[ 12] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 203 This major hurdle can be circumvented by two strategies: (i) restoring nucleolytic activity by the conjugation of suitable pendant groups or by incorporating an RNase H-competent window (mixed backbone oligonucle- otide) or (ii) increasing the efficiency of steric blockade. In this respect, the simple assay described in this article has the advantage of discriminating true translation arrest from target RNA destruction. Because natural mRNA are used in this assay, both untranslated and coding regions can be examined. Finally, the stimultaneous translation of three mRNA provides a convenient, and important, internal control. 25 W. F. Lima, V. Driver-Brown, M. Fox, R. Hanecak, and T. W. Bruice, J. Biol. Chem. 272, 626 (1997). 26 O. Matveeva, B. Felden, S. Audlin, R. F. Gesteland, and J. F. Atkins, Nucleic Acids Res. 25, 5010 (1997). 27 O. Matveeva, B. Felden, A. Tsodikov, J. Johnston, B. P. Monia, J. F. Atkins, R. F. Gesteland, and S. M. Freier, Nature Biotechnol. 16, 1374 (1998). [ 12] Purification of Antisense Oligonucleotides By RANJIT R. DESHMUKH, DOUGLAS L. COLE, and YOGESH S. SANGHVI Introduction Despite ongoing and significant advances in synthesis chemistry and reactor/reagent delivery systems design, 1 crude products of antisense phos- phorothioate oligonucleotide (AO) solid-phase syntheses still typically con- tain only about 75% full-length oligomer. While this could be seen as an excellent overall yield for a 19-step synthesis, the products must be purified to a much higher full-length oligomer content prior to use as antisense drugs. Oligonucleotide purification research has thus been a key part of successful antisense drug development. / Selective separation methods are required for the preparation of high- purity AO for antisense discovery research and clinical development and for the development of meaningful analytical control procedures. Finally, preparative separation methods must be created and scaled up to support AO manufacture for therapeutic use. 1 M. Andrade, A. S. Scozzari, D. L. Cole, and V. T. Ravikumar, Nucleosides Nucleotides 16, 1617 (1997). 2 S. T. Crooke, in "Antisense Research and Application" (S. T. Crooke, ed.), p. 1. Springer- Verlag, Berlin, 1998. Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 313 0076-6879/99 $30.00
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`204 GENERAL METHODS [ 12] Concern for synthetic oligonucleotide impurity types and quantities can vary, depending on the target application. High-purity oligonucleotides are required for diagnostic probe and antisense drug applications, whereas lower purity oligomers may be acceptable as oligonucleotide primers. Mi- crogram to gram AO quantities are often sufficient for in vitro and pharma- cological screening purposes, whereas kilograms are required for AO drug clinical trials. Actual and potential AO drug markets range from kilogram to metric ton levels. A single broad purification strategy could have utility over this range of scale and purity requirements, but the optimal systems differ for specific AO compounds. This article first broadly reviews the available methods for AO purification and then stresses techniques suitable for milligram, gram, and kilogram scale purification. This article discusses methods for purifying crude AO by two key chro- matographic techniques, reversed phase (RP) and anion exchange (AX). Other currently available techniques are also summarized briefly. For RP and AX techniques, small-scale results are presented first, followed by a discussion of large-scale methods. Examples presented are directed exclu- sively to the phosphorothioate oligonucleotides, first-generation antisense oligonucleotide drugs. General Purification Strategies for Oligonucleotides Characteristic properties of synthetic oligonucleotides and their process- related impurities, such as polarity, charge, and size, may be exploited for their purification. 3-5 For example, a hydrophobic protecting group left attached at the 5'-O-oligonucleotide terminus, such as 4,4'-dimethoxytrityl (DMT), allows effective use of RP-high-performance liquid chromatogra- phy (HPLC) 6'7 for purification. Hydrophobicity of the heterocyclic bases at mild pH also aids RP-HPLC purification, although selectivity of this effect is most significant for short oligonucleotides. Both hydrophobic effects are 3 W. Haupt and A. Pingoud, J. Chromatogr. 260, 419 (1983). 4 G. Zon, in "High-Performance Liquid Chromatography in Bioteclmology" (W. Hancock, ed.), p. 301. Wiley, New York, 1990. 5 G. Zon and J. A. Thompson, BioChromatography 1, 22 (1986). 6 y. S. Sanghvi, M. Andrade, R. R. Deshmukh, L. Holmberg, A. N. Scozzari, and D. L. Cole, in "Manual of Antisense Methodology" (G. Hartman and S. Endres, eds.), p. 3. Kluwer Academic Publishers, Norwell, 1999. 7 W. J. Warren and G. Vella, in "Protocols for Oligonucleotide Conjugates" (S. Agrawal, ed.), p. 233. Humana Press, Totowa, NJ, 1994.
