Automated pilot scale purification of synthetic phosphorothioate oligonucleotides

Transcription

1 application note Automated pilot scale purification of synthetic phosphorothioate oligonucleotides Summary This application note describes a convenient d simple protocol for the purification of synthetic phosporothioate oligonucleotides using ion exchge chromatography on automated ÄKTA TM explorer 1 system. UNICORN TM, the software controlling all ÄKTAdesign systems, contains a pre-programmed method template that fully supports the purification procedure used. Sample: Tritylated phosphorothioate 21-mer oligonucleotide in 25% ammonia Sample load: 6 ODU (corresponding to 222 mg oligonucleotide) Column: FineLINE Pilot 35, packed with 3 ml of SOURCE 15Q, bed height 3.1 cm. Eluent A: 1 mm NaOH Eluent B:.4% TFA in distilled water Eluent C: 1 mm NaOH + 2 M NaCl Flow rate: 3 cm/h (48 ml/min) System: ÄKTAexplorer 1 AU 26 nm ms/cm The purification of nucleotides, 21 d 25 nts respectively, was performed on a pre-packed RESOURCE TM Q 1 ml column. The column contains SOURCE TM 15Q, a polymerbased, 15µm diameter, monosized ion exchge bead. The protocol was also scaled up 3-fold using the same preprogrammed method template. Crude material (.2 g) was applied to a FineLINE TM Pilot 35 column, (i.d. 35 mm) packed with 3 ml SOURCE 15Q. The yield obtained was 1 mg d the purity, checked by capillary electrophoresis, was 97.3%. These results correspond with those obtained on the RESOURCE Q1 ml column. These data show that ÄKTAexplorer 1, controlled by UNICORN, is a convenient way of obtaining reliable d reproducible results, which c also be scaled-up A 26 nm min Sample A 26 nm Introduction The potential use of oligonucleotides as drugs will dramatically increase the production demd for highly purified synthetic oligonucleotides. Today, most purification procedures are based on either reversed phase chromatography (RPC), ion exchge chromatography, or sequential combinations of both. In our experience, ion exchge chromatography is the best choice for large scale separation of the desired product from n-1 d n-2 mers. The resolution obtained with ion exchge is often superior to RPC d the technique has the additional advtage of not requiring orgic solvents or other high cost eluents. In the case of phosphorothioate oligonucleotides, ion exchge chromatography on SOURCE 15Q has been shown to readily separate fully thiolated oligonucleotides from incompletely thiolated oligonucleotides of the same length (1) Fig fold scale-up of the purification of a 21-mer tritylated phosphorothioate oligonucleotide shown in Figure 2. Lower curves show alysis by capillary electrophoresis of crude sample (left) d product pool (right) AC, p1

2 The method described is based on that trityl-on phosphorothioate oligonucleotides bind more strongly to SOURCE 15Q th the corresponding trityl-off alog. It is thus possible to capture d purify oligonucleotides directly from the crude ammonia solution obtained after cleavage of the oligonucleotide from the synthesis support. The capture d initial purification is followed by on-column cleavage of the trityl group from the oligonucleotide. A final gradient elution is performed to elute d further purify the target oligonucleotides. No programming is required as the entire one-column purification procedure is pre-programmed in UNICORN, the control system of ÄKTAexplorer. To run the automated procedure, simply attach the column, specify the column volume d flow rate, d start the run. The overall results of this study demonstrate the speed d reliability of developing purifications on ÄKTAexplorer as well as the value of using ion exchge chromatography on SOURCE 15Q in purification schemes for oligonucleotides. Results d Discussion Method description SOURCE 15Q is stable in mild alkali, which allows purification under denaturing conditions at ph 12. Aggregation of self-complementary or GC-rich oligonucleotides during purification is thus avoided. AU 26 nm Sample: Tritylated phosphorothioate 21-mer oligonucleotide in 25% ammonia Sample load: 2 ODU (corresponding to 7.4 mg oligonucleotide) Column: RESOURCE Q 1 ml, 3 cm bed height Eluent A: 1 mm NaOH Eluent B:.4% TFA in distilled water Eluent C: 1 mm NaOH + 2 M NaCl Regeneration: 2 M NaCl in 3% ispropol Flow rate: 3 cm/h (1.6 ml/min) System: ÄKTAexplorer Synthesis in trityl-on mode min 5 Loading onto column ms/cm The development of this methodology has been described earlier (2). The method contains the following steps; please refer to Figure 2. 1 The crude mixture from the synthesis is applied to the column. Recommended loading is 5 7 mg of crude oligonucleotide/ml medium Removal of non-tritylated failure sequences On-column cleavage of trityl group Re-equilibration Final purification by linear salt gradient elution 2 Non-tritylated failure sequences, which bind less strongly to the ion exchger, are removed by washing with 2 column volumes of 1 mm NaOH with 2 M NaCl. 3 On-column cleavage of the trityl group is performed in 3 minutes using.4% TFA in distilled water at a low flow rate. This fully detritylates the bound oligonucleotides. 4 The column is re-equilibrated with 1 mm NaOH. Column regeneration Fig. 2. Description of the one-column purification procedure. The chromatogram shows purification of a 21-mer tritylated phosphorothioate oligonucleotide on RESOURCE Q 1 ml. The results from alysis by capillary electrophoresis is shown in Figure AC, p2

