product and impurity are eluted in less than 5 minutes. The method of clause 19, wherein the peak of the mRNA product does not substantially overlap with the peak of the impurity. The method of any one of the preceding clauses, further comprising applying a step gradient to the mobile phase. The method of clause 21, wherein the step gradient comprises the steps of:

(i) applying a mixture of about 50% (v/v) of the first eluent and about 50% of the second eluent (v/v);

(ii) applying, after (i), 100% (v/v) of the second eluent; and

(iii) applying, after (ii), a mixture of about 50% (v/v) of the first eluent and about 50% (v/v) of the second eluent. The method of clause 21 , wherein

(ii) at a second time point, increasing (e.g., linearly) the percent volume of the second eluent over a time span of about 1.7 to 2.1 minutes, until reaching 100% volume of the second eluent;

(iii) maintaining, for about 50-70 seconds, 100% volume of the second eluent;

(iv) at a third time point, decreasing (e.g., linearly) the percent volume of the second eluent over a time span of less than 15 seconds until reaching 50% volume of the second eluent; (v) maintaining a mixture of 50% of the first eluent and 50% of the second eluent.

24. The method of clause 21 wherein the step gradient comprises:

(i) applying, at an initial time, a mixture of 52% (v/v) of the first eluent and 48% (v/v) of the second eluent;

(ii) applying, at a time of 6.6 minutes, a mixture of 47% (v/v) of the first eluent and 53% (v/v) of the second eluent;

(iii) applying, at a time of 8.6 minutes, 100% (v/v) of the second eluent;

(iv) applying, at a time of 9.6 minutes, 100% (v/v) of the second eluent; and

(v) applying, at a time of 9.8 minutes, a mixture of 52% (v/v) of the first eluent and 48% (v/v) of the second eluent.

25. The method of clause 21, wherein the step gradient comprises:

26. The method of any one of the preceding clauses, further comprising detecting analytes of the sample using a UV detector with a range of 260 nm. The method of any one of the preceding clauses, wherein the column is an analytical column or a preparative column. The method of any one of the preceding clauses, wherein the column comprises particles (e.g., porous resin particles). The method of clause 28, wherein the particles are hydrophobic or comprise hydrophobic functional groups. The method of clause 28 or 29, wherein the particles have a diameter ranging from 2 um to about 10 um, about 2 um to 6 um, or about 4 pm. The method of any of clauses 28 to 30 wherein the particles comprise pores having a diameter ranging from about 100 A to about 3000 A, from about 100 A to about 2000 A, or from about 500 A to about 2000A. The method of any one of the preceding clauses, wherein a temperature of the column is about 53°C to about 65°C, about 55°C to about 63°C, about 57°C to about 61°C, or about 59°C. The method of any one of the preceding clauses, wherein a flow rate of the column is about 0.2 to 0.5 mL/min, 0.3 to 0.4 mL/min, or about 0.35 mL/min. The method of any one of the preceding clauses, wherein dimensions of the column are about 2.1 x 100 mm. The method of any one of the preceding clauses, wherein a chromatographic run length of the column is less than about 20 minutes or about 15 minutes. The method of any one of the preceding clauses, further comprising the step of performing a blank subtraction. The method of any one of the preceding clauses, wherein the lipid nanoparticle (LNP) comprises at least one cationic or ionizable lipid, preferably a cationic or ionizable amino lipid.

38. The method of any one of the preceding clauses, wherein the RNA has a length of at least 200 bp.

39. The method of any one of the preceding clauses, wherein the RNA has a GC content comprising one or more of: codon “GCC” with a frequency of 0.40, codon “GCT” with a frequency of 0.28, codon “GCA” with a frequency of 0.22, or codon “GCG” with a frequency of 0.10.

40. Use of the method of any one of the preceding clauses as a quality control during or after manufacturing of a lipid nanoparticle (LNP)-formulated mRNA product.