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`[ 121 PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 205 useful in the preparative hydrophobic interaction chromatographic (HIC) purification of oligonucleotides, s Oligonucleotide backbone phosphates impart a strong negative charge to the molecule, with the mass to charge ratio related (but not linearly so) to the number of nucleotides in the oligomer. Anion-exchange chromatog- raphy is thus a selective separation method for synthetic oligonucleotide separations and is widely used in both analysis and purification. 7'9-16 To produce very pure oligonucleotides, highly specific affinity Watson- Crick base pairing can be exploited for purification under nonequilibrium conditions in the affinity chromatography mode. 17-I9 Gel permeation chromatography (GPC) ~° is useful for the size-based separation of oligonucleotides from one another or for gross size-class chemical separations such as the isolation of total isolated oligonucleotide from contaminating protein. This technique is not particularly useful for the purification of synthetic oligonucleotides less than 50 bases in length. Two or more stationary phase-solute interaction mechanisms may be combined in mixed-mode chromatographies in order to enhance separation selectivity. For example, by the addition of ion-pairing agents to RP-HPLC mobile phasesy 1-24 hydrophobic and charge-charge interactions may be combined in a single separation. Direct interaction of the stationary phase with oligonucleotide phosphate diester groups and other stationary phase- 8 p. Puma, in "HPLC: Practical and Industrial Applications" (J. Swadesh, ed.), p. 81. CRC Press, Boca Raton, FL, 1997. 9 W. A. Ausserer and M. L. Biros, BioTechniques 19, 136 (1995). J0 B. J. Bergot and W. Egan, J. Chromatogr. 599, 35 (1992). ll B. J. Bergot, U.S. Patent 5,183,885 (1993). 12 R. R. Drager and F. E. Regnier, Anal Biochem. 145, 47 (1985). 13 V. Metelev and S. Agrawal, Anal Biochem. 200, 342 (1992). 14 j. R. Thayer, R. M. McCormick, and N. Avdalovic, Methods Enzymol. 271, 147 (1996). 15 R. R. Deshmukh, W. E. Leitch II, and D. L. Cole, J. Chromatogr. A 806, 77 (1998). 16 j. Liautard, C. Ferraz, J. S. Widada, J. P. Capony, and J. P. Liautard, J. Chromatogr. 476, 439 (1989). 17 S. Agrawal and P. C. Zamecnik, PCT International WO 90/09393 (1990). is H. Orum, P. E. Nielsen, M. Jorgensen, C. Larsson, C. Stanley, and T. Koch, BioTechniques 19, 472 (1995). 19 H. Schott, H. Schrade, and H. Watzlawick, J. Chromatogr. 285, 343 (1984). 20 K.-I. Kasai, J. Chromatogr. 618, 203 (1993). 21 p. j. Oefner, C. G. Huber, F. Umlauft, G.-N. Berti, E. Stimpfl, and G. K. Bonn, Anal, Biochem. 223, 39 (1994). 22 A. P. Green, J. Burzynski, N. M. Helveston, G. M. Prior, W. H. Wunner, and J. A. Thompson, Bio Techniques 19, 836 (1995). 23 C. Huber, P, Oefner, and G. Bonn, Anal Biochem. 212, 351 (1992). 24 C. G. Huber, J. Chromatogr. A 806, 3 (1998).
