Methods of preparing oligonucleotide compositions using ultrafiltration/diafiltration
Disclosed herein are methods for preparing compositions containing oligonucleotides. Methods of this disclosure involve subjecting an aqueous solution of a oligonucleotide to ultrafiltration/diafiltration (UF/DF) in order to form a retentate containing the oligonucleotide, where the ultrafiltration/diafiltration (UF/DF) is carried out using an aqueous buffer solution containing one or more salts. Also disclosed herein are oligonucleotide-containing compositions obtained by these methods.
1 . A method for preparing a composition comprising an oligonucleotide, the method comprising subjecting an aqueous solution of the oligonucleotide to ultrafiltration/diafiltration (UF/DF) to form a retentate comprising the oligonucleotide, wherein the ultrafiltration/diafiltration (UF/DF) is carried out using an aqueous buffer solution and wherein the aqueous buffer solution comprises i) sodium acetate and ammonium acetate or ii) sodium acetate and potassium acetate.
2 . The method of claim 1 , wherein the total concentration of sodium acetate and ammonium acetate or the total concentration of sodium acetate and potassium acetate in the buffer solution is in the range of 10 mM to 200 mM.
3 . The method of claim 2 , wherein the total concentration of sodium acetate and ammonium acetate or the total concentration of sodium acetate and potassium acetate in the buffer solution is 40 mM.
4 . The method of claim 1 , wherein
the aqueous buffer solution comprises sodium acetate and ammonium acetate, and the total concentration of sodium acetate and ammonium acetate ranges from 20 mM to 100 mM; or
the aqueous buffer solution comprises sodium acetate and potassium acetate, and the total concentration of sodium acetate and potassium acetate ranges from 20 mM to 100 mM.
5 . The method of claim 1 , wherein the molar ratio of sodium acetate to ammonium acetate or the molar ratio of sodium acetate to potassium acetate in the buffer solution ranges from 1:20 to 20:1.
6 . The method of claim 5 , wherein:
the aqueous buffer solution comprises sodium acetate and ammonium acetate, and the molar ratio of sodium acetate to ammonium acetate in the aqueous buffer solution is 17:3; or
the aqueous buffer solution comprises sodium acetate and potassium acetate, and the molar ratio of sodium acetate to potassium acetate in the aqueous buffer solution is 17:3.
7 . The method of claim 1 , wherein the aqueous buffer solution comprises 34 mM of sodium acetate and 6 mM of ammonium acetate or the aqueous buffer solution comprises 34 mM of sodium acetate and 6 mM of potassium acetate.
8 . The method of claim 1 , wherein the ultrafiltration/diafiltration (UF/DF) is carried out with a permeate flux ranges from 5 L·m −2 ·hr −1 to 25 L·m −2 ·hr −1 .
9 . The method of claim 1 , wherein
the ultrafiltration/diafiltration (UF/DF) is carried out with a diavolume from 3 to 10.
10 . The method of claim 1 , wherein the concentration of the oligonucleotide in the retentate is from 30 g/L to 150 g/L.
11 . The method of claim 1 , further comprising subjecting the retentate to lyophilization to form a lyophilized composition comprising the oligonucleotide.
12 . The method of claim 11 , wherein
i) the weight percentage of sodium in the lyophilized composition ranges from 2% to 10%.
13 . The method of claim 11 , wherein the weight percentage of acetate in the lyophilized composition is less than 3%.
14 . The method of claim 1 , wherein the oligonucleotide is an antisense oligonucleotide having 16 to 30 nucleotides.
15 . The method of claim 1 , wherein the oligonucleotide is nusinersen.
16 . The method of claim 1 , wherein
the aqueous buffer solution comprises sodium acetate and ammonium acetate, and the total concentration of sodium acetate and ammonium acetate ranges from 30 mM to 60 mM; or
the aqueous buffer solution comprises sodium acetate and potassium acetate, and the total concentration of sodium acetate and potassium acetate ranges from 30 mM to 60 mM.
17 . The method of claim 1 , wherein the molar ratio of sodium acetate to ammonium acetate or the molar ratio of sodium acetate to potassium acetate in the buffer solution ranges from 1:1 to 19:1.
18 . The method of claim 1 , wherein the molar ratio of sodium acetate to ammonium acetate or the molar ratio of sodium acetate to potassium acetate in the buffer solution ranges from 5:1 to 19:1.
19 . The method of claim 1 , wherein the concentration of the oligonucleotide in the retentate is from 70 g/L to 125 g/L.
20 . The method of claim 1 , wherein the ultrafiltration/diafiltration (UF/DF) is carried out using a membrane having a molecular weight cutoff (MWCO) in the range from 1 kDa to 7 kDa.
21 . The method of claim 1 , the ultrafiltration/diafiltration (UF/DF) is carried out by a tangential flow filtration.
22 . The method of claim 11 , wherein the weight percentage of the sodium in the lyophilized composition is 5.2%±0.9%.