41. The method of any one of the preceding clauses comprising a multivalent formation of mRNA.

42. The method of clause 41, wherein the multivalent formulation comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more different mRNAs, each mRNA encoding a different antigen from one or more organisms, bacteria, or viruses.

43. The method of any one of the preceding clauses, wherein the mRNA comprises a first mRNA encoding a first antigen and a second mRNA encoding a second antigen.

44. The method of clause 43, wherein a first mRNA encodes an influenza antigen and a second mRNA encodes a SARS-COV-2 antigen.

45. The method of clause 43, wherein the length of the first mRNA ranges from about IkB to about 4 kB or from about 1 kB to about 2 kB and the length of the second mRNA ranges from about 3kB to about 5 kB.

46. The method of any one of the preceding clauses, wherein mRNA concentration ranges from about 1 ug/ml to about 20 ug/ml.

47. The method of any one of the preceding clauses wherein the mRNA concentration is about 1%.

48. The method of any one of the preceding clauses, wherein the percent (%) volume of the well-defined peak corresponding to the impurity is determined based on the proportion between the total area of the mRNA (sum of the mRNA signal and mRNA-lipid adduct signal) and the area of the impurity (the area of the mRNA-lipid adduct signal).

49. The method of any one of the preceding clauses, wherein the percent (%) volume of the well-defined peak in a multivalent formulation corresponds to the cumulative value of the contributions of each mRNA-lipid adduct.

50. The method of any preceding clause, wherein the well-defined peak (i) is suitable for integration to determine the percent (%) volume of the mRNA lipid adduct to within 1 to 16% of the mRNA sample.

51. The method of any preceding clause, wherein the lipid nanoparticle (LNP)-formulated mRNA product comprises a mixture of different LNPs encapsulating different mRNA products.

52. The method of any preceding clause, wherein the lipid nanoparticle (LNP)-formulated mRNA product comprises a first type of LNP encapsulating a first mRNA and a second type of LNP encapsulating a second mRNA different from the first mRNA.

For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

The term “about” is used when determinants or values do not need to be identical, i.e. 100% the same. Accordingly, “about” means, that a determinant or values may diverge by 1% to 20%, for example by 1% to 10%; in particular, by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. The skilled person knows that for example certain parameters or determinants can slightly vary based on the method of how the parameter has been determined. For example, if a certain determinants or value is defined herein to have for example a length of “about 100 nucleotides”, the length may diverge by 1% to 20%. Accordingly, the skilled person knows in that specific example, the length may diverge by 1 to 20 nucleotides. Accordingly, a length of “about 100 nucleotides” may encompass sequences ranging from 80 to 120 nucleotides.

For purposes herein, ranges may be expressed as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, an embodiment includes from the one particular value and/or to the other particular value. Another embodiment includes an approximation of the one particular value (e.g., plus or minus 5%, 4%, 3%, 2% or 1% of the value) to an approximation of the other particular value (e.g., plus or minus 5%, 4%, 3%, 2% or 1% of the value).

For purposes herein, the term “a” or “an” refers to one or more of an entity; for example, “an LNP-mRNA product” refers to one or more of those compounds or at least one such compound. As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Accordingly, the terms “a” or “an,” “one or more,” and “at least one” can be used interchangeably unless indicated otherwise.

Further, the terms “comprising,” “having,” and “including” may be used interchangeably unless indicated otherwise. Furthermore, a compound “selected from the group consisting of’ refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element or elements without necessarily excluding any other elements. Thus, a composition “comprising” X may consist exclusively of X or may include something additional (e.g., X + Y).

The word “substantially” does not exclude “completely” for example a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus, components can be mixed in any order. Where there are three components, the two components can be combined with each other, and then the combination may be combined with the third component, etc.

Percentages in the context of numbers should be understood as relative to the total number of the respective items. In other cases, and unless the context dictates otherwise, percentages should be understood as percentages by weight (wt %).

