High-Throughput Oligonucleotide Analysis on a Small Particle Pellicular Anion-Exchanger

Transcription

1 High-Throughput Oligonucleotide Analysis on a Small Particle Pellicular Anion-Exchanger J.R. Thayer*, C.A. Pohl, D. Jamieson, and X. Liu; Thermo Fisher Scientific, Sunnyvale, CA, USA

2 Overview Purpose: Demonstrate an oligonucleotide separation column capable of increased throughput with comparable resolution, and improved resolution with comparable throughput, to the industry-leading Thermo Scientific DNAPac PA2 pellicular anion exchanger. Methods: Pack a 4 µm diameter resin in SST-clad PEEK hardware, and apply a stationary phase (nanobead) harboring DNAPac PA2 chemistry. Compare columns prepared with the 4µm substrate resin to columns prepared with the 8 µm substrate to verify improved performance. Results: The new RS (Rapid Separation) column is available in 4.6 x 5 mm, 4.6 x 5 mm and 4.6 x 25 mm formats. The 4.6 x 25 mm RS (4 µ) format delivers better oligonucleotide resolution using gradient times comparable to the 25 mm (8 µ) DNAPac PA2. The 4.6 x 5 mm RS format delivers resolution comparable to the 4. x 25 mm (8 µ) DNAPac PA2, but with shorter elution times (improved throughput) and the 4.6 x 5 mm RS format provides very fast oligonucleotide separations (very high throughput) where the highest resolution, or resolution of isomers, is not needed. Introduction Pellicular Anion-Exchange chromatography has provided industry-leading oligonucleotide (ON) resolution for over 2 years. Its introduction in the early 99s then provided new options for control of ON selectivity, allowing facile separations of closely-related sequences, even of the same lengths. In 24, introduction of the DNAPac PA2 improved ON resolution, promoted improved throughput, and also increased column longevity, especially at high ph and temperatures []. We describe the DNAPac PA2 RS (Rapid Separation) column, further improving resolution where the very highest ON resolution is required [2 4], or throughput; where good resolution has been achieved, but improved throughput is needed. For separations where high ON resolution is readily achieved, a 4.6 x 5 mm column allows even higher throughput separations. Methods Sample Preparation Oligonucleotide samples were acquired from Midland Certified Reagent Company, Inc. (dt 2-6, Midland, TX) or Integrated DNA Technologies Inc. (all others, Coralville, IA). Dry ONs were suspended in deionized (DI) water, typically to.5 or 6 mg/ml, and diluted in DI water as needed. Liquid Chromatography Separations were performed on a Thermo Scientific Dionex UltiMate 3 BioRS chromatograph, consisting of Biocompatible LPG-34RS pump, Biocompatible WPS- 3RS split-loop sampler, TCC-3RS thermal compartment and DAD3RS diode-array detector. Data Analysis Chromatographic system control, data acquisition and peak integration employed Thermo Scientific Dionex Chromeleon software version High-Throughput Oligonucleotide Analysis on a Small Particle Pellicular Anion-Exchanger