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`206 GENERAL METHODS [ 121 solute charge interactions are available simultaneously on hydroxyapatite columns. 25'26 A nucleic acid-specific chromatographic media, RPC-5, 27'28 was used to simultaneously exploit charge interaction and hydrophobicity for the purification of nucleic acids, oligonucleotides, and tRNAs. RPC-5 solid support is not currently available commercially, however. Most of these separation techniques have been used at microgram to gram scales. RP, AX, and HIC are the only approaches that have been demonstrated at scales much greater than 100 g loading of AO. These three techniques are therefore the focus of the remainder of this article. Experimental Oligonucleotides All AO used in this study were synthesized in-house at various scales. The Milligen 8800 (PE Biosystems, Framingham, MA) ohgonucleotide synthesizer was used for synthesis of ISIS 2105 and ISIS 5132/CGP69846A. OligoPilot II and OligoProcess synthesizers (both from Amersham Phar- macia Biotech, Piscataway, NJ) were used to manufacture ISIS 2302. All these AO are 20-mer phosphorothioate oligodeoxyribonucleotides. Leaving the final DMT group on the 5'-nucleotide after completing oligomerization generates DMT-on crude oligomers. DMT-off crude oligomers are obtained by removing the final DMT as the final oligomerization step. All crude products are cleaved from solid support and base- and phosphate-deblocked in concentrated aqueous ammonia. Ammonia is removed by rotary evapo- ration in vacuo, and the resulting aqueous oligonucleotide solution is used for chromatography. DMT-on crude products are stored in a 1% triethyl- amine (v/v) solution to maintain basic pH. Chemicals and Buffers All general chemicals and buffers are ACS grade unless stated other- wise. Buffers are prepared from dry chemicals in most cases. A stock solution of 1 M NaOH is made and then used to make dilute NaOH solutions. The list of buffers used is given in Table I. Y. Yamasaki, A. Yokoyama, A. Ohnaka, Y. Kato, T. Murotsu, and K.-I. Matsubara, J. Chromatogr. 467, 299 (1989). 26 G. Bernardi, Methods Enzymol. 21, 95 (1971). 27 R. L. Pearson, J. F. Weiss, and A. D. Kelmers, Biochem. Biophys. Acta 228, 770 (1971). 28 R. D. Wells et aL, Methods Enzymol. 65, 327 (1980).
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`[19.] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 207 TABLE I BUFFERS Buffer system Composition pH AX buffer I A 20 mM NaOH 12.0 B 20 mM NaOH + 2.5 M NaC1 12.0 AX buffer II A 50 mM KOH 12.6 B 50 mM KOH + 2.5 M KC1 12.6 RP buffer I A 2.5 M sodium acetate 7.2 B Methanol C Deionized water RP buffer II A 2.5 M sodium acetate 7.2 B Methanol C Deionized water D 1% trifluoroacetic acid in water Chromatography Instrumentation Preparative liquid chromatography experiments are conducted on Bio- Cad workstations (PE Biosystems, Framingham, MA). The BioCad 20 provides a 20-ml/min maximum flow rate. The BioCad 60 and BioCad 250 have maximum flow rates of 60 and 250 ml/min, respectively. These chromatographic workstations have six buffer ports and automated method programming. Columns up to 600 ml volume are eluted on these chromato- graphs. At production scale, the KiloPrep KP100 (Biotage Division of Dyax Corp., Cambridge, MA) is used for RP-HPLC. Analytical AX analysis is conducted on a Waters Associates (Bedford, MA) system comprising a Waters 600E chromatograph with Waters 717 autosampler, 991 photodiode array detection system, and the Millennium 2.01 operating system. A ther- mostatted heating block is used to heat analytical columns. Chromatographic Columns and Media Unless otherwise stated, columns smaller than 5 ml packed volume are purchased prepacked from vendors. Larger volume anion-exchange columns from 5 to 200 ml packed volume are slurry packed in-house. A 200-ml AP-5 glass column (Waters Corp., Milford, MA, 50 mm i.d. × 100 mm length) is also used. All RP columns are packed at high pressure by the vendors. Production-scale radial compression cartridges are used in
