Polyinosinic—polycytidylic acid compositions and methods of making same
The present invention relates to compositions comprising polyinosinic (poly(I)) and polycytidylic acid poly(C) molecules, or a salt and/or solvate thereof, comprising double-stranded polyribonucleotides formed by the respective single-stranded poly(I) and poly(C) molecules and methods of making same. The present invention further relates to compositions wherein the disclosed respective poly(I) and poly(C) single-stranded molecules are annealed to thereby form double-stranded poly(I:C) molecules.
1. Polyinosinic-polycytidylic acid [poly(I:C)] molecules, or a salt and/or solvate thereof, comprising double-stranded polynucleotides formed by annealing together respective single-stranded poly(I) and poly(C) molecules, wherein
(a) at least 40% of the double-stranded polyribonucleotides comprise at least 850 base pairs, and
(b) at least 50% of the double-stranded polyribonucleotides comprise between 400 and 5000 base pairs.
2. The poly(I:C) molecules of claim 1 , wherein
(a) at least 60% of the double-stranded polyribonucleotides have at least 850 base pairs;
(b) at least 70% of the double-stranded polyribonucleotides have between 400 and 5000 base pairs; and
(c) between 20% and 45% of the double-stranded polyribonucleotides have between 400 and 850 base pairs.
3. The poly(I:C) molecules of claim 1 , wherein
(a) between 5% and 60%, preferably between 10% and 30%, of the double-stranded polyribonucleotides have less than 400 base pairs;
(b) between 20% and 45% or between 15% and 30%, preferably between 20% and 30%, of the double-stranded polyribonucleotides, have between 400 and 850 base pairs;
(c) between 20% and 70%, preferably between 50% and 60%, of the double-stranded polyribonucleotides have between 850 and 5000 base pairs; and
(d) between 0% and 10%, preferably 1% or less, of the double-stranded polyribonucleotides have more than 5000 base pairs.
4. The poly(I:C) molecules of claim 1 , wherein
(a) between 7% and 57% of the double-stranded polyribonucleotides have less than 400 base pairs;
(b) between 20% and 45% of the double-stranded polyribonucleotides have between 400 and 850 base pairs;
(c) between 20% and 70% of the double-stranded polyribonucleotides have between 850 and 5000 base pairs; and
(d) between 0% and 9% of the double-stranded polyribonucleotides have more than 5000 base pairs.
5. A method for producing the poly(I:C) molecules of claim 1 , comprising:
(a) dissolving the poly(I) molecules in PBS to form a first solution;
(b) separately dissolving the poly(C) molecules in PBS to form a second solution;
(c) filtering each respective solution obtained from step (a) and step (b) using a membrane;
(d) concentrating each respective permeate obtained for each respective filtration obtained in step (c) for each respective solution over a membrane to form a respective retentate for each respective solution;
(e) mixing each respective retentate obtained for each respective solution (a) and (b) with a PBS solution;
(f) determining the optical density of each respective retentate obtained in step (e) for each respective solution (a) and (b) and adjusting the total volume to a 1:1 stoichiometry;
(g) combining the respective poly(I) solution with the poly(C) solution obtained at step (e) using heat;
(h) cooling the resulting combined solution to room temperature;
(i) filtering the solution obtained from step (h); and
(j) purifying and desalting the resulting solution.
6. The method of claim 5 , wherein at step (a) and step (b), the poly(I) molecules and the poly(C) molecules are respectively provided as a powder.
7. The method of claim 5 , wherein at step (a) and step (b), the poly(I) molecules and the poly(C) molecules are respectively dissolved at 50° C.
8. The method of claim 5 , wherein at step (c), the filtering membrane comprises a 300 kDa cut-off or a 500 kDa cut-off.
9. The method of claim 5 , wherein the membrane of step (d) is a 30 kDa membrane.
10. The method of claim 5 , wherein the mixing in step (e) is performed using a 10X PBS solution.
11. The method of claim 5 , wherein the combining step (g) is performed at 55-62° C.
12. The method of claim 5 , wherein the filtering step (i) is performed using a G3 glass filter, optionally having a pore size of about 15 μm to 40 μm.
13. The method of claim 5 , wherein the desalting of step (j) is performed using isopropanol.
14. The method of claim 5 , wherein the solution in step (j) is freeze-dried and/or lyophilized at room temperature.
15. The method of claim 5 , wherein
(a) the poly(C) solution of step (b) is heated to a temperature of between 61° C. to 66° C. prior to mixing with the poly(I) solution of step (a); and
(b) the combining of step (g) is performed at a temperature of 55° C. to 58° C., after which the combined mixture is filtered over a 0.2 μm membrane.
16. The composition according to claim 1 , wherein
(a) the single-stranded poly(I) molecules comprise:
(i)<400 bases consist of 80-95% poly(I) molecules,
(ii) 400-850 bases consist of 5-20% poly(I) molecules,
(iii) 850-5000 bases consist of 0-5% poly(I) molecules, and
(iv)>5000 bases consist of 1% or less poly(I) molecules; and
(b) the single-stranded poly(C) molecules comprise:
(i)<400 bases consist of 20-82% poly(C) molecules,
(ii) 400-850 bases consist of 15-40% poly(C) molecules,
(iii) 850-5000 bases consist of 3-50% poly(C) molecules, and
(iv)>5000 bases, consist of 1% or less of poly(C) molecules.