Method for determining at least one quality parameter of an RNA sample
The present invention relates to a method for quality control analysis of an RNA sample comprising n different RNA molecule species using reverse transcription and a polymerase chain reaction (PCR) based assay, dPCR, preferably ddPCR, thereby determining at least one quality parameter. Moreover, the RNA molecules in the RNA sample may be in complexed form. In particular, the method is suitable for use in quality control during or following production of RNA samples for pharmaceutical use.
1. A method for quality control analysis of an RNA sample comprising n different RNA molecule species using reverse transcription and a polymerase chain reaction (PCR) based assay, wherein each of the n different RNA molecule species comprises one or more coding RNA molecules of synthetic origin, wherein n is an integer in the range of 3 to 200, comprising the following steps:
a) simultaneously reverse transcribing the one or more coding RNA molecules of at least one RNA molecule species of the n different RNA molecule species in a single reaction vessel, thereby providing a cDNA sample comprising at least one cDNA molecule species, wherein each cDNA molecule species corresponds to one of the at least one RNA molecule species,
b) subjecting the cDNA sample to the PCR based assay, and
c) determining the at least one quality parameter of the RNA sample,
wherein the PCR based assay is digital PCR (dPCR), and wherein the at least one quality parameter is selected from the group consisting of (i) quantity of the one or more coding RNA molecules of the at least one RNA molecule species, (ii) presence of the one or more coding RNA molecules of the at least one RNA molecule species, (iii) integrity of the one or more coding RNA molecules of the at least one RNA molecule species, and (iv) quantitative ratio between the one or more coding RNA molecules of at least two RNA molecule species.
2. The method according to claim 1 , wherein the RNA sample is a pharmaceutical RNA sample and further comprises a pharmaceutically acceptable carrier.
3. The method according to claim 1 , wherein the digital PCR is droplet digital PCR (ddPCR).
4. The method according to claim 1 , wherein n is an integer of 3 to 100.
5. The method according to claim 1 , wherein step b) comprises a step of
b1) simultaneous analysis of all cDNA molecule species in a single reaction vessel, or,
b2) simultaneous analysis of two or more cDNA molecule species in a single reaction vessel, or
b3) analysis of each cDNA molecule species in a separate reaction vessel.
6. The method according to claim 1 , wherein the PCR based assay employs a detectable label.
7. The method according to claim 1 , wherein the PCR based assay employs a sequence specific detectable label.
8. The method according to claim 5 , wherein in step b1) for each cDNA molecule species a different detectable label is used, or wherein in step b2) for each cDNA molecule species to be analyzed in a single reaction vessel a different detectable label is used.
9. The method according to claim 1 , wherein the one or more coding RNA molecules are obtained by an in vitro method.
10. The method according to claim 9 , wherein the in vitro method is in vitro transcription or chemical RNA synthesis.
11. The method according to claim 1 , wherein prior to step a) the one or more coding RNA molecules are purified using a method selected from the group consisting of high-performance liquid chromatography (HPLC), tangential flow filtration, oligo d(T) purification, ion exchange chromatography, hydroxyapatite chromatography, core bead flow-through chromatography, and combinations thereof.
12. The method according to claim 1 , wherein the one or more coding RNA molecules of each RNA molecule species encode a different amino acid sequence.
13. The method according to claim 1 , wherein the one or more coding RNA molecules of each of the n different RNA molecule species encode for one of n different peptides.
14. The method according to claim 1 , wherein the RNA sequences of the one or more coding RNA molecules of each of the n different RNA molecule species are at least 80% identical to each other.
15. The method according to claim 1 , wherein the n different RNA molecule species are selected from a group consisting of n different RNA molecule species encoding n different proteins or peptides derived from different serotypes or strains of a pathogen, n different RNA molecule species encoding n different antigens from one pathogen, n different RNA molecule species encoding n different antigens from different pathogens, n different RNA molecule species encoding n different isoforms or variants of an antigen, preferably a cancer antigen, n different RNA molecule species encoding n different epitopes of an antigen, n different RNA molecule species encoding n different cancer specific and/or patient specific cancer antigens, n different RNA molecule species encoding n different antibodies or antibody chains, n different RNA molecule species encoding n different proteins of one or more metabolic pathways, n different RNA molecule species encoding for n different isoforms of a protein for molecular therapy, n different RNA molecule species encoding for n different therapeutically active RNA molecule species, and combinations thereof.
16. The method according to claim 1 , wherein at least one of the one or more coding RNA molecules is present in complexed form with at least one carrier compound, thereby forming at least one RNA-carrier complex.
17. The method according to claim 16 , wherein the at least one RNA-carrier complex comprises more than one of the one or more coding RNA molecules and/or comprises one or more coding RNA molecules of different RNA molecule species.
18. The method according to claim 16 , wherein the at least one carrier compound is a member selected from the group consisting of peptides, polymers, carbohydrates, cholesterol, polyethylene glycol (PEG), lipids, phospholipids, PEGylated lipids, cationic and polycationic compounds, and combinations thereof.
19. The method according to claim 16 , wherein the at least one RNA-carrier complex comprises a member selected from the group consisting of liposome, lipid nanoparticle (LNP), and PEGylated peptide-based polymer complex and mixtures thereof.
20. The method according to claim 1 , wherein n is an integer in the range of 4 to 50.
21. The method according to claim 4 , wherein the n different RNA molecule species encode n different proteins derived from different serotypes or strains of a pathogen.
22. The method according to claim 21 , wherein the n different proteins comprise hemagglutinin (HA) antigens from influenza viruses.
23. The method according to claim 22 , wherein the n different proteins comprise HA antigens from influenza A and B viruses.
24. The method according to claim 23 , wherein n is 3.
25. The method according to claim 4 , wherein the n different RNA molecule species differ from each other in length by no more than 5%.
26. The method according to claim 25 , wherein n different RNA molecule species are at least 80% identical to each other.
27. The method according to claim 25 , wherein prior to step a) the one or more coding RNA molecules are purified by tangential flow filtration.
28. The method according to claim 27 , the RNA molecules are complexed with lipid nanoparticles (LNPs).
29. The method according to claim 27 , the RNA molecules are mRNA molecules.
30. The method according to claim 27 , wherein the n different RNA molecule species encode n different proteins derived from different serotypes or strains of a pathogen.
31. The method according to claim 30 , wherein the n different proteins comprise hemagglutinin (HA) antigens from influenza viruses.
32. The method according to claim 31 , wherein the n different proteins comprise HA antigens from influenza A and B viruses.
33. The method according to claim 32 , wherein n is 3.
34. The method according to claim 27 , wherein the digital PCR is droplet digital PCR (ddPCR).