The term “antigen” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a substance which may be recognized by the immune system, for example by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by formation of antibodies and/or antigenspecific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. Also fragments, variants and derivatives of peptides or proteins comprising at least one epitope are understood as antigens.

The terms “coding sequence” or “coding region” and the corresponding abbreviation “cds” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a sequence of several nucleotide triplets, which may be translated into a peptide or protein. A coding sequence in the context of the present invention may be an RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon and which for example terminates with a stop codon. The term “derived from” as used throughout the present specification in the context of a nucleic acid, i.e. for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares for example at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e. for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined. For example, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, for example in order to increase RNA stability even further and/or to prolong and/or increase protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins) the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares for example at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.

The term “fragment” as used throughout the present specification in the context of a nucleic acid sequence (e.g., RNA or a DNA) or an amino acid sequence may typically be a shorter portion of a full-length sequence of a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment typically consists of a sequence that is identical to the corresponding stretch within the full-length sequence. A particular fragment of a sequence in the context of the present invention, consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e. full- length) molecule from which the fragment is derived (e.g., a virus protein). The term “fragment” as used throughout the present specification in the context of proteins or peptides may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence, N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original protein. The term “fragment” as used throughout the present specification in the context of RNA sequences may, typically, comprise an RNA sequence that is 5 ’-terminally and/or 3 ’-terminally truncated compared to the reference RNA sequence. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore for example refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide. Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides.

The terms “heterologous” or “heterologous sequence” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence refers to a sequence (e.g., RNA, DNA, amino acid) that is derived from another gene, another allele, or another species or virus. Two sequences are typically understood to be “heterologous” if they are not derivable from the same gene or from the same allele, i.e., although heterologous sequences may be derivable from the same organism or virus, in nature, they do not occur in the same nucleic acid or protein.

The term “identity” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, nucleic acid sequences or amino acid (aa) sequences as defined herein, for example, the aa sequences encoded by the nucleic acid sequence as defined herein or the aa sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same residue as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using an algorithm, an algorithm integrated in the BLAST program. Sequence identity can be determined by using the EMBOSS Water sequence alignment tool at the EMBL-EBI website https://www.ebi.ac.uk/Tools/psa/emboss_water/ with the parameters gap open=12, gap extend=l and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA/RNA sequences, or by using the EMBOSS Needle sequence alignment tool at the EMBL-EBI website https://www.ebi.ac.uk/Tools/psa/emboss_needle/ with default parameters (e.g., gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10 and end gap extend=0.5 and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA/RNA sequences). Unless specified otherwise, where the application refers to sequence identity to a particular reference sequence, the identity is intended to be calculated over the entire length of that reference sequence. The terms “immunogen” or “immunogenic” will be recognized and understood by the person of ordinary skill in the art, and are intended to refer to a compound that is able to stimulate/induce an (adaptive) immune response. An immunogen may be a peptide, polypeptide, or protein.

The term “immune response” will be recognized and understood by the person of ordinary skill in the art, and is intended to refer to a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.

A lipidoid compound, also simply referred to as lipidoid, is a lipid-like compound, i.e. an amphiphilic compound with lipid-like physical properties. In the context of the present invention, the term lipid is considered to encompass lipidoid compounds.

The terms “nucleic acid” or “nucleic acid molecule” as used herein, will be recognized and understood by the person of ordinary skill in the art. The terms “nucleic acid” or “nucleic acid molecule” particularly refers to DNA (molecules) or RNA molecules). The term is used synonymously with the term polynucleotide. For example, a nucleic acid or a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers that are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The terms “nucleic acid” or “nucleic acid molecule” also encompasses modified nucleic acid (molecules), such as base -modified, sugar-modified or backbone-modified DNA or RNA (molecules) as defined herein.

The terms “nucleic acid sequence”, “DNA sequence”, “RNA sequence” will be recognized and understood by the person of ordinary skill in the art, and refer to a particular and individual order of the succession of its nucleotides.