3 5 The target oligonucleotide is further purified from shorter sequences by elution with a sodium chloride gradient. 6 The column is regenerated with 3% isopropol in 2 M NaCl. Separation on RESOURCE Q 1 ml The purification procedure was first run on RESOURCE Q 1 ml using the pre-programmed method template contained in UNICORN. Figures 2, 3 d 4 show the results from the purifications of the 21-mer d 25-mer oligonucleotide. Fractions were alysed by capillary electrophoresis. The yield from the purification of the 21-mer oligonucleotide was 3.1 mg with a purity of 97.6%, d the purification of the 25-mer oligonucleotide resulted in a yield of 2.6 mg with a purity of 98.4%. Scale-up A 3-fold scale-up was performed by increasing the column diameter while keeping other parameters such as linear flow rate, sample load/ml media d column bed height constt. The same chromatography system, ÄKTAexplorer, d the same pre-programmed method template were used for this scaled-up run. A FineLINE Pilot 35 column (35 mm inner diameter) was packed with 3 ml of SOURCE 15Q to a bed height of 3.1 cm. The mechical packing method, also performed on ÄKTAexplorer, is a simple one-step procedure that takes less then 3 minutes. Figure 1 shows the results of the purification of the 21-mer tritylated phosphorothioated oligonucleotide from a sample load of 7.4 mg/ml medium. The final yield was 1 mg, d the purity was 97.3%, which corresponds well to the result achieved with the 1 ml pre-packed column (see Table 1) d clearly demonstrates the scaleability of the method. Column volume (ml) Purity (%) Yield (mg) Table 1. Analytical results from the purification of a 21-mer tritylated phosphorothioate oligonucleotide at a sample load of 7.4 mg crude oligonucleotide/ml SOURCE 15Q. A 26 nm A 26 nm A 26 nm a) b) c) Fig. 3. Analysis by capillary electrophoresis from the separation of the 21-mer oligonucleotide, shown in Figure 2. a) Crude sample, b) the eluted non-tritylated failure sequences d c) a pool of the purified target oligonucleotide AC, p3

