METHOD FOR DETECTING OLIGONUCLEOTIDE CONJUGATES

Abstract

The present invention relates to a method for detecting at least one oligonucleotide conjugate of interest in solution, wherein the oligonucleotide conjugate of interest is composed of a nucleic acid entity and of a nonpolar entity, wherein the nucleic acid entity is chemically linked to the nonpolar entity, and wherein the method comprises the steps of providing a liquid sample comprising the oligonucleotide conjugate of interest; separating the oligonucleotide conjugate of interest from the liquid sample by analytical means under conditions including the presence of at least one cyclodextrine in solution; and detecting the oligonucleotide conjugate of interest by means of qualitative or quantitative analysis.

Claims

1. A method for detecting at least one oligonucleotide conjugate of interest in solution, wherein the oligonucleotide conjugate of interest is composed of a nucleic acid entity and of a nonpolar entity, wherein the nucleic acid entity is chemically linked to the nonpolar entity, and wherein the method comprises the steps of: a) providing a liquid sample comprising the oligonucleotide conjugate of interest; b) separating the oligonucleotide conjugate of interest from the liquid sample by analytical means under conditions including the presence of at least one cyclodextrin in solution; c) detecting the oligonucleotide conjugate of interest by means of qualitative or quantitative analysis.

2. The method of claim 1, wherein the analytical means of step b) is selected from the group consisting of anion exchange high performance liquid chromatography (AEX-HPLC), size exclusion liquid chromatography (SEC-LC), reverse phase high performance liquid chromatography (RP-HPLC), ion pairing reversed phase high performance liquid chromatography (IP-RP-HPLC) and capillary gel electrophoresis (CGE).

3. The method of claim 1, wherein the nucleic acid entity of the oligonucleotide conjugate is composed of DNA or RNA nucleotides or any combination thereof, preferably wherein the nucleic acid entity is a chemically synthesized oligonucleotide, more preferably a chemically synthesized oligonucleotide comprising or consisting of modified DNA nucleotides and/or modified RNA nucleotides.

4. The method of claim 1, wherein the nucleic acid entity has a length of from 6 to 150 nucleotides, preferably of from 10 to 80 nucleotides, more preferably of from 12 to 50 nucleotides.

5. The method of claim 1, wherein step c) further includes the detecting of impurities of the oligonucleotide conjugate of interest, preferably wherein the impurities are composed of or consist of at least one non-full length nucleic acid entity, more preferably in the form of one or more non-full length synthesis product(s), even more preferably with a length or structure different to the full-length synthesis product, or any combination thereof.

6. The method of claim 1, wherein the nonpolar entity is a lipophilic or a hydrophobic entity, preferably wherein the nonpolar entity is selected from the group consisting of cholesterol, tocopherol and fluoroquinolone, more preferably wherein the nonpolar entity is cholesterol.

7. The method of claim 1, wherein i) the anion exchange high performance liquid chromatography (AEX-HPLC) is performed at a temperature of from 10 C. to 90 C., preferably at a temperature of from 30 C. to 75 C., preferably at ambient temperature; ii) the size exclusion high performance liquid chromatography (SEC-HPLC) is performed at a temperature of from 10 C. to 50 C., preferably at a temperature of from 20 C. to 40 C.; iii) the reverse phase high performance liquid chromatography (RP-HPLC) is performed at a temperature of from 10 C. to 100 C., preferably at a temperature of from 40 C. to 70 C.; iv) the ion pairing reverse phase high performance liquid chromatography (IP-RP-HPLC) is performed at a temperature of from 10 C. to 100 C., preferably at a temperature of from 30 C. to 85 C.; v) the capillary gel electrophoresis (CGE) is performed at a temperature of from 10 C. to 60 C., preferably at a temperature of from 30 C. to 50 C.

8. The method of claim 1, wherein the at least one cyclodextrin is selected from the group consisting of alpha, beta, gamma or delta variants of cyclodextrin, preferably wherein the at least one cyclodextrin is in the form of methyl-beta cyclodextrin.

9. The method of claim 1, wherein the at least one cyclodextrin in solution is present at a final concentration of from 0.01 mM to 50 mM, preferably at a final concentration of from 0.5 mM to 25 mM, more preferably at a final concentration of from 10 mM to 25 mM, most preferred at a final concentration of 20 mM.

10. The method of claim 1, wherein the at least one cyclodextrin is added to the liquid sample before carrying out step b).

11. The method of claim 1, wherein the detecting in step c) is carried out by means of UV readout, by means of fluorescence readout or by means of mass spectrometry (MS), or any method alike.

