JPWO2021209601A5 - - Google Patents

Description

In certain embodiments of the method of the present invention, washing with the first salt solution may be performed at an ionic strength ranging from 0.5M to 12.0M , or from 1M to 10M , or from 2M to 8M , or from 4M to 6M .

Example 2. Removal of contaminants from an in vitro transcription solution containing DNA, protein and mRNA by means of a high salt wash The conditions of Example 1 were repeated, except that the DNA sample described in Example 1 was replaced by an in vitro transcription mixture. Unbound contaminants passed through the anion exchanger immediately after injection. Bound contaminants were dissociated from the column by a high salt wash, which allowed them to be eluted. Portions of the chromatograms representing the main fractions of the column equilibration, sample injection, wash with equilibration buffer and high salt wash are shown in FIG . 2. Contaminants removed by the high salt wash are indicated by the dark grey shaded areas. As can be seen, the amount of contaminants removed by the wash is greater than the amount of DNA removed in Example 1. This is an indication of a higher contaminant content of the in vitro transcription mixture containing DNA, dsRNA and protein.

Example 3. Persistence of ssRNA binding to strong anion exchanger during high salt washes The conditions of Examples 1 and 2 were repeated, except that a sample containing a ssRNA ladder containing ssRNA ranging in size from 200b to 6000b was substituted. The ssRNA bound at the time of injection remained bound during the equilibration buffer wash and also during the high salt wash . The UV profile is strictly parallel to the baseline profile, in contrast to Examples 1 and 2, where removal of contaminants was evident in the shaded peaks . The ssRNA was then eluted with a salt gradient at 65°C.

Except to the extent inconsistent with an explicit teaching herein, all references cited herein are incorporated by reference in their entirety.
Exemplary embodiments of the present invention are described below.
<1>
applying a sample containing single-stranded RNA to an anion exchanger;
washing the anion exchanger with a first salt solution having a first ionic strength in the range of 0.5 M to 12.0 M at a first temperature in the range of 18° C. to 25° C.; and
Eluting the single-stranded RNA with a second salt solution having a second ionic strength at a second temperature in the range of 35° C. to 80° C.
Including,
Provided that the first ionic strength is at least 0.5 M higher than the second ionic strength.
A method for purifying single-stranded RNA using an anion exchanger.
＜2＞
The method according to <1>, wherein the washing with the first salt solution is carried out at an ionic strength in the range of 1M to 10M, or 2M to 8M, or 4M to 6M.
＜3＞
The method according to <1> or <2>, wherein the washing with the first salt solution is carried out using a chaotropic salt.
＜4＞
The method according to <3>, wherein the chaotropic salt is selected from the group consisting of a guanidine salt, a perchlorate, a thiocyanate, or a mixture thereof.
＜5＞
The method according to any one of <1> to <4>, wherein the washing with the first salt solution contains a chelating agent at a concentration in the range of 1 mM to 1000 mM, or 5 mM to 500 mM, or 10 mM to 100 mM, or 20 mM to 50 mM.
＜6＞
The method according to any one of <1> to <5>, wherein the washing with the first salt solution contains one or more chelating agents selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and tris(2-aminoethyl)amine (TREN).
＜７＞
The method according to any one of <1> to <6>, wherein the second salt solution used for eluting the single-stranded RNA is applied at the second temperature in the range of 45° C. to 70° C., or 50° C. to 70° C., or 55° C. to 70° C., or 60° C. to 70° C.
＜8＞
The method according to any one of <1> to <7>, wherein the anion exchanger is selected from the group consisting of a strong anion exchanger having a quaternary amino ion exchange group, a weak anion exchanger having a tertiary amino ion exchange group, a weak anion exchanger having a secondary amino ion exchange group, a weak anion exchanger having a primary amino group, and an anion exchanger having two or more types of amino groups.
＜9＞
The method according to any one of <1> to <8>, wherein the size of the RNA is in the range of 1,000 bases to 25,000 bases.