The term “stabilized RNA” refers to an RNA that is modified such, that it is more stable to disintegration or degradation, for example, by environmental factors or enzymatic digest, such as by exo- or endonuclease degradation, compared to an RNA without such modification. Preferably, a stabilized RNA in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell. The stabilization effect may also be exerted outside of cells (e.g., in a buffer solution etc.) for storage of a composition comprising the stabilized RNA.

The term “variant” as used throughout the present specification in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is intended to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence, for example, a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from. The variant is a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from. A “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides of such nucleic acid sequence.

The term “variant” as used throughout the present specification in the context of proteins or peptides is intended to refer to a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s)/substitution(s), such as one or more substituted, inserted and/or deleted amino acid(s). Suitably, these fragments and/or variants have the same, or a comparable specific antigenic property (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined, for example, using CD spectra (circular dichroism spectra). A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Alternatively, a “variant” of a protein or polypeptide may have from 1 to 20, for example from 1 to 10 single amino acid mutations compared to such protein or peptide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 or 20 single amino acid mutations. For mutations we mean or include substitution, insertion or deletion. In one embodiment, a variant of a protein comprises a functional variant of the protein, which means, in the context of the invention, that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity as the protein it is derived from.

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention is not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods, which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

The following examples demonstrate a method of resolving an impurity peak (a Late Eluting sPecies - LEP) from the mRNA product. The impurity may comprise lipid-mRNA adducts which can be generated by electrophilic impurities derived from the ionizable cationic lipid component through covalent addition to the mRNA nucleobases.

Example 1. mRNA Formulations

The LNP-encapsulated mRNA formulations in the present examples were produced according to techniques known in the art as referenced herein. mRNA products include mRNA molecules that are unmodified and modified (e.g., 5 -prime capped, 3 -prime poly-adenylated sequences, modified uridine chemistry (N1 -methyl -pseudouridine)) that are suitable for use as vaccines and other therapeutics. mRNA products include those that encode an antigen fragment, a full-length protein, or a variant or derivative thereof.

The methods provided herein may be used with final bulk or drug product formulations. For example, the concentrated mRNA/LNP bulk comprised mRNA expressing an antigen at a concentration of 1230 ug/mL (and may cover any suitable range 1 mg/mL or more and may further be encapsulated in LNPs comprising DSPC, ALC-0315, cholesterol and ALC-0159 at a molar ratio of about 47.4: 10.0:40.9: 1.7 (DSPC:ALC-0315:cholesterol:ALC-0159)), and buffered in Na2HPC>4 12.3 mM, NaCl 92.25 mM, Sucrose 150 mM, pH 7.4. As another example, the mRNA/LNP drug product (e.g., 100 ug/mL or 50 ug/mL) may comprise Na2HPC>4 1 mg/mL, NaCl 7.5 mg/mL, Sucrose 150 mM, pH 7.4.

Present embodiments may be applied to any suitable LNP formulation for encapsulation of mRNA.

Example 2. Preparation of mobile phase A (DBAA 0.05 M/TEAA 0.1 M in water)

30 mL of DBAA solution 0.5 M was added to a glass graduated cylinder. Then, 30 mL of TEAA solution 1 M was added. Ultrapure water was added up to a volume of 300 mL. The solution was mixed slowly to homogenize the solution, and then the solution was transferred to a glass bottle.

Example 3. Preparation of mobile phase B (DBAA 0.05 M/TEAA 0.1M in 50/50 H2O/ACN (v/v) 30 mL of DBAA solution 0.5 M was added to a glass graduated cylinder. Then, 30 mL of TEAA solution 1 M was added. Ultrapure water was added up to a volume of 150 mL. 150 mL of acetonitrile was added to the solution containing DBAA/TEAA/water. The solution was mixed slowly to homogenize the solution.