12. The method of claim 1, wherein the method is used for analytical or preparative purposes, preferably i) wherein, if the method is used for analytical purposes, the quality of the synthesis product is determined in step c), preferably by determining the degree of impurities; or ii) wherein, if the method is used for preparative purposes, the yield of the full-length synthesis product is optimized in step c) in that liquid fractions containing the oligonucleotide conjugate of interest are collected.

13. The method of claim 12, wherein, if the method is used for analytical purposes, the quality of the synthesis product is defined by the amount and/or by the ratio of the full-length synthesis product versus the amount and/or the ratio of the non full-length synthesis products, preferably wherein the non full-length synthesis products are intermediate and/or irregular synthesis products or any combination thereof, more preferably wherein the intermediate synthesis products lack one or more nucleotides at either ends or at both ends, most preferably wherein the intermediate synthesis products have the form of n-1, n-2, n-3, n-4, n-5, n-6, n-7, n-8, n-9, n-10, or alike.

14. A method for evaluating the quality of chemically synthesized oligonucleotides, wherein the method comprises the steps of: a) providing a liquid sample containing or suspected of containing at least one oligonucleotide conjugate of interest, wherein the at least one oligonucleotide conjugate of interest is composed of a nucleic acid entity and of a nonpolar entity, wherein the nucleic acid entity is chemically linked to the nonpolar entity, and wherein the nucleic acid entity is a chemical oligonucleotide synthesis product; b) separating the at least one oligonucleotide conjugate of interest from the liquid sample by analytical means under conditions including the presence of at least one cyclodextrine in solution; c) detecting the at least one oligonucleotide conjugate of interest by means of qualitative or quantitative analysis; d) collecting liquid fractions; e) analysing the collected fractions containing or suspected of containing the oligonucleotide conjugate of interest, characterized in that the nucleic acid entity of the oligonucleotide conjugate of interest is composed or consists of the at least one full-length synthesis product.

15. (canceled)

Description

FIGURE LEGENDS

[0063] FIG. 1: SEC-HPLC Column: GE Healthcare Superdex 75 Increase 10/300 GL. Temperature: Room Temperature 25 C. (non-denaturing: Duplex stays intact during chromatography). Eluent: 1PBS in 15% ACN with 1 mM Methyl--Cyclodextrin or 1PBS in 15% ACN w/o Methyl--Cyclodextrin. Flow rate: 0.9 mL/min. Black trace: Duplex analyzed in presence of Methyl--Cyclodextrin. Blue trace: Duplex analyzed in absence of Methyl--Cyclodextrin (duplex peak does not elute from column, no peak).

[0064] FIG. 2: Single strand analysis of X32755K1 by AEX-HPLC: Column: ThermoFisher Scientific DNA Pac PA200; 4250 mm Temperature: 85 C. Eluent A: 25 mM TRIS; 1 mM EDTA in 25% Acetonitrile at pH=8; Eluent B 500 mM sodium perchlorate in Eluent A; The eluents are prepared with or without presence of 5 mM Methyl--Cyclodextrin. Flow rate: 1.0 mL/min. Compounds are eluted by gradient of eluent B from 24.5% after one minute increased to 37% at 33 minutes. Black trace: X32755K1 analyzed in presence of 5 mM Methyl--Cyclodextrin in eluent A and eluent B. Blue trace: X32755K1 analyzed in absence of Methyl--Cyclodextrin in eluent A and eluent B.

[0065] In the presence of Methyl--Cyclodextrin, the main peak is more symmetric, has much smaller peak width at baseline (0.92 vs. 0.62 min) resulting in a greater peak height. Greater peak height corresponds to higher sensitivity for detection of the main peak. Resolution of the impurity peaks from main peak is improved, e.g. the resolution according to the USP (US Pharmacopeia) of later eluting impurity peak to the main peak is increased from 1.48 in absence of Methyl--Cyclodextrin to 3.63 in presence of 5 mM Methyl--Cyclodextrin in the eluents.

[0066] FIG. 3: Single strand analysis of X32755K1 by CGE: CapillaryeCAP DNA Capillary (65 cm total length; 100 m I.D.), Beckman Coulter, No.: 477477; Temperature: 35 C. Run Buffer: 1TRIS Borate Buffer with 10 mM Methyl--Cyclodextrin or 1TRIS Borate Buffer w/o 10 mM Methyl--Cyclodextrin. Separation Voltage: 30 kV. Blue trace: X32755K1 analyzed in presence of 10 mM Methyl--Cyclodextrin. Black trace: X32755K1 analyzed in absence of Methyl--Cyclodextrin (single strand peak does not elute from capillary, no peak).