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`208 OENEgAL ~ETHODS [ 121 the Biotage KP100 chromatograph. A list of oligonucleotide separation chromatographic media typically available in our laboratory and discussed in this article is given in Table II. Analytical Methods to Support Purification Development From each purification, column fractions or pools are assayed for AO content by UV absorbance. Chromatographic impurity profiles are deter- mined by analytical AX HPLC to quantify sulfurization-related phosphoro- thioate oligomer impurities, and length-altered impurities are determined by quantitative capillary gel electrophoresis (QCGE). These methods are described later in greater detail. Identity of the oligonucleotide product in crude material is determined by electrospray-mass spectrometry (ES-MS). In addition to these routine methods, additional analytical techniques may be used to completely characterize oligonucleotide products and their impu- rities, as summarized previously. 6 Quantitative UV Spectroscopy. A solution concentration of oligonucleo- tides is determined by measuring absorbance at 266 nm, Fractions are diluted appropriately to bring absorbance into the linear response range. The measured OD/ml can be suitably converted into mg/ml values by using the factor 25.7 OD = 1 mg for most 20-mer phosphorothioate deoxyoligo- nucleotides. TABLE II CHROMATOGRAPHIC COLUMNS AND MEDIA Media Nominal Ligand Bead particle Trade name Manufacturer chemistry chemistry size (/zm) BondaPak HClsHA Waters Corp. (Milford, Cls Silica 37-55 MA) Oligo R3 PE BioSystems (Framing- Cls PSDVB a 40 ham, MA) Q HyperD F BioSepra, Inc. (Marlbor- AX (Q) Ceramic-coated 35 ough, MA) silica with gel inside pores Poros HQ/50 PE Biosystems AX (Q) PSDVB 50 Poros HQH PE Biosystems AX (Q) PSDVB 10 Resource Q Amersham Pharmacia AX (Q) PSDVB 15 Biotech (Piscataway, N J) a Polystyrene divinylbenzene.
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`[12] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 209 Analytical AX Chromatography. Analytical AX HPLC is used to quan- tify partial phosphodiester oligonucleotide impurities in the phosphorothio- ate AO used in this work. In such phosphorothioate oligonucleotides, each backbone phosphorous carries a nonbridging sulfur substituent. Synthesis- related partial phosphodiester impurities have one or more backbone phos- phorous atoms substituted only by oxygen. These impurities are commonly denoted as (P=O)1, (P=O)2, and so on, where the subscript gives the number of phosphate diesters present in the backbone. A strong anion- exchange (SAX) column, POROS HQ/H (4.6 mm i.d. × 100 mm length) is used for most analyses. Preparative chromatography fractions are loaded directly onto the analytical column without a sample preparation step. The buffer system used is buffer A (20 mM NaOH) and buffer B (2.5 M NaC1 in buffer A). A linear gradient from 0 to 100% B over 20 min is used at 70 °. The Resource Q 1-ml column is used for some SAX analyses, using a similar elution profile. Other detailed protocols for analytical SAX HPLC of synthetic oligonucleotides are given by Srivatsa et aL29 and Thayer et aL 14 Quantitative Capillary Gel Electrophoresis (QCGE). QCGE is the most effective separation-based method for determining length-variant impuri- ties in sequentially assembled synthetic oligonucleotides, such as (n-l), (n- 2), and so on deletions, where n-mer is the desired full-length product. The QCGE method has been discussed in detail by Srivatsa et aL 3° A P/ACE 5000 (Beckman Coulter, Fullerton, CA) instrument is used for QCGE analysis. A gel-filled eCap ssDNA 100 capillary (Beckman Coulter) with a separation length of 40 cm is used at 40 °. A 100 mM Tris-borate/7 M urea electrolyte is used as supplied by the vendor. The sample is injected electrokinetically at 10 kV for 5 to 20 sec, followed by separation at 14.1 kV (300 V/cm). Samples for QCGE are prepared according to the following procedure. Fractions from AX eluates are desalted using Centricon (Amicon/Milli- pore) microcentrifuge cartridge 3SR modules. These cartridges have a mo- lecular weight cutoff of 3000. Of the sample to be desalted, 1.5 ml is put in the holder and deionized water is added until the level reaches the fill- up mark (about 3 ml). This is centrifuged at 6000 rpm for 60 min. The permeate is then discarded and the retentate is again refilled with water to the fill-up mark. This is repeated three times. After the final spin, the permeate is discarded and the cartridge is inverted. It is centrifuged again at 300 rpm for 5 min to push the retentate into a collection vial. Finally, 29 G. S. Srivatsa, P. Klopchin, M. Batt, M. Feldman, R. H. Carlson, and D. L. Cole, J. Pharm. Biomed. AnaL 16, 619 (1997). 30 G. S. Srivatsa, M. Batt, J. Schuette, R. Carlson, J. Fitchett, C. Lee, and D. L. Cole, Z Chromatogr. A 680, 469 (1994).