3 Table. Oligonucleotides used (* indicates 2,5 -linkage, S indicates PS linkage) RNA Dio-: 5 -AUG AAC UUC AGG GUC AGC UUG -3 Dio-6: 5 -AUG AAC UUC A*G*G GUC AGC UUG -3 Dio-9: 5 -AUG AAC UUC AGG GUC* AGC UUG -3 FP-S: 5 -AGC UGA s CCC UGA AGU UCA UdCdT -3 DNA dt 2-6 : 5 - (TTT TTT TTT TTT) -5 ON34: 5 -TAG GTT CTC TAA CGC TGA CTG ATT GTA GGT GTT C- 3 ON35: 5 -GTA GGT TCT CTA ACG CTG ACT GAT TGT AGG TTC TC- 3 ON44: 5 -TAC TGA TTG TAG GTT CTC TAA CGC TGA CTG ATT GTA GGT TCT C - 3 ON45: 5 -CTG ACT GAT TGT AGG TTC TCT AAC GCT GAC TGA TTG TAG GTT CTC - 3 ON54: 5 -TCT GTA ACG CTG ACT GAT TGT AGG TTC TCT AAC GCT GAC TGA TTG TAG GTT CTC - 3 ON55: 5 -TTC TGT AAC GCT GAC TGA TTG TAG GTT CTC TAA CGC TGA CTG ATT GTA GGT TCT C - 3 Results I. Applications Performance Our goal is to offer a column that: i) produces resolution better than the DNAPac PA2 (8 µ), and with significantly higher throughput, and ii) delivers considerably better resolution in a time frame comparable to the standard DNAPac columns. Our tests compare gradient column performance between a 4. x 25 mm DNAPac PA2 and DNAPac PA2 RS 4.6 mm ID columns in 5, 5 and 25 mm lengths. A: Length-based Separations Most ON separations attempt to resolve related sequences on the basis of length, and the DNAPac performs this function well. DNAPac PA2 RS (4.6x5mm) 55 ma DNAPac PA2 (4.x25mm) 3 ma 26 ON34 ON35 ON34 ON35 ON44 ON45 ON44 ON45 ON54 ON55 ON54 ON55 Conditions: Column: DNAPac PA2 RS (4.6 x 5 mm, Top) DNAPac PA2 (4. x 25 mm, Bottom) Eluents: A: 4mM Tris, ph 8 B: A =.25M NaCl Samples: ON34, ON35, ON44, ON45, ON54, ON55 Detection by absorbance at 26nm Temperature at 3 C Gradients: Top (4.6x5mm): Time %A %B Flow (ml/min) Bottom (4. x 25 mm) FIGURE. Length-based Oligonucleotide resolution. Oligonucleotides composed of 34, 35, 44, 45, 54 and 55 bases were injected and eluted as described. Linear velocity was.2mm/sec. In this comparison, the DNAPac PA2 RS column provides comparable resolution for all three pairs of ONs in less than 75% of the time required for the standard DNAPac PA2. Due to the smaller particle size, the RS column also delivers better detection sensitivity. Thermo Scientific Poster Note PN2949_HPLC_24_E_5/4S 3

4 2) Phosphorothioate The DNAPac colum FIGURE 4. Phosph diastereoisomers of in three lengths; 5.5 min, the 5 m completed the sepa increased throughput oughput, to the xchanger. apply a stationary ns prepared with the fy improved Comparison of c mm, 4.6 x 5 mm etter oligonucleotide c PA2. The mm (8 µ) DNAPac x 5 mm RS format ere the highest 55 ma26 g oligonucleotide (ON) ded new options for quences, even of the resolution, promoted high ph and 6 33 Conditions and eluents as in Figure Each of these 4. phosphorothioat format generate the power of the Column rugge There are few h exhibit degradat PA2 RS opera in Table 2 DNAP Gradient (4.6 x 5 mm) 37 PS linkage) Inj # 5 Figure 2B: Resolution of deoxythymidine oligonucleotides on the DNAPac PA2 RS (4 µ), using a curved gradient (Curve 3). Sample: dt 2-6. In Figure 2A (above), the DNAPac PA2 (8 µ) 4. x 25 mm column partially resolves 57 ONs by length, and 2 ON variants eluting between the major peaks, in less than 5 minutes using a curved gradient optimized for the separation. C - 3 CTC - 3 TTG TAG GTT CTC - 3 ATT GTA GGT TCT C - 3 DNAPac PA2 RS Flow:.2 ml/min 3 BioRS mpatible WPSD3RS diode-array mployed Thermo DNAPac PA2 RS Flow:.2 ml/min 25 Absorbance ma26 Company, Inc. (dt2e, IA). Dry ONs were d in DI water as Flow:.2 ml/min improving resolution ere good resolution where high ON throughput 22 DNAPac PA2 RS In contrast, the DNAPac PA2 RS column (Figure 2B) resolves 58 ONs by length and 26 ON variants eluting between the major peaks, in less than minutes, using a similarly optimized gradient. These examples demonstrate improved performance for length-based separations and improved resolution of identical length variants by the new, small-particle DNAPac PA2 RS column in 4.6 x 5 mm format. B: Resolution of Oligonucleotide Isomers ) Linkage isomers Annealing of single stranded RNAi sequences into duplexes is known to generate 2,5 linkages, and phosphorothioate linkages generate diastereoisomers [2,3]. Resolution of these forms is often for Therapeutic ON developments. Analysis on a critical Small Particle Pellicular Anion-Exchanger 4 High-Throughput Oligonucleotide Sam Na Ave Rel.S Table 2 reveals R respectively. We o Conclusion We confirm the DN In 5 mm f