4 Sample: Tritylated phosphorothioate 25-mer oligonucleotide in 25% ammonia Sample load: 2 ODU (corresponding to 7.4 mg oligonucleotide) Column: RESOURCE Q 1 ml, 3 cm bed height Eluent A: 1 mm NaOH Eluent B:.4% TFA in distilled water Eluent C: 1 mm NaOH + 2 M NaCl Regeneration: 2 M NaCl in 3% ispropol Flow rate: 3 cm/h (1.6 ml/min) System: ÄKTAexplorer 1 AU ms/cm 12 A 26 nm A 26 nm A 26 nm a) b) c) d) min 4 26 nm Fig. 4. Purification of a 25-mer tritylated phosphorothioate oligonucleotide. a) The elution pattern from the chromatographic separation, b) capillary electrophoresis of the crude sample, c) the eluted non-tritylated failure sequences d d) the product pool. The one column purification procedure described here has also been used for preparative purification of phosphorothioate oligonucleotides using SOURCE 3Q, a polymer-based 3 µm diameter spherical ion exchge bead with properties as SOURCE 15Q except for the bead size. In this case, a.8 litre SOURCE 3Q column was used to purify 1 56 mg full length product. The purification was performed on a BioProcess Chromatography System (2). Experimental Materials ÄKTAexplorer 1, UNICORN control system, RESOURCE Q 1 ml, SOURCE 15Q, SOURCE 3Q, FineLINE Pilot 35, OligoPilot II DNA/RNA Synthesizer, phosphoramidates, NAP 1 Columns d FPLCdirector are all products of Amersham Biosciences. Oligonucleotide synthesis The oligonucleotides used in this study were synthesized on OligoPilot II DNA/RNA Synthesizer using its trityl-on mode. The 21-mer d 25-mer triylated phosphorothioate oligonucleotides were synthesized according to a stdard procedure (see Tables 2). The crude reaction mixture obtained after cleavage from the synthesis support was applied directly to the ion exchger without y pretreatment. Chromatography ÄKTAexplorer 1 with UNICORN control system was used for chromatography. Experimental details are described in the figures. Instrument: OligoPilot II DNA/RNA Synthesizer Sequence: 21-mer 25-mer Synthesis scale: 13 µmole 14 µmole Phosphorothioate amidite: 1.5 equivalents 1.5 equivalents Coupling time: 4. min 4. min Cycle time: 21 min 21 min Waste/cycle: 16 ml 16 ml Coupling efficiency: 98.3% 98.5% Table 2. Conditions for the synthesis of a 21-mer d a 25-mer tritylated phosphorothioate oligonucleotides AC, p4

5 Analysis Capillary electrophoresis was used for alysis during this study. All samples were desalted on NAP 1 Columns with distilled water prior to alysis. Experimental details are described in Table 3. Capillary: Buffer: Running conditions: Sample application: Data collection: ecap ssdna 1 Gel Column (Beckm) Tris-borate/Urea 1kV/3 min 7 kv/5 s FPLCdirector Table 3. Running conditions for capillary electrophoresis alysis. Ordering information Designation Code No. ÄKTAexplorer 1 (Fraction collector not included) ÄKTAexplorer ÄKTAexplorer 1 Air (includes air sensors) Fraction Collector Frac FineLINE Pilot RESOURCE Q 1ml RESOURCE Q 6 ml SOURCE 15Q PE 4.6/ SOURCE 15Q 5 ml ml ml Related product literature References 1. Application Note: Pilot scale purification of phosphorothioate DNA oligonucleotides. Code No Johsson, Hs J., Svensson, M. Poster presented at Nucleic Acid-Based Therapeutics, June , S Diego, USA. CodOrdering information Product Code No. ÄKTAexplorer System Series Data File FineLINE Pilot 35 Data File SOURCE 15Q, SOURCE 15S, Data File RESOURCE Q d RESOURCE S Strategies for large scale purification of synthetic oligonucleotides Appl Note AC, p5

6 to order: Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: Fax: Belgium Tel: Fax: Cada Tel: Fax: Central, East, South East Europe Tel: Fax: Denmark Tel: Fax: Finld Tel: Fax: Frce Tel: Fax: Germy Tel: Fax: Italy Tel: Fax: Jap Tel: Fax: Latin America Tel: Fax: Middle East d Africa Tel: +3 (1) Fax: +3 (1) Netherlds Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russi Federation Tel: +7 (95) , Fax: +7 (95) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerld Tel: Fax: UK Tel: Fax: USA Tel: Fax: ÄKTA, UNICOR N, R ESOUR CE, SOUR CE, FineLINE, OligoPilot, NAP d FPLCdirector are trademarks of Amersham Biosciences Limited or its subsidiaries. Amersham d Amersham Biosciences is a trademark of Amersham plc. Amersham Biosciences AB Björkgat 3, SE Uppsala, Sweden. Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, Engld. Amersham Biosciences Inc 8 Centennial Avenue, PO Box 1327, Piscataway, NJ 8855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9, D Freiburg, Germy. All goods d services are sold subject to the terms d conditions of sale of the compy within the Amersham Biosciences group that supplies them. A copy of these terms d conditions is available on request. Amersham Biosciences AB 1999 All rights reserved AC, p6 Produced by Wikströms, Sweden , Oct., 1999 Printed matter. Licence