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`210 GENERAL METHODS [ 121 the desalted solution is diluted to a concentration of 0.20D/ml and loaded on the QCGE autosampler. Purification of Crude DMT-On Phosphorothioate Oligodeoxyribonucleotide AO Phosphorothioate AO synthesized by the phosphoramidite approach on solid supports are usually cleaved from support with the DMT-protecting group from the final synthon intact at the 5' terminus. The acid-labile, hydrophobic DMT group quite effectively permits the purification of syn- thetic oligonucleotides by preparative reversed-phase chromatography. RP-HPLC has thus become the most frequently used technique for purify- ing crude AO products. HIC and, to a lesser degree, AX can also make use of the DMT group as an aid in purifying the full-length product. RP-HPLC has been used for synthetic oligonucleotide purification over a wide range of scales. The reversed-phase separation of formerly capped failure sequences without the DMT function ("DMT-off") from the DMT- on product pool is straightforward. This procedure is convenient for small- scale syntheses 4 and has been shown very effective at a 100-g column loading scale for the purification of therapeutic antisense oligonucleotides. 6 A dis- tinct advantage of RP methods is that similar protocols may be used for a variety of structurally modified synthetic oligonucleotide crude products. The RP method used for the purification of DMT-on synthetic phosphodies- ter DNA oligomers, for example, is very similar to the method typically used for DMT-on O,O-linked phosphorothioate 2'-O-alkyI-RNA/DNA hybrid oligomers. The same method, with minor modifications, can be used for crude oligonucleotide products protected with other 5'-O-ether groups, such as MMT. Examples of such purification are discussed next, with pro- tocols. RP Purification of a 20-mer Phosphorothioate Oligodeoxyribonucleotide A O DMT-on crude ISIS 5132/CGP 69846A is purified on a polymeric RP column, Oligo R3 (Fig. 1), using buffer RP-I (Table I). A crude product solution (200 /zl) is diluted in 200 mM sodium acetate solution and is injected on the column. The separation gradient used is shown in Fig. 1. At low loading, such as used in this experiment, two distinct peaks are seen. The first two are failure sequence peaks containing oligomers without the DMT group and the third peak is the DMT-on oligomer-containing peak. Typically, benzamide generated by the deprotection of adenine or cytosine base is seen as a distinct peak eluting between the main peaks. 4
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`[ 1 9.] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 211 E 3.0- 2.5- 2.0 1.5 1.0 0.5 o.o.- t'~ o --I gradient (%B) e / 5 10 15 20 25 30 Time, mln 0.6 -0.5 0.4 O 3 0.3 0.2 -0.1 1 ~00 35 FIG. 1. RP-HPLC purification of DMT-on crude ISIS 5132/CGP 69846A (20-mer phospho- rothioate oligodeoxyribonucleotide). Peaks a, b, and c are failure sequences without the DMT group. Peak d is the benzamide peak. Peak e is the main product peak with DMT group and peaks f are failure sequences with DMT group. Sample: DMT-on crude ISIS 5132/CGP 69846A, 1 mg/ml, 4-ml injection; Column: Oligo R3 (PE BioSystems), 10 mm i.d. × 100 mm length, column volume (CV) = 7.85 ml. Buffer A: 50 mM NaOH, Buffer B: Methanol. Flow rate: 10 ml/min. Gradient program: Equilibration: 93% A + 7% B, for 4 CV, Load 4 ml sample, Wash* 97% A + 3% B for 20 CV, Elute: 20% B to 70% B in 17 CV, Hold 2 CV. (*The long wash used for this example is not necessary, 4 CV wash is sufficient).
