PROCESS FOR SEPARATION OF OLIGONUCLEOTIDE OF INTEREST FROM A MIXTURE

Abstract

A method for separation of an oligonucleotide from a mixture using a biphasic mobile phase/stationary phase liquid-liquid chromatography system. A first mobile phase contains the oligonucleotide and the stationary phase contains an exchanger substance that removably binds to the target oligonucleotide. The mobile phase is caused to flow in contact with the stationary phase in a liquid-liquid chromatography apparatus such that the oligonucleotide becomes bound to the exchanger substance in the liquid stationary phase. The oligonucleotide is then displaced from the liquid stationary phase into a second liquid mobile phase by means of a displacer substance able to displace the oligonucleotide from the stationary phase into the second mobile phase.

Claims

1. A method for the separation of a target oligonucleotide from a mixture of the target oligonucleotide and one or more impurity comprising: providing a biphasic mobile phase-stationary phase liquid-liquid chromatography system comprising a first liquid mobile phase, and a liquid stationary phase containing at least one exchanger substance that removably binds to the target oligonucleotide; causing the first liquid mobile phase to carry the target oligonucleotide in a flow relative to and in contact with the liquid stationary phase in the column of a liquid-liquid chromatography apparatus such that the target oligonucleotide becomes bound to the exchanger substance in the liquid stationary phase; then displacing the target oligonucleotide from the liquid stationary phase into a second liquid mobile phase flowing relative to and in contact with the stationary phase through the column by means of a displacer substance able to displace the target oligonucleotide from the liquid stationary phase into the second mobile phase, characterized in that the exchanger substance is a salt of an organic secondary, tertiary or quaternary amine with a counter anion, of the general formula:
R.sup.1R.sup.2R.sup.3R.sup.4N.sup.+X.sup.− wherein through the sequence secondary, tertiary and quaternary respectively two, three or four of the groups R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently C.sub.1-20 alkyl or substituted alkyl such as fluoro or trifluoromethyl substituted alkyl, or benzyl and the remainder are hydrogen, and X.sup.− is a halide anion.

2. A method according to claim 1 characterised by the steps of: (1) providing a first liquid phase containing the target oligonucleotide and one or more impurity in solution, and a second liquid phase containing the exchanger substance that removably binds to the target oligonucleotide, the first and second liquid phases forming two distinct phases when in contact with each other; (2) introducing the second liquid phase into a centrifugal partition chromatography apparatus as a stationary liquid phase therein; (3) introducing the first liquid phase containing the target oligonucleotide and one or more impurity in solution into the centrifugal partition chromatography apparatus as a first mobile phase and causing this first liquid phase to flow through the centrifugal partition chromatography apparatus in contact with the second liquid stationary phase such that the target oligonucleotide becomes removably bound to the exchanger substance in the second liquid stationary phase; (4) introducing a third liquid phase which forms a distinct phase when in contact with the second liquid phase and which contains in solution at least one displacer substance able to displace the target oligonucleotide from the second liquid phase into the centrifugal partition chromatography apparatus, as a second mobile phase and causing this second mobile phase to flow through the centrifugal partition chromatography apparatus in contact with the second liquid phase such that the target oligonucleotide becomes displaced from the stationary phase and enters solution in the second mobile phase; (5) isolating the displaced target oligonucleotide from the second mobile phase.

3. A method according to claim 1 characterised in that the target oligonucleotide comprises 10-30 bases.

4. A method according to claim 3 characterised in that the target oligonucleotide is the 20 base oligonucleotide which has the sequence 5′-UCAAGGAAGAUGGCAUUUCA-3′.

5. A method according to claim 1 characterised in that the stationary phase comprises a mixture of one or more organic liquid which is substantially immiscible with water and one or more organic liquid which is miscible with water.

6. A method according to claim 5 characterised in that the first and second mobile phases comprise a mixture of one or more organic liquid which is miscible with water, and water.

