Analytical HPLC methods

Patent metadata

Field	Value
Publication number	US-11866696-B2
Application number	US-201816639265-A
Country	US
Kind code	B2
Filing date	Aug 17, 2018
Priority date	Aug 18, 2017
Publication date	Jan 9, 2024
Grant date	Jan 9, 2024

Abstract

Aspects of the disclosure relate to liquid chromatography (e.g., HPLC) methods which enable high resolution separations of polynucleotides of various lengths, sequences, and/or base compositions in a highly tunable manner. In some embodiments, the disclosure describes liquid chromatographic methods for separating a nucleic acid (e.g., a polyadenylated nucleic acid, such as an mRNA) from a complex mixture by using multiple ion pairing agents in the same mobile phase system. Accordingly, in some embodiments methods described by the disclosure are useful for assessing the quality of pharmaceutical preparations comprising nucleic acids.

First claim

What is claimed is:

1. A method of separating a first messenger ribonucleic acid mRNA from a mixture, the mixture comprising the first m RNA and at least a second mRNA, and further comprising one or more impurities or at least a third nucleic acid, comprising the steps:
i. contacting a stationary phase of a reverse phase chromatography column with the mixture; and
ii. eluting the first m RNA with a mobile phase, wherein the mobile phase comprises one or more solvents and two or more ion pairs, wherein at least one ion pair is a size-selective ion pair comprising four or more carbon atoms per alkyl chain, and at least one ion pair is a composition-selective ion pair having three or fewer carbon atoms per alkyl chain, such that the first mRNA traverses the column with a retention time that is different than a retention time of the second mRNA or different than a retention time of the one or more impurities or different than a retention time of the third nucleic acid of the mixture thereby isolating the first mRNA from the second mRNA or isolating the first m RNA from the one or more impurities or isolating the first m RNA from the third nucleic acid of the mixture or isolating the first m RNA from the second mRNA, the third mRNA, and the one or more impurities.

2. The method of claim 1 , wherein the stationary phase comprises particles.

3. The method of claim 1 , wherein the mobile phase is:
(i) one solvent;
(ii) a mixture of a first solvent and a second solvent; or
(iii) a mixture of a first solvent solution and a second solvent solution.

4. The method of claim 1 , wherein the mobile phase is a mixture of (i) a first solvent solution and (ii) a second solvent solution, wherein the first solvent solution comprises one or more solvents and one or more ion pairs, and wherein the second solvent solution comprises one or more solvents and one or more ion pairs.

5. The method of claim 4 , wherein the first solvent solution and/or the second solvent solution comprises one or more solvents independently selected from the group consisting of water, polar aprotic solvents, C 1-4 alkanols, C 1-6 alkanediols, and C 2-4 alkanoic acids.

6. The method of claim 1 , wherein step (ii) comprises: passing the mobile phase through the column, wherein the mobile phase is a mixture of a first solvent solution and a second solvent solution, and wherein the first solvent solution and the second solvent solution independently comprise one or more ion pairs selected from the group consisting of a triethylammonium salt.

7. The method of claim 1 , wherein the first mRNA comprises 5′ and 3′ UTRs; a 5′ 7-methylguanosine group or a 5′ 7-methylguanosine cap analog; a 3′-polyadenosine tail; or a combination thereof.

8. The method of claim 1 , further comprising the step of
detecting or isolating the second mRNA and/or the one or more impurities or third nucleic acid.

9. The method of claim 1 , wherein said eluting is isocratic with respect to a solvent composition of the mobile phase.

10. The method of claim 9 , wherein said eluting is isocratic or gradient with respect to a concentration of the two or more ion pairs.

11. The method of claim 10 , wherein the mobile phase comprises two ion pairs, and a relative concentration of the ion pairs is varied during the eluting.

12. The method of claim 1 , wherein said eluting is gradient with respect to a solvent composition of the mobile phase.

13. The method of claim 12 , wherein said eluting is isocratic or gradient with respect to the concentration of one or more ion pairs.

14. The method of claim 1 , wherein:
(i) the at least one size-selective ion pair is selected from the group consisting of a tetrabutylammonium salt, hexylammonium salt, and dibutylammonium salt; and
(ii) the at least one composition-selective ion pair is a triethylammonium salt.

15. The method of claim 14 , wherein:
(i) the at least one size-selective ion pair is selected from the group consisting of tetrabutylammonium phosphate (TBAP), hexylammonium acetate (HAA), and dibutylammonium acetate (DBAA); and
(ii) the at least one composition-selective ion pair is triethylammonium acetate (TEAA).

16. The method of claim 4 , wherein each of the first and second solvent solutions comprises at least one of the two or more ion pairs in a concentration of about 1 mM to about 100 mM.

Assignees

• Modernatx Inc

Inventors

• Issa William
• Packer Meredith

Classifications

• C12N15/101 Primary

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title

• A61K48/0091 Primary

  Purification or manufacturing processes for gene therapy compositions · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title

• G01N2030/027

  Liquid chromatography · CPC title