Method for producing RNA molecule compositions

1. A method for producing a ribonucleic acid (RNA) pharmaceutical composition comprising at least two different RNA molecule species, the method comprising the following steps:

I) performing simultaneous RNA in vitro transcription of a mixture of at least two different deoxyribonucleic acid (DNA) molecule species in a single reaction vessel, wherein each of the at least two different DNA molecule species encode the at least two different RNA molecule species thereby generating the at least two different RNA molecule species;

II) obtaining an RNA molecule composition comprising the at least two different RNA molecule species generated in step I);

III) purifying the RNA from the RNA molecule composition; and

IV) formulating the at least two different RNA molecule species in a pharmaceutical formulation to produce the RNA pharmaceutical composition,

wherein the at least two different RNA molecules vary in length from each other by no more than 100 nucleotides and wherein the amounts of each of the at least two different RNA molecules are not more than 20% different from each other in the RNA molecule composition.

2. The method according to claim 1 , further comprising prior to step a step of:

1) generating the mixture of at least two different DNA molecule species using bacterial amplification,

2) generating the mixture of at least two different DNA molecule species using polymerase chain reaction (PCR), and/or

3) generating the mixture of at least two different DNA molecule species using enzymatic amplification.

3. The method according to claim 2 , wherein step 1) comprises a step of:

i) transforming a bacterial cell culture with at least one single DNA plasmid species of the mixture of at least two different DNA plasmid species, wherein each DNA plasmid species encodes one of the at least two different RNA molecule species.

4. The method according to claim 2 , wherein step 1) comprises a step of:

i)transforming at least two single bacterial cell cultures each with a single DNA plasmid species of the at least two different DNA plasmid species, wherein the single DNA plasmid species encodes one of the at least two different RNA molecule species.

5. The method according to claim 3 , further comprising a step of:

ii) isolating at least one single bacterial cell clone for each DNA plasmid species of the mixture of at least two different DNA plasmid species, and

iii) MHO growing each of the at least one single bacterial cell clone isolated in step ii) in a separate bacterial cell clone culture.

6. The method according to claim 4 , further comprising after step i) the following steps:

ii) isolating at least one single bacterial cell clone of each of the at least two single bacterial cell cultures transformed in step i),

iii) growing each of the single bacterial cell clones isolated in step ii) in a separate bacterial cell culture, and

iv) selecting at least one bacterial cell clone culture for each of the at least two different DNA plasmid species.

7. The method according to claim 5 , further comprising:

iv) determining at least one parameter of growth kinetics and/or amount of plasmid DNA of the at least one single bacterial cell clone culture, and

v) selecting one bacterial cell clone culture for each of the at least two different DNA plasmid species depending on the parameter determined in step iv).

8. The method according to claim 7 , wherein step iv) comprises a step of:

determining a parameter of growth kinetics by measuring the optical density of the bacterial cell clone culture after a time interval, and/or

determining the amount of plasmid produced per volume and time of bacterial cell culture.

9. The method according to claim 7 , wherein the selected bacterial cell clone culture for each of the at least two different DNA plasmid species exhibits similar or identical growth kinetics and/or similar or identical DNA production levels.

10. The method according to claim 7 , wherein step 1) further comprises a step of:

inoculating and growing an amount of at least one of the one or more bacterial cell clone cultures selected for each of the at least two different DNA plasmid species in a single reaction vessel, or

inoculating and growing an amount of at least one of the one or more bacterial cell clone cultures selected for each of the at least two different DNA plasmid species in one or more separate reaction vessels for each of the at least two different DNA plasmid species.

11. The method according to claim 10 , wherein equal amounts of each bacterial cell clone culture are inoculated.

12. The method according to claim 10 , wherein the amount of each bacterial cell clone culture used for inoculating is selected so that equal or similar amounts of each of the at least two different DNA plasmid species are obtained.

13. The method according to claim 1 , wherein the amount of each of the at least two different RNA molecule species in the RNA molecule composition is proportional or at least 90% proportional to the amount of the corresponding DNA molecule species in the mixture of at least two different DNA molecule species.

14. The method according to claim 1 , wherein the DNA sequences of the at least two different deoxyribonucleic acid (DNA) molecule species are at least 90% identical to each other.

15. The method according to claim 1 , wherein the RNA sequences of the at least two different RNA molecule species are at least 90% identical to each other.

16. The method according to claim 1 , wherein each of the at least two different DNA molecule species encodes for different RNA molecule species, wherein each of the at least two different RNA molecule species encodes for an antigen of different serotypes or strains of a same pathogen.

17. The method according to claim 16 , wherein each of the at least two different RNA molecule species encodes for an influenza antigen.

18. The method of claim 1 , wherein the at least two different RNA molecule species encode different variants of the same target peptide or protein, wherein said composition comprises the at least two different RNA molecule species in identical amounts.

19. The method according to claim 2 , wherein step 1) comprises a step of:

i)transforming a single bacterial cell culture with a mixture of at least two different DNA plasmid species, wherein each DNA plasmid species encodes one of the at least two different RNA molecule species.

20. The method according to claim 19 , further comprising after step i) the following steps:

ii) isolating at least at least two single bacterial cell clones, and

iii) growing each of the at least two single bacterial cell clones isolated in step ii) in a separate bacterial cell clone culture,

iv) determining the identity of the DNA plasmid species of each of the at least at least two single bacterial cell clone cultures grown in step iii), and

v) selecting at least one single bacterial cell clone culture for each of the at least two different DNA plasmid species.

21. The method of claim 1 , further comprising producing a RNA molecule composition comprising at least three different RNA molecule species.

22. The method of claim 21 , further comprising producing a RNA molecule composition comprising at least four different RNA molecule species.

23. The method according to claim 1 , wherein each of the at least two different RNA molecule species encodes for tumor antigen.

24. The method of claim 1 , wherein the at least two different DNA molecule species comprise at least two different DNA plasmid species.

25. The method according to claim 24 , wherein the DNA plasmid species have the same plasmid backbone.