7. A method according to claim 5 characterised in that the stationary and mobile phases comprise the respective two equilibrium phases of: (i) a liquid system containing C.sub.1-6 alkyl C.sub.1-6 alkanoate ester, C.sub.1-8 alkanol and water; or (ii) a liquid system containing C.sub.1-8 alkanol and water; or (iii) a liquid system containing C.sub.1-8 alkanol, di-(C.sub.1-8 alkyl) ketone and water; or (iv) a liquid system containing di-(C.sub.1-8 alkyl) ketone and water; or (v) a liquid system containing C.sub.1-6 alkyl C.sub.1-6 alkyl ether or a C.sub.4-10 cyclic ether, C.sub.1-8 alkanol and water.

8. A method according to claim 1 characterised in that the halide anion of the exchanger substance is chloride.

9. A method according to claim 8 characterised in that the exchanger substance is selected from a mixture of tri-(n-octyl) methyl ammonium chloride and tri-(n-decyl) methyl ammonium chloride; and benzalkonium chloride.

10. A method according to claim 1 characterised in that the exchanger substance is selected from cetyltrimethylammonium bromide, methyltrioctylammonium chloride, a mixture of alkylbenzyldimethylammonium chlorides of various alkyl chain lengths, benzyltrimethylammonium chloride, tetrabutylammonium chloride, and a high molecular weight, oil soluble secondary amine supplied as a liquid in the free-base form in protonated form.

11. A method according to claim 1 characterised in that the second liquid mobile phase has the same composition of liquids as the first mobile phase.

12. A method according to claim 11 characterised in that the stationary phase comprises a mixture of predominantly C.sub.1-8 alkanol and C.sub.1-6 alkyl C.sub.1-6 alkanoate ester, and the second mobile phase comprises a mixture of water and C.sub.1-8 alkanol.

13. A method according to claim 11 characterised in that the stationary phase comprises a mixture of predominantly C.sub.1-6 cyclic ether and C.sub.1-8 alkanol, and the second mobile phase comprises a mixture of predominantly water and C.sub.1-8 alkanol.

14. A method according to claim 1 characterised in that the displacer substance is selected from alkali metal halides, sulphates and oxalates, Saccharin sodium salt, Sunset Yellow and Amaranth.

15. A method according to claim 1 characterised in that the concentration of the displacer substance in the second mobile phase is 5-30 mM.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] The present invention will now be described by way of example only with reference to the following drawings.

[0074] FIG. 1 shows a UV chromatogram of the second mobile phase exiting from the column from Experiment 8.

[0075] FIG. 2 shows a Chromatogram reconstruction after fractions analysis from Experiment 8.

[0076] FIG. 3 shows a Chromatogram reconstruction after fractions analysis from Experiment 21.

[0077] FIG. 4 shows a UV chromatogram of the second mobile phase exiting from the column from Experiment 4.

[0078] FIG. 5 shows a Chromatogram reconstruction after fractions analysis from Experiment 18.

[0079] A number of experiments were performed as set out in the table below. Two experiments listed in the table as Experiments 8 and 21 are described in detail below.

Experiment 8

[0080] The liquid phases were prepared according to the following procedure. Ethyl acetate, 1-butanol and water were mixed in volume ratios 3:2:5. The bi-phasic system was left to settle until two clear phases were obtained, then these phases were separated.

[0081] To the top phase layer (ethyl acetate/butanol with a minor proportion of water), was added Aliquat 336™ (exchanger substance) to obtain a concentration of 40 mM and the container was labeled as “stationary phase”, i.e. the second liquid phase.

[0082] The bottom phase (water/butanol with a minor proportion of ethyl acetate), was divided into two equal portions. To the first portion was added sodium hydroxide to obtain a concentration of 10 mM, and the container was labeled as “first mobile phase”, i.e. the first liquid phase.

[0083] To the second portion of bottom phase was added sodium hydroxide to obtain a concentration of 10 mM and potassium iodide (displacer substance) until a concentration of 13.3 mM was reached, and the container was labeled “second mobile phase”, i.e. the third liquid phase.