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`212 GENERAL METHODS [ 121 In Fig. 1, absorption is monitored at 266 and 290 nm. The absorbance ratio is quite distinct for benzamide, allowing its distinction from the 1 : 3 absorbance ratio in oligonucleotide-containing peaks. It should be noted that even in the absence of separation of the benzamide peak from the DMT-off oligomer peak, good separation of the DMT-on peak from the DMT-off peak is readily achieved. The purity of a broad DMT-on heart cut is typically between 93 and 97% full-length by QCGE area-% analysis. Even higher purity material may be obtained by narrower fractionation. At higher loadings (>20 mg/ml CV), the chromatogram is more complex, but good purification is still possible with careful subfractionation of the DMT-on peak. Silica-based C18 reversed-phase columns give good capacity and excel- lent selectivity for these crude synthetic oligonucleotide separations. The only disadvantage is that the eluent pH must be near neutrality for column stability so high pH cannot be used to denature occasionally significant secondary or tertiary structures to minimize chromatographic band widths. There are a large number of protocols for the elution of silica-based RP columns with near-neutral buffers. 4'7,31 Three commonly used buffers for RP-HPLC of AO include two volatile buffer systems: (1) 100 mM TEAA/ ACN and (2) 100 mM ammonium acetate/methanol. A typical nonvolatile buffer system is (3) 200 mM sodium acetate/methanol. At large scale, sodium acetate buffer systems provide a highly useful combination of selec- tivity and capacity. In addition, this buffer system yields the sodium salt of the product oligonucleotide. Sodium is a preferred countercation for therapeutic antisense oligonucleotides, for physiological reasons, and gives products of manageable hygroscopicity and excellent photostability. For mass spectrometric characterization of product oligomers, volatile buffers may yield more useful salts. 32'33 Other polymeric columns with surface chemistry similar to that of the Oligo R3 column include PRP-1 (Hamilton, Reno, NV) and Source 30 RPC (Amersham Pharmacia Biotech, Piscataway, N J). These may be used under appropriately but slightly modified elution conditions and loading capacities. RP-HPLC with On-Column Detritylation A key feature of polymeric RP stationary phases is that eluent buffers may have extreme pH, as in the previous example wherein a high pH buffer 31 L. W. McLaughlin and N. Piel, in "Oligonucleotide Synthesis: A Practical Approach" (M. J. Gait, ed.), p. 117. Oxford Univ. Press, New York, 1990. 32 A. Apffel, J. A. Chakel, S. Fisher, K. Lichtenwalter, and W. S. Hancock, Anal. Chem. 69, 1320 (1997). 33 L. L. Cummins, M. Winniman, and H. J. Gaus, Bioorg. Med. Chem. Lett. 7, 1225 (1997).
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`[12] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 213 was used to eliminate reversible secondary structure contributions to band width to improve resolution. Similarly, polymeric RP columns can tolerate contact with strongly acidic solutions for on-column product detritylation preparatory to anion-exchange LC purification. Removal of DMT postpuri- fication requires acid treatment, cold ethanol precipitation, and physical isolation, e.g., by centrifugation. Little net processing time is typically saved, and final product purities are no greater than by reversed-phase DMT-on purification, but any potential low-level contamination by DMT-on material in detritylated product can be avoided. An example of such an alternative protocol is shown in Fig. 2. In this strategy, the DMT-off failure peak is first eluted while retaining the DMT-on peak on column. Absorbed material is then exposed to a dilute TFA solution for a well-controlled contact time, converting most absorbed oligomer to DMT-off product, which is then recovered by continued organic elution. In typical cases, 1-2% (v/v) trifluo- roacetic acid (TFA) in water is satisfactory, with contact times of 5 to 10 min. This approach can reduce depurination to levels detectable, if at all, only by mass spectrometry. An advantage