5 B: Resolution of Oligonucleotide Isomers ) Linkage isomers Annealing of single stranded RNAi sequences into duplexes is known to generate 2,5 linkages, and phosphorothioate linkages generate diastereoisomers [2,3]. Resolution of these forms is often critical for Therapeutic ON developments. DNAPac PA2 RS (4.6 x 25 mm) Flow:.3 ml/min Dio- [-] 8. Dio-6 [/] Dio-9 [5] ma M NaCl: 28. % DNAPac PA2 (4. x 25 mm) 5 5 Dio- [-] Flow:.85 ml/min Dio-6 [/] Dio-9 [5] 8. ma M NaCl: 28. % 5 5 FIGURE 3. Linkage isomer separations. Three 2-base identical sequence ONs with, or without 2,5 -linkages in specific positions within the sequence were chromatographed with a gradient of mm NaCl over 4 column volumes in Tris buffered eluent at ph 8 and 3 C. Top Panel: DNAPac PA2 RS 4.6 x 25 mm. Bottom Panel: Standard DNAPac PA2 4. x 25 mm. Pressure for RS column: 88 psi (6 bar) The 4.6 x 25 mm DNAPac PA2 RS, at a faster relative flow rate, demonstrates better peak width and resolution at slightly higher throughput even with identical column lengths. In this case the throughput for the RS column is ~% higher than for the DNAPac PA2. Thermo Scientific Poster Note PN2949_HPLC_24_E_5/4S 5

6 2) Phosphorothioate diastereoisomers The DNAPac columns can resolve phosphorothioate linkage diastereoisomers [4]. FIGURE 4. Phosphorothioate diastereoisomer separations. Separation of the diastereoisomers of a 2-base ON (FP-S) chromatographed on the DNAPac PA2 RS in three lengths; 5, 5 and 25 mm. The 25 mm column completed the separation in.5 min, the 5 mm column resolved the isomers within 7.5 min and the 5 mm column completed the separation in 2.6 minutes. Conditions as in Figure 3 (25 mm long column). Comparison of column length. 4.6 x -5, -5, -25 mm 55 DNAPac PA2 RS: 4.6 x 5 mm FP-A Flow:.2 ml/min FP-B 2 3 ma DNAPac PA2 RS 4.6 x 5 mm FP-A Flow:.2 ml/min FP-B DNAPac PA2 RS 4.6 x 25 mm FP-A Flow:.2 ml/min FP-B 5 5 Each of these 4.6 mm ID columns resolved the two diastereoisomers arising from the phosphorothioate linkage at position 6 in the FP-S sequence. Even the 5 mm long format generated a resolution > 4 for these isomers, and does so in < 3 minutes, showing the power of the pellicular anion-exchange approach. 3. Column ruggedness There are few high performance columns designed for oligonucleotides, and most of those exhibit degradation within a relatively low number of sample injections. The DNAPac PA2 RS operates at elevated pressures (up to, psi, (~7 bar) so we demonstrate in Table 2 DNAPac PA2 RS column longevity using the diastereoisomer separation. FP-A FP-B Inj # Sample Ret.Time Asym(EP) Ret.Time Asym(EP) Name min min 