Example 4a. Triton X- 100 10% solution in Rnase free water

A 10% Triton solution was made by adding 0.5 mL of Triton X-100 to 4.5 mL of Rnase free water in a Rnase free tube, and was mixed well. Example 4b. Brij-35 5% solution in Rnase free water

A 5% Brij-35 solution was made by adding 1 mL of Brij-35 10% solution to 1 mL of Rnase free water in a Rnase free tube, and was mixed well.

Alternatively a 5% Brij-35 solution was made by weighting 100 mg of solid Brij-35 and adding 2 mL of Rnase free water in a Rnase free tube, and was mixed well.

Example 5a. Pre-treatment by addition of surfactant

A liquid sample comprising an mRNA product (e.g., encapsulated in a LNP) stored at -70° C was thawed at room temperature at least 30 minutes before use, and the sample was mixed by gentle inversion. In an aspect, the sample was diluted to have a final concentration in mRNA of 80 pg/mL (± 40 pg/mL) in at least 500 pL using Rnase free water, though any suitable concentration range may be used (e.g., 1 pg/mL up to 100 pg/mL or more).

In an Rnase free tube, 150 pL of sample + 50 pL of Triton X-100 10% solution was added. Samples were slightly vortexed to homogenize the solution.

Example 5b. Pre-treatment by addition of surfactant

For low concentrations, a liquid sample comprising a mRNA product (e.g., encapsulated in a LNP) stored at -70° C was thawed at room temperature at least 30 minutes before use, and the sample was mixed by gentle inversion. In an aspect, the sample was diluted to have a final concentration in mRNA of between 3 and 20 pg/mL in at least 300 pL, though any suitable concentration range may be used (e.g., 1 pg/mL up to 100 pg/mL or more). For the dilution, a dilution buffer of Tris 25mM pH 7.4 - NaCl 6mM in Rnase free water was used. In an RNAse free tube, 150 pL of sample + 50 pL of Brij-35 5% solution was added.

Samples were prepared in duplicate. Samples were slightly vortexed to homogenize the solution. Example 6. Column set up

The initial flow rate through the column (e.g., the DNAPac column) at 0. 1 mL/min was set to the initial conditions of the gradient. The column temperature was set to 60°C. The flow rate was increased progressively up to 0.4 mL/min by 0.1 mL/min steps. The column was equilibrated at 0.4 mL with the column temperature at 60°C. Once the column temperature was stabilized at 60°C and the flow rate of 0.4 mL/min was reached, one or more gradient cycles applying the chromatographic working conditions was performed.

Example 7. RNA extraction from mRNA-LNPs and binaries

RNA extraction is performed (optional with regard to the present method) according to techniques known in the art (Packer et al.). For example, mRNA is extracted from the mRNA- LNP formulation or lipid binary mixture by isopropanol precipitation. 100 pL of mRNA-LNP or binary is diluted 10-fold in 900 pL ammonium acetate (60 mM) in isopropanol, vortexed briefly, and centrifuged at 14,000 g for 15 min at 4 °C. The supernatant is discarded and the pellet is washed with 1 mb isopropanol, vortexed, and centrifuged at 4 °C; the pellet is dried in vacuo and resuspended in 100 pL Rnase-free water at room temperature. The resuspended drug product may be analyzed by the present chromatographic methods.

Present chromatographic techniques are compatible with sample extraction techniques of the LNP-mRNA product (e.g., liquid - liquid mRNA extraction techniques).

Example 8. Reversed phase HPLC

Sample (e.g., subjected to pre -treatment) separation was performed on a DNA Pac RP HPLC column with dimensions 2.1 * 100 mm from Thermo Fisher Scientific, wherein the stationary phase had a particle size of 4 pm and the flow rate was 0.35 mL/minute, with a column temperature of 59 °C. Elution is performed in gradient mode using a mixture of DBAA, TEAA, water and acetonitrile.