[0084] The Centrifugal Partition Chromatography (CPC) apparatus used was a commercially available 200 ml Armen CPC instrument. Typically such apparatus comprise a rotor made of ca. 20 circular partition discs. Typically such apparatus can be adjusted from 200-2000 rpm, generating a centrifugal force of ca. 120 g at 1000 rpm and 480 g at 2000 rpm). The Kromaton FCPC200 apparatus was set on descending mode. The column was rotated at 1200 rpm. The column was filled with the solution from the “stationary phase” container (second liquid phase plus Aliquat 336™) at a flow rate of 10 mL/min., following the specification operating conditions for the apparatus. Typically commercially available Dionex P580HPG 4-way binary high pressure gradient pumps (Sunnyvale, Calif., USA) may be used to introduce the stationary and mobile phases into the apparatus, typically via low-pressure injection valves (such as Upchurch, CIL, Cluxeau, Saint-Foy-La-Grande, FR), typically equipped with a 21 ml sample loop.

[0085] A sample of protected target oligonucleotide, being a 20-mer RNA oligonucleotide 5′-uca agg aag aug gca uuu ca-3′, but known to be contaminated with impurities possibly including 17-, 18-, 19- and/or 21-mer impurities (400 mg) was dissolved in the first mobile phase, (10 mL). Once dissolved, 10 mL of the stationary phase liquid (but containing no exchanger substance) was added to this solution of the oligonucleotide in the mobile phase to form a two phase mixture.

[0086] The mixture of the solution of target oligonucleotide dissolved in the first mobile phase and the stationary phase liquid was injected into the column, and this mixture was pumped through the column at a flow rate of 5 mL/min for 20 min. At this time equilibration had been reached, i.e. no more stationary phase was being stripped out of the column by the flowing mobile phase, and no target oligonucleotide was coming out of the column, as detected by analyzing the eluent exiting from the column using HPLC.

[0087] Then the second mobile phase (third liquid phase) was pumped at a similar flow rate for 100 min. Fractions of exiting second mobile phase were collected every minute and analysed off-line.

[0088] Referring to FIG. 1, this follows the introduction of the first and second mobile phases into the column starting at time=zero. During the first ca. 17 minutes a small quantity of stationary phase was displaced from the column. At ca. 20 minutes equilibrium was reached and very little stationary phase was displaced by the flowing first mobile phase. The second mobile liquid phase containing displacer substance was introduced after 20 minutes. Thereafter the peak represents elution of substances displaced by the second mobile phase from the stationary phase. Analysis of fractions from this peak indicated that the impurities present in the introduced oligonucleotide were tightly stacked at the beginning and end of this peak. FIG. 2 shows a Chromatogram reconstruction after fractions analysis from Experiment 8.

Experiment 21

[0089] The solvent system was composed of Me-THF/n-BuOH/Water in volume ratios (3:1:4) respectively. These three liquids were mixed and allowed to settle into two phases, which were separated.

[0090] To the upper organic phase was added Aliquat 336 (exchanger substance) to obtain a concentration of 40 mM, and this organic phase was designated for use as the stationary phase.

[0091] The lower aqueous phase was divided into two portions. To one portion was added sodium hydroxide to produce a concentration of 10 mM, and this portion was designated as the first mobile phase. To the second portion of the lower phase was added sodium hydroxide to produce a concentration of 10 mM, and Amaranth (displacer substance) to produce a concentration of 4.4 mM. This portion was designated the second mobile phase.

[0092] The crude sample of oligonucleotide (400 mg containing approximately 88% of target oligonucleotide in 35 mL of aqueous ammonia) was combined with 5 mL of the portion of upper phase of the solvent system designated as the stationary phase (containing no exchanger substance).

[0093] The column was rotated at 1200 rpm and filled with stationary phase in descending mode. Then the sample as prepared above was loaded onto the column while pumping mobile phase 1 at 5 mL/min. The first mobile phase was pumped for 10 minutes, followed by the second mobile phase also at a rate of 5 mL/min for 120 min. Fractions were collected every minute.

[0094] FIG. 3 shows the HPLC peaks of the eluate from Experiment 21, indicating “N DMT-off” as the target nucleotide without its DMT protecting group, “N+1” and “N-1” as the target nucleotide+/−one base also without the DMT protecting group, and imp 1-10 as traces corresponding respectively to ten impurities. It is seen that elution of impurities imp 1-10 and N−1 occurs virtually together and is largely complete before purified target oligonucleotide is eluted by the second mobile phase, and during the time period from ca. 81 to 95 minutes after introduction of the second mobile phase>95% pure target oligonucleotide is eluted. An N+1 impurity eluted mainly toward the end of the target peak.