is that the procedure can be automated at small scale on a programmable chromatographic workstation. We have scaled up the procedure for purification of up to 10 g of crude oligonucleotide per run, but sample loading must be reduced to maintain robust separation on larger columns. Gram Scale (1-10 g) Purification of Phosphorothioates on RP-HPLC Phosphorothioate DNA oligonucleotide ISIS 2105 DMT-on crude prod- uct was purified on a 600-ml Oligo R3 column (50.4 mm i.d. x 300 mm length) in a sodium acetate/methanol buffer system. The chromatogram is shown in Fig. 3. The elution profile is very similar to that seen at smaller scales. In this case, 4.5 g of crude oligonucleotide is loaded on the column at 7.5 mg/ml of CV. Purification of the full-length product is good for this relatively heavy sample loading, with the .benzamide peak clearly evident. Because of the high load, however, the DMT-on peak is split into multiple apparent peaks. These are analyzed by analytical SAX HPLC to determine pooling strategy. The selected product pool contains approximately 1.6 g of oligonucleotide at >95 area-% DMT-on purity. Production Scale RP-HPLC Purification of 10-100 g Oligonucleotide Methodology used under GMP conditions in our facility for 100-g pre- parative HPLC runs is similar to the method described in the previous section, except that gradient conditions are optimized to maximize sample loading and product purity. Sample loading and flow rates are linearly scaled from small column conditions. To ensure comparable physical bed
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`214 GENERAL METHODS [ 12] 2.0 E pH 1.5- 1.0- o.5. gradient (%B) a b O.OL • . -. .................. o 5 1'0 1'5 I t ~J J-- acid --J ii d !i 3'5 3'0 3'5 10 -r FIG. 2. On-column detritylation of 20-mer phosphorothioate AO. Peak a is the failure peak and peak b is the benzamide peak. Both these peaks elute before acid is introduced. Peaks c and d elute after acid treatment. Peak c is the detritylated product peak and peak d is the undetritylated portion of the DMT-on starting material. The solid line indicates the %B gradient profile and the dashed line indicates the pH profile. Sample: DMT-on ISIS 5132/ CGP 69846A, 1 mg/ml, 4 ml injection; Column: Oligo R3 (PE BioSystems), 10 mm i.d. × 100 mm length, column volume (CV) = 7.85 ml. Buffer A: 50 mM NaOH, Buffer B: Methanol, and Buffer C: 2% TFA in water. Flow rate: 10 ml/min. Gradient program: Equilibration: 93% A + 7% B, for 4 CV, Load 4 ml sample, Elution 1: 7% B to 35% B in 10 CV, wash: 3% B for 2 CV, Detritylation: 100% C for 5 min (flow rate 1 ml/min for last 4 min), neutralization: wash 3% B for 5 CV, Elution 2: 20% B to 70% B in 17 CV, hold 2 CV.
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`[12] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 215 5.0 ¸ 25 "/5 / H ~H iH ~H 5O Time, mln Fro. 3. RP purification of phosphorothioate AO at gram scale. The elution was monitored at 290 nm (thick trace) and 265 nm (thin trace). Vertical lines indicate the manual fraction points, p indicates the pooling for the product. Sample: DMT-on crude ISIS 2105 4.5 g injec- tion; Column: Oligo R3 (PE BioSystems), 50.4 mm i.d. × 600 mm length, column volume (CV) = 600 ml. Buffer A: 2.5 M sodium acetate, pH 7.2, Buffer B: Methanol, and Buffer C: DI water. Flow rate: 50 ml/min. Elution 20 to 30% in 0.5 CV, 30 to 70% in CV, wash 90% B in 2 CV. 4% A was maintained through the process, diluted with Buffer C. integrity among small-scale and large-scale columns, radial compression columns are used. The KP 100 (Biotage Division, Dyax Corp., Cambridge, MA) high-pressure automated chromatography equipment is used. The radial compression columns are prepacked with BondaPak HC18HA silica- based media (Waters Corp.). Figure 4 shows an example of RP purification of a 35-g crude 21-mer phosphorothioate oligonucleotide. Note that separa- tion of DMT-on and DMT-off peaks is excellent and not compromised when a large column is used. Product purity and yield are therefore high and similar to small-column performances. A further increase in column scale successfully allows 100-g scale purifications per run.