[0067] FIG. 4: Single strand analysis of X32755K1 by CGE: CapillaryeCAP DNA Capillary (65 cm total length; 100 m I.D.), Beckman Coulter, No.: 477477; Temperature: 35 C. Run Buffer: 1TRIS Borate Buffer with 10 mM Methyl-6-Cyclodextrin or 1TRIS Borate Buffer with 20 mM Methyl--Cyclodextrin or 1TRIS Borate Buffer w/o 10 mM Methyl--Cyclodextrin. Separation Voltage: 30 kV. Pink trace: X32755K1 analyzed in presence of 10 mM methyl--cyclodextrin; Blue trace: X32755K1 analyzed in presence of 10 mM Methyl--Cyclodextrin. Black trace: X32755K1 analyzed in the absence of methyl--cyclodextrin (single strand peak does not elute from capillary, no peak).

[0068] FIG. 5: Structure of immobilized cholesterol.

[0069] FIG. 6: About 1 mg of crude material was purified via HPLC using the Source 15Q resin at ambient temperature. Buffer contained 30% acetonitrile (ACN).

[0070] FIG. 7: About 1 mg of crude material was purified via HPLC using the Source 15Q resin at ambient temperature. Buffer contained 25% ACN and 20 mM Methyl--cyclodextrin (MbCD).

[0071] FIG. 8: About 100 g of crude material was purified via HPLC using the Source 15Q resin at 60 C. Buffer contained 30% ACN only, no MbCD was added.

[0072] FIG. 9: About 8 mg of crude material was HPLC purified using the TSK Gel resin at ambient temperature. NaBr gradient, 20 mM Na-phosphate, pH 7.8 in 15% ACN containing 20 mM MbCD. Flow rate was 1 mL/min and the gradient was programmed to start from 0% buffer B to reach 40% buffer B in 60 minutes.

[0073] FIG. 10: About 8 mg of crude material was HPLC purified using the Source 15Q resin at ambient temperature. NaBr gradient, 20 mM Na-phosphate, pH 7.8 in 15% ACN containing 20 mM MbCD. Flow rate was 1 mL/min and the gradient was programmed to start from 0% buffer B to reach 40% buffer B in 60 minutes.

[0074] FIG. 11: About 8 mg of crude material was HPLC purified using the TSK Gel resin at 60 C. Material was eluted using a NaBr gradient, 20 mM Na-phosphate, pH 7.8 in 15% ACN containing 20 mM MbCD. Flow rate was 1 mL/min and the gradient was programmed to start from 0% buffer B to reach 10% buffer B in 5 minutes and subsequently the slope of the gradient was changed to reach 40% B in 60 minutes.

[0075] FIG. 12: About 8 mg of crude material was HPLC purified using the Source 15Q resin at 60 C. NaBr gradient, 20 mM Na-phosphate, pH 7.8 in 15% ACN containing 20 mM MbCD. Flow rate was 1 mL/min and the gradient was programmed to start from 0% buffer B to reach 10% buffer B in 5 minutes and subsequently the slope of the gradient was changed to reach 40% B in 60 minutes.

[0076] FIG. 13: Analytical results are shown. Pooled fractions correspond to the area of the FLP peak marked with the two-headed arrow (.Math.) in FIGS. 9 to 12, respectively.

EXAMPLES

Example 1: Methyl--Cyclodextrin as Additive in SEC-LC

[0077] Goal: Development of a SEC-LC method for the analysis of a cholesterol-conjugated oligonucleotide duplex.

[0078] Background: Usually, cholesterol-modified oligonucleotides do not elute from an SEC-column. Addition of methyl--cyclodextrin to the SEC Buffer masks the cholesterol of the oligonucleotide and thus allows for the compound eluting as a peak from the SEC column.

[0079] Test Sample: siRNA-duplex CD-10452K1:

TABLE-US-00001 DuplexXD-10452K1 Abbreviation AxoID Sequence FLPs X32755K1 5-(Chol4)GGAUGAAGUGGAGAUUAGUdTdT-3 FLPas X02812K3 5-ACUAAUCUCCACUUCAUCCdTdT3

[0080] SEC-HPLC Column: GE Healthcare Superdex 75 Increase 10/300 GL. The SEC-LC was performed at room temperature to achieve non-denaturing conditions, so that the siRNA-duplex stays intact during chromatography. The eluents were composed of 1PBS in 15% ACN with 1 mM methyl--cyclodextrin or 1PBS in 15% ACN without methyl--cyclodextrin and a flow rate: of 0.9 mL/min was applied. The result in FIG. 1 shows, that the duplex peak can only be observed in presence of 1 mM methyl--cyclodextrin (Black trace), but not in absence (blue trace), as then no peak elutes and the material is strongly bound to the SEC column surface.