The mobile phase comprised a first eluent and a second eluent. The first eluent comprised or consisted of triethylammonium acetate (TEAA) (100 mM; Sigma- Aldrich) and dibutylammonium acetate (DBAA) (50 mM; TCI America) in water. The second eluent comprised or consisted of triethylammonium acetate (TEAA) (100 mM) and dibutylammonium acetate (DBAA) (50 mM) in 50% water and 50% acetonitrile (EEO/MeCN 50:50) (Sigma- Aldrich). In aspects, separation was accomplished by step-gradient according to the parameters of Table 1 or Table 2. mRNA was detected by UV at 260 nm, and the LEP was quantified as a percent of the total chromatographic peak area.

Table 1. Parameters for resolution of LEP by RP-UPLC

In aspects, blank subtraction was performed, in which a sample without analytes (a blank sample) was injected into the reverse phase HPLC. The profile from the blank sample was then subtracted from a profile generated by a sample with analytes. Blank subtraction was helpful for reducing background noise and /or accounting for signal drift.

Table 2. Parameters for resolution of LEP by RP-UPLC.

The obtained chromatograms were evaluated using software and the relative peak area was determined in percent (%) as commonly performed in the art. The relative peak area indicates the amount of RNA that was not affected by formation of lipid-RNA adduct. Late eluting sPecies (i.e. lipid-RNA adduct) was quantified as relative percentage of the total chromatographic peak area. Given that the mobile phase gradient is optimized to produce a well-defined mRNA product peak and mRNA-lipid adduct peak, the present method is specific for determination of the amount of mRNA-lipid adduct.

In other aspects, to evaluate mRNA total amount in the analysis, a standard curve may be prepared using not encapsulated mRNA that is previously quantified with an orthogonal method and is injected at different concentrations. Total recovery is extrapolated by linear regression calculation executed by the software.

Example 9. Treatment with surfactant

In one aspect, a sample of the composition comprising the ionizable amino-lipid (and optionally other lipids to form an LNP) and the RNA (suitably mRNA), was treated with a surfactant (e.g., about 2-3% Triton X-100 or about 2.5% Triton X-100 or Brij-35) to disrupt or lyse the LNPs. Following preparation of the RNA sample with surfactant, analytical RP-

HPLC was performed to determine the integrity of RNA. Example 10. Software Parameters

Integration may be performed manually or with software. Software parameters were as follows:

Example 11 (Low Dose mRNA)

Present techniques are compatible with low dose formulations of mRNA. In aspects, solutions of the first eluant and second eluant were made according to Examples 2 and 3. Pre-treatment of a sample comprising a low dose of mRNA/LNP (e.g.. l-20 ug/ml ) is treated with Brij-35 according to Examples 4b and 5b. The sample was then analyzed based on the chromatographic conditions provided in the examples herein.

Present techniques are compatible with any suitable LNP or mixtures of different LNPs, any suitable modification (e.g., uracil modification, 3’ poly A tail addition, 5’ capping, etc.) and is independent of nucleotide composition and presence of one or more untranslatable regions (UTRs). Present techniques are also suitable for quantification of a sample comprising up to 1%, 5%, 10%, 16% of mRNA-lipid adduct.

Example 12 (Results obtained with different concentrations of Brij-35)

In order to investigate the effect of Brij-35 concentration on the resolution and integrity of main peak, different Brij35 percentages were explored. To evaluate the robustness of variations of the concentration of the surfactant, samples were prepared at a concentration of 20 pg/ml based on mRNA content, with three final different percentages of Brij-35: 0.75%, 1.25% and 1.75%. Different types of constructs were selected for analysis, including Covid- 19 constructs and a SI V.

Constructs were each diluted in Tris-based buffer.

Preliminary results confirmed the absence of impact of Brij-35 concentration on the detection and stability of mRNA since no relevant changes in integrity and retention times were observed. The mRNA product peak was about 3.110 minutes and the adduct was about 3.412 minutes (data not shown) with no observed overlap between the curves. Additionally, the efficiency of the surfactant was also confirmed at lower concentrations (e.g., about 0.75%- about 1.25%), and even when reduced to Brij-35 0.75%, no significant differences in main peak and mRNA-lipid adduct peak areas were observed.