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`3. 2,5- 15- I-- C --I 8 sY _j 216 GENERAL METHODS [ 12] [-- d --I Time, mln FIG. 4. Production scale (10-100 g) RP-HPLC purification of phosphorothioate AO. The x axis indicates the absorbance in arbitrary units. Peaks a, b indicate DMT-off failure containing peaks, c denotes the product cut and d indicates DMT-on containing failures. Sample: DMT-on crude 21-mer phosphorothioate; Column: Radial compression module, containing BondaPak HClsHA media (Waters Corp.), buffer system same as in Fig. 3. Use of AX Chromatography for Purification of D MT-On Oligonucleotides The analytical HPLC trace of DMT-on oligonucleotides (Fig. 5) shows that in this chromatographic mode the DMT-on product peak is well sepa- rated from the DMT-off failure sequences peak. It is therefore feasible to isolate DMT-on material from crude product mixtures by this method. However, the affinity of the DMT-on peak for anion exchanger media is very high, with up to 3 M salt being reported necessary for the complete elution of DMT-on material, 34 so greater care is needed to clean and regen- erate the column after each separation. It is also possible to load the entire DMT-on peak onto the anion- exchange resin and detritylate the material-on column. For this procedure, 34 H. J. Johansson and M. A. Svensson, "Nucleic Acid-Based Therapeutics." IBC Conference, San Diego, 1995.
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`[ 12] PURIFICATION OF ANTISENSE OLIGONUCLEOTIDES 217 ~..00-- 0.90- 0,80. 0.70- O. 60- ~o.5o- 0.40- 0,30- 0.20- O, 10- 0.00- -- 0.00 <~ DMT-off peaks ~> [ ~ , L i [ lo.oo 20.00 Time, min DMT-on peak i 30!00 i i FIG. 5. SAN analysis of DMT-on crude phosphorothioate AO. Sample: DMT-on ISIS 2105. Column: POROS HQH (PE BioSystems), 4.6 × 100 mm length. Buffer A: 20 mM NaOH, Buffer B: 2.5 M NaC1 in A. Flow rate: 1 ml/min. Temperature: 70 °. Gradient program: Elution 0-100% B in 20 min. the DMT-on peak is first isolated from a reversed-phase column, then loaded directly to an anion-exchange column. The bound material is detrity- lated by a procedure similar to that in Fig. 2. While removal of DMT- cation and residual DMT-on material is difficult, the method can be used for reducing the partial phosphodiester content of synthetic phosphorothioate oligonucleotides. Purification of DMT-On Phosphorothioates on Hydrophobic Interaction Media Hydrophobic interaction chromatography (HIC) offers another way to purify crude DMT-on phosphorothioate oligonucleotides. 8'35 HIC has the advantage that the ammoniacal oligonucleotide cleavage solution can be loaded directly to the column and large organic solvent volumes are unnec- 35 Z. Zhang and J.-Y. Tang, Curr. Opin. Drug Disc. Dev. 1, 304 (1998).
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`218 GENERAL METHODS [ 121 essary. The method has been used at a 100-g scale, but because considerable effort is necessary to optimize the method for each sequence, it is most practical for large-scale use. HIC purification product pooling usually fol- lows protocols similar to those used for RP-purified oligonucleotides. Purification of Crude DMT-Off Phosphorothioate Oligodeoxyribonucleotide AO Starting the purification process with DMT-off crude oligonucleotide has the advantage that detritylation need not be carried out after purifica- tion. At large scale, detritylation and subsequent precipitation are more difficult than at small scales. As the examples in this section indicate, high purity can be obtained without recourse to the DMT group to facilitate hydrophobic purification. Anion-exchange (AX) chromatography is conve- nient for the purification of DMT-off crude products. RP does have applica- tion for the purification of DMT-off crude products, but has been mainly useful for short oligonucleotides. For this reason, only AX chromatography is discussed in this section for the purification of DMT-off oligomers. At larger scales, AX processes avoid the use of large quantities of organic solvent, can use widely available low-pressure chromatography equipment, and can give product yields at least equivalent to those from RP chromatog- raphy of DMT-on oligomers. AX can also be used as an orthogonal purification step following the RP-HPLC purification of DMT-on AO crudes. The product pool post-RP purification or post-HIC purification is detritylated and precipitated and the oligonucleotide is then redissolved in water and purified by AX HPLC. This method increases purity of the RP purified at the expense of full- length oligom