Example 2: Methyl--Cyclodextrine as Additive in AEX-HPLC

[0081] Goal: Development of AEX-HPLC method for the analysis of cholesterol-conjugated oligonucleotides.

[0082] Background: Add Methyl--cyclodextrine to the different HPLC Buffers to mask the cholesterol of the oligonucleotide and thus, changing the properties of interaction with the column material.

[0083] Test Sample: X32755K1 single stranded oligonucleotide:

TABLE-US-00002 Abbreviation AxoID Sequence FLPs X32755K1 5-(Chol4)GGAUGAAGUGGAGAUUAGUdTdT-3 ThermoFisherScientificDNAPacPA200,4250mm Column (Thermo;Art.No.063000) Bufferwithoutbeta- EluentA:25%ACN,1mMEDTA,25mMTrispH8 cyclodextrin EluentB:Awith500mMNaC104 Bufferwithbeta- EluentA:25%ACN,1mMEDTA,25mMTrispH8and5mM cyclodextrin cyclodextrine EluentB:Awith500mMNaC104 ColumnTemp. 85C. Flow: 1.00ml/min

TABLE-US-00003 TABLE 1 Gradient Gradient Table Time Flow [ml/min] % A % B 0.5 1.0 75.5 24.5 0.0 1.0 75.5 24.5 1.0 1.0 75.5 24.5 33.0 1.0 63.0 37.0 33.2 1.0 0 100.0 33.7 1.0 0 100.0 34.0 1.0 75.5 24.5 39.0 1.0 75.5 24.5

TABLE-US-00004 TABLE 2 Results for AEX-HPLC Analysis of Y32755K1 Peak Width Relative Resolution at baseline Retention Time to Main peak Description [min] to Main peak (according to USP) AEX-HPLC of X32755K1 with 5 mM cyclodextrine Peak1 0.62 0.921 2.48 Peak2 0.36 0.940 2.46 Peak3 1.88 0.990 0.15 Main Peak 0.52 1.000 / Peak5 0.38 1.091 3.63 AEX-HPLC of X32755K1 without 5 mM cyclodextrine Peak1 n.a. 0.983 n.a. (only Peak-Shoulder) Peak2 n.a. 0.991 n.a. (only Peak-Shoulder) Peak 3 n.a. Not detected, n.a. Co-elution with main peak Main Peak 0.92 1.000 n.a. Peak4 1.11 1.050 1.48

[0084] With 5 mM beta-cyclodextrine in AEX-HPLC Buffers the following was observed (FIG. 2). The peak width at baseline is significantly reduced form 0.92 min to 0.52 min. The decrease in peak width results in a significant increase in the resolution of peaks eluting just before and after the main peak. The peaks are symmetric in presence of 5 mM beta-Cylcodextrine and not in the absence of this additive. The results of FIG. 2 show the following:

A) Peak No. 3 is only detected when analyzing in presence of 5 mM beta-cylcodextrine and co-elutes with the main peak in absence of beta-cylcodextrine.
B) Peak No. 2 is resolved with resolution of 2.46 by USP compared to no resolution, as peak only results in a small shoulder on the main peak, but no separation
C) Peak No. 5 is separated with a resolution of 3.68 in presence of 5 mM beta-cylcodextrine and only 1.48 in absence of beta-cylcodextrin.

Example 3: Methyl--Cyclodextrin as Additive in CGE

[0085] Goal: Development of a Capillary Gel Electrophoresis Method (CGE) for the Analysis of Cholesterol-conjugated oligonucleotides. All work was conducted on a PA800plus CE instrument from Beckman Coulter. Background: CGE does not work for cholesterol-modified oligonucleotides as compounds are strongly retained by CGE gel and no peaks eluted from the capillary. Addition of 10 mM or more Methyl--cyclodextrin to the separation gel and to the separation buffers of the CGE system mobilizes the cholesterol modified strand and sharp peaks can be observed.

[0086] Test Sample: single strand X32755K1 (sense strand in AHA1-Duplex XD-10452K1):

TABLE-US-00005 Abbreviation AxoID Sequence FLPs X32755K1 5-(Chol4)GGAUGAAGUGGAGAUUAGUdTdT-3

TABLE-US-00006 TABLE 3 Conditions of Capillary Gel Electrophoresis (CGE) Capillary eCAP DNA Capillary (65 cm total length; 100 pm I.D.), Beckman Coulter, No.: 477477 Buffer without 1x TRIS-Borate Buffer beta-cyclodextrine Buffer with 1x TRIS-Borate Buffer with 10 mM beta-cyclodextrine beta-cyclodextrin Capillary Temp. 35 C. Separation Voltage 30 kV

[0087] FIGS. 3 and 4 show that X32755K1 can only be analysed in presence of 10 or 20 mM methyl--cyclodextrin (blue trace in FIG. 3 and blue and pink trace in FIG. 4), whereas in the absence of methyl--cyclodextrin, no peak can be detected.