The following table shows the reported values of mRNA-lipid adduct percentage for different products at different concentrations of Brij-35. The samples are valid, since the duplicate injections were highly reproducible. The SD between the values of mRNA-lipid adduct percentages obtained for the different concentrations of Brij-35 are also reported. The deviation was significantly lower than 1 for all the samples.

Example 13 (Multivalent formulation)

Low dose multivalent formulations are also suitable with the present techniques. In aspects, solutions of the first and second eluant were made according to Examples 2 and 3. Pre- treatment of a sample comprising a low dose of a multivalent mRNA LNP sample (e.g., 1-20 pg/ml ) is treated with Brij-35 according to Examples 4b and 5b. The sample was then analyzed based on the chromatographic conditions provided in the examples herein. In other examples, the present method was tested on two products containing different constructs (e.g., covid and flu) mixed together, and was also tested on samples of flu containing different constructs (with different sizes) (data not shown). Multivalent formulations may comprise two or more mRNAs, encapsulated in any suitable LNP (or combination of different LNPs) with any suitable modification (e.g., uracil modification, 3’ poly A tail addition, 5 ’capping, etc.) and is independent of nucleotide composition and presence of one or more untranslatable regions (UTRs). Present techniques are suitable for quantification of a sample comprising up to 1%, 5%, 10%, 16% of mRNA- lipid adduct. The percentage of mRNA-lipid adduct is determined based on the value of relative area of the adduct calculated over the total area of mRNA (sum of mRNA product peak and mRNA-lipid adduct peak). In multivalent formulations, percentage of mRNA-lipid adduct is provided as the cumulative value from the contributions of the one or more constructs. Present techniques may be applied to compositions with any suitable LNP for encapsulating one or more mRNAs.

Suitably, any surfactant which is UV silent at the measured 260 nm range may be used with the embodiments provided herein.

Conclusion

Development and optimization of a novel analytical method were performed (see, FIG. 2A- 9B) allowing quantitative measurement of the LEP. The chromatographic parameters and results reported in FIGs. 2A-9B showed a significant and substantial improvement in eluting the LEP into a single, well-defined peak that was resolved (e.g., measured quantitatively, for example, by integration). The % of LEP present was determined based on the proportion between the Total Area of the mRNA (sum of the mRNA product peak and mRNA-lipid adduct peak) and the LEP Area (the area of the mRNA-lipid adduct peak). In multivalent formulations, the percentage of mRNA-lipid adduct is the cumulative value from the contributions of each mRNA. Elution of the LEP occurred more rapidly than art-disclosed techniques, with the peak very sharp and well-defined, facilitating measurement and avoiding baseline issues (see, FIGs.

2A-9B).

Accordingly, the present method may be used to measure different amounts of impurities such as the LEP, from various LNP -encapsulated mRNA products with different mRNA as well as different lipids.

Claims

2. The method of claim 1, wherein the impurity is a lipid-mRNA adduct.

3. The method of claim 1 or claim 2, wherein pre-treating the sample comprises:

(i) diluting or mixing the sample with a surfactant (e.g., Triton, SDS, Tween 20, Brij-35), wherein the surfactant is present at a concentration of 1-10%, or

(ii) extracting the mRNA and lipid-mRNA adduct from the sample, before contacting the column with the sample.

4. The method of any of claims 1 to 3, wherein a volume percentage of the first eluent in the mobile phase is 0% to 55% (v/v) and a volume percentage of the second eluent in the mobile phase is 45% to 100% (v/v).

5. The method of any one of the preceding claims, wherein the first eluent comprises at least two ion pairs in a first solvent or first solvent solution, and wherein the second eluent comprises at least two ion pairs in a second solvent or second solvent solution.

6. The method of claim 5, wherein the at least two ion pairs of the first eluent are a triethylammonium salt and a dibutylammonium salt and the at least two ion pairs of the second eluent are a triethylammonium salt and a dibutylammonium salt.

7. The method of claim 6, wherein the first solvent or first solvent solution comprises water, and wherein the second solvent or second solvent solution comprises acetonitrile in water (e.g., 50% acetonitrile in H2O).

8. The method of claim 6 or claim 7, wherein the triethylammonium salt in the first and second eluent is triethylammonium acetate, the dibutylammonium salt in the first and second eluent is dibutylammonium acetate, the first solvent solution is water, and the second solvent solution is TbO/acetonitrile 50:50.

9. The method of claim 8, wherein, for the first eluent, a concentration of triethylammonium acetate is about 75mM - 125 mM (e.g., about 100 mM) and a concentration of dibutylammonium acetate is about 25-75 mM (e.g., about 50 mM), and wherein, for the second eluent, a concentration of triethylammonium acetate is about 75mM - 125 mM (e.g., about 100 mM) and a concentration of dibutylammonium acetate is about 25-75 mM (e.g., about 50 mM).

10. The method of any one of the preceding claims, wherein the first retention time of the mRNA product is less than the second retention time of the impurity, and wherein both the mRNA product and impurity are eluted in less than 5 minutes.

11. The method of claim 10, wherein the peak of the mRNA product does not substantially overlap with the peak of the impurity.

12. The method of any one of the preceding claims, further comprising applying a step gradient to the mobile phase.

13. The method of claim 12, wherein the step gradient comprises the steps of:

(ii) applying, after (i), 100% (v/v) of the second eluent; and

(iii) applying, after (ii), a mixture of about 50% (v/v) of the first eluent and about 50% (v/v) of the second eluent.

14. The method of claim 12, wherein the step gradient comprises the steps of:

15. The method of any one of the preceding claims, wherein the column is an analytical column or a preparative column and the column comprises particles (e.g., porous resin particles) that are hydrophobic or comprise hydrophobic functional groups.

16. The method of claim 15, wherein the particles have a diameter ranging from 2 pm to about 10 pm, about 2 pm to 6 pm, or about 4 pm or wherein the particles comprise pores having a diameter ranging from about 100 A to about 3000 A, from about 100 A to about 2000 A, or from about 500 A to about 2000A.

17. The method of any one of the preceding claims, wherein a temperature of the column is about 53°C to about 65°C, about 55°C to about 63°C, about 57°C to about 61°C, or about 59°C; wherein a flow rate of the column is about 0.2 to 0.5 mL/min, 0.3 to 0.4 mL/min, or about 0.35 mL/min; and wherein dimensions of the column are about 2.1 x 100 mm.

18. The method of any one of the preceding claims, wherein the lipid nanoparticle (LNP) comprises at least one cationic or ionizable lipid, preferably a cationic or ionizable amino lipid.

19. The method of any one of the preceeding claims, wherein the RNA has a GC content comprising one or more of: codon “GCC” with a frequency of 0.40, codon “GCT” with a frequency of 0.28, codon “GCA” with a frequency of 0.22, or codon “GCG” with a frequency of 0.10.

20. Use of the method of any one of the preceding claims as a quality control during or after manufacturing of a lipid nanoparticle (LNP)-formulated mRNA product.

21. The method of any preceding claim comprising a multivalent formation of mRNA.

22. The method of claim 21 wherein a first mRNA encodes for an influenza antigen and a second mRNA encodes for a SARS-COV-2 antigen.

23. The method of claim 21 or 22 wherein the length of the first mRNA ranges from about IkB to about 4 kB, to about 1.5 kB to about 2 kB and the length of the second mRNA ranges from about 3kB to about 5 kB.

24. The method of any one of the preceding claims wherein the mRNA has a total concentration of up to 1%.

25. The method of any one of the preceding claims, wherein the percent (%) volume of the well-defined peak corresponding to the impurity is determined based on the proportion between the total area of the mRNA and the area of the impurity.