Further Experiments

[0095] Details of experiments 1-30 are presented in tabular form below. Experiment 1 was a control in which no exchanger or retainer substance was used. In experiment 12 it is believed no stationary phase was retained because the two component system 2-butanol-water did not form two phases with a difference in density such that they easily formed two distinct phases with little tendency to emulsify.

[0096] In the column “oligo type” P═O refers to phosphodiester forms of the oligonucleotide, and P═S refers to phosphorothioateforms of the oligonucleotide. Al336 denotes Aliquat 336 and BACI denoted Benzalkoniun chloride. In presenting the data from these experiments a target purity was set and the amount of target material recovered with that purity was calculated. Isotachic train refers to the time in minutes during which the target oligonucleotide is eluted by the second mobile phase at the set purity. “MIBK” is an abbreviation for methylisobutylketone. “MtBE” is an abbreviation for methyltertiarybutyl ether. “MeTHF” is an abbreviation for 2-methyltetrahydrofuran.

[0097] FIG. 4 shows a UV chromatogram of the second mobile phase exiting from the column from Experiment 3, and FIG. 5 shows a Chromatogram reconstruction after fractions analysis from Experiment 18. In each of these the envelope representing the target oligonucleotide shows that for substantial periods of flow of the second mobile phase, considerably purified target oligonucleotide is being eluted and can be isolated from the second mobile phase using conventional techniques.