Example 4: Methyl--Cyclodextrin for IEX HPCL Purifications

[0088] Sequence: A 20mer consisting of alternating RNA nucleotides and 2-O-Methyl nucleotides was extended by a DNA nucleotide and a cholesterol ligand on the 3-end. The sequence was assembled on a controlled pore glass (CPG) solid support loaded with cholesterol. The pore size was 500A and the cholesterol loading was 85 mol/g. The solid support was obtained from Prime Synthesis (Aston, Pa. 19014, USA). The structure of the immobilized cholesterol is shown in FIG. 5.

[0089] The oligonucleotide sequence was prepared employing the well established phosphoramidite based oligomerization chemistry. RNA phosphoramidites, 2-O-Methylphosphoramidites as well as ancillary reagents were purchased from SAFC Proligo (Hamburg, Germany). Specifically, the following amidites were used: (5-O-dimethoxytrityl-N.sup.6-(benzoyl)-2-O-t-butyldimethylsilyl-adenosine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5-O-d imethoxytrityl-N.sup.4-(acetyl)-2-O-t-butyldimethylsilyl-cytidine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5-O-d imethoxytrityl-N.sup.2-(isobutyryl)-2-O-t-butyldimethylsilyl-guanosine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5-O-dimethoxytrityl-2-O-t-butyldimethylsilyl-uridine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. 2-O-Methylphosphoramidites carried the same protecting groups as the regular RNA amidites. All amidites were dissolved in anhydrous acetonitrile (100 mM) and molecular sieves (3 ) were added. 5-Ethyl thiotetrazole (ETT, 500 mM in acetonitrile) was used as activator solution. Coupling times were 8 minutes for RNA residues and 6 minutes for 2-O-methyl residues.

[0090] The support bound cholesterol conjugated oligonucleotide was cleaved from the solid phase and deprotected according to published procedures (Wincott, F. et al. Synthesis, deprotection, analysis and purification of RNA and ribozymes (Nucleic Acids Res. 23, 2677-2684, 1995). Typical crude materials contained the desired full length product (FLP) in a range of 70-80%.

[0091] To investigate different conditions for HPLC purification of the crude cholesterol conjugated oligonucleotide small scale columns with 5 mm diameter and 50 mm bed height were used. These 1 mL columns were packed with anion exchange resins typically used to purify oligonucleotides. Specifically, two different AEX beads were tested. Source 15Q (15 m beads) available from GE Healthcare and TSKgel SuperQ-5PW (20 m beads) available from Tosoh were selected. Purifications were carried out on an AKTA Purifier 100 (GE Healthcare).

[0092] For elution, the following buffers were used: Buffer A was made of 20 mM Tris, pH 8. Buffer B had the same composition as buffer A, but contained additionally 500 mM sodium perchlorate (NaClO.sub.4) or 1.4 M Sodium bromide (NaBr). Moreover, because of the hydrophobic nature of the cholesterol ligand (each failure sequence is composed of a 3-cholesterol due to the chemical synthesis starting from the 3-end) buffers contained 20-30% acetonitrile (ACN) as well.

[0093] For purifications at elevated temperatures a column oven (CO30 from Torrey Pines Scientific, Carlsbad, Calif., USA) and a mobile phase pre-heater (TL-600 available from Timberlein instruments, Boulder, Colo., USA) was used. Both devices were set to the same temperature (e.g. 60 C.).

[0094] The addition of MbCD to the elution buffers has been demonstrated to alter the elution profile in a predictable manner and enables purifications at ambient temperature as (truncated) cholesterol conjugated oligonucleotides elute in distinct peaks (see FIGS. 6 and 7). When no MbCD was added, a temperature of 60 C. is needed to obtain distinct peaks for cholesterol conjugated oligonucleotides (see FIG. 8).

[0095] Taken together, the addition of MbCD to the elution buffers allows for IEX HPLC purification of cholesterol conjugated oligonucleotides at ambient temperature (see FIGS. 9 to 12). In addition, the amount of ACN modifier in the mobile phase can be reduced significantly.

[0096] These features render capital investments into mobile phase pre-heaters and column ovens or jacketed columns unnecessary. In addition, organic solvents/waste can be cut at least in half.