TABLE-US-00001 % Target oligo purity by HPLC peak area Number Retainer Displacer Exp Oligo in crude of Loading Loading Instrument Conc Conc number type material bases (mg) vol (mL) Column Solvent system Name (mM) Name (mM) 1 Protected 88% 20 100 13.7 FCPC200 Me-THF:n-BuOH: Al336 0 Saccharin 0 2′OMe-RNA Water P = 5 3:1:4 2 Protected 89% 20 200 10 Armen EtOAc:n-BuOH: Al336 20 KI 20 2′OMe-RNA CPC Water 3 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 40 KI 20 2′OMe-RNA CPC Water 4 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 40 KI 20 2′OMe-RNA CPC Water 5 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 40 KI 13.3 2′OMe-RNA CPC Water 6 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 40 KI 10 2′OMe-RNA CPC Water 7 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 24 KI 6 2′OMe-RNA CPC Water 8 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 40 KI 13.3 2′OMe-RNA CPC Water 9 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 20 KI 6.7 2′OMe-RNA CPC Water 10 Protected 89% 20 400 20 Armen EtOAc:n-BuOH: Al336 20 KI 6.7 2′OMe-RNA CPC Water 11 Protected 88% 20 400 40 FCPC200 1-pentanol/ Al336 40 KI 13.3 2′OMe-RNA water 12 Protected 88% 20 400 40 FCPC200 2-butanol/ Al336 40 NaI 13.3 2′OMe-RNA water 13 Protected 88% 20 400 40 FCPC200 1-BuOH/MIBK/ Al336 40 NaI 13.3 2′OMe-RNA water P = 5 1:3:4 14 Protected 88% 20 400 40 FCPC200 Me-THF:n-BuOH: Al336 40 NaI 13.3 2′OMe-RNA Water P = 5 3:1:4 15 Protected 89% 20 400 40 FCPC200 MIBK/water Al336 40 NaI 13.3 2′OMe-RNA P = 5 16 Protected 83% 20 400 40 FCPC200 1-pentanol/MIBK/ Al336 40 NaI 13.3 2′OMe-RNA water P = 5 1:3:4 17 Protected 83% 20 400 40 FCPC200 1-butanol/water Al336 40 NaI 13.3 2′OMe-RNA P = 5 18 Protected 81% 20 approxi- 10 FCPC200 Me-THF:n-BuOH: Al336 40 NaI 13.3 DNA mately Water P = O 400 3:1:4 19 Protected 88% 20 400 40 FCPC200 Me-THF:n-BuOH: Al336 40 Saccharin 13.3 2′OMe-RNA Water P = 5 3:1:4 20 Protected 89% 20 approxi- 10 FCPC200 EtOAc:n-BuOH: Al336 40 KI 13.3 2′OMe-RNA mately Water P = 5 400 3:2:5 21 Protected 83% 20 400 40 FCPC200 Me-THF:n-BuOH: Al336 40 Amaranth 4.4 2′OMe-RNA Water P = 5 3:1:4 22 Protected 88% 20 400 40 FCPC200 Me-THF:n-BuOH: CTAB 40 Saccharin 13.3 2′OMe-RNA Water P = 5 3:1:4 23 Protected 92% 10 200 32 FCPC200 EtOAc:n-BuOH: Al336 20 NaI 3.3 DNA Water 24 Protected 83% 20 400 40 FCPC200 Me-THF:n-BuOH: BACl 40 Sunset 13.3 2′OMe-RNA Water Yellow P = 5 3:1:4 25 Protected 88% 20 100 Me-THF:n-BuOH: Al336 8 Saccharin 4 2′OMe-RNA Water P = 5 3:1:4 26 Protected 88% 20 1000  100 FCPC200 Me-THF:n-BuOH: Al336 50 Saccharin 25 2′OMe-RNA Water P = 5 3:1:4 27 Protected 88% 20 550 30 FCPC200 Me-THF:n-BuOH: Al336 29 Saccharin 14.5 2′OMe-RNA Water P = 5 3:1:4 28 Protected 88% 20 550 30 FCPC200 Me-THF:n-BuOH: Al336 29 Saccharin 14.5 2′OMe-RNA Water P = 5 3:1:4 29 Protected 39% 15 100 34 FCPC200 Me-THF:n-BuOH: Al336 10.4 Amaranth 1.15 LNA gapmer Water P = O 3:1:4 30 Protected 63% 30 100 25 FCPC200 Me-THF:n-BuOH: Al336 13 Amaranth 1.44 DNA Water 31 Protected 84% 20 100 25 FCPC200 Me-THF:n-BuOH: Al336 15 NaI 5 siRNA Water .sup.1Recovered target Mobile Isotactic material Base phase train Purity of Yield at Exp Conc flow rate Rotation (min) pooled specified number Name (mM) (mL/min−1) speed Duration fractions purity 1 NaOH 10 5 1200 No separation 2 — 5 1200 5 >94% 0% 3 — 5 1200 9 >94% 0% 4 NaOH 10 5 1200 9 >94% 34% 5 NaOH 10 5 1200 23 >94% 59% 6 NaOH 10 5 1200 9 not determined 7 NaOH 10 5 1200 36 not determined 8 NaOH 10 5 1200 13 >94% 70% 9 NaOH 10 5 1200 30 not determined 10 NaOH 10 5 1200 30 not determined 11 NaOH 10 5 1200 20 >94% 43% 12 NaOH 10 5 1200 no stationary phase retention 13 NaOH 10 5 1200 20 >94% 52% 14 NaOH 10 5 1200 17 >94% 74% 15 NaOH 10 5 1200 17 >93% 30% 16 NaOH 10 5 1200 23 >92% 49% 17 NaOH 10 5 1200 18 >92% 43% 18 NaOH 10 5 1200 10 >94% 68% 19 NaOH 20 5 1200 18 >94% 88% 20 — — 5 1200 10 >93% 60% 21 NaOH 10 5 1200 17 >94% 83% 22 NaOH 10 5 1200 8 >93% 54% 23 NaOH 10 5 1200 20 >94% 86% 24 NaOH 10 5 1200 15 >94% 28% 25 NaOH 10 5 1500 13 >94% 76% 26 NaOH 20 5 1500 22 >94% 84% 27 NaOH 20 3.5 1200 30 >94% 72% 28 NaOH 20 3.5 1200 30 >94% 71% 29 NaOH 10 5 1200 9 >80% 90% 30 NaOH 10 5 1200 13 >90% 68% 31 NaOH 10 5 1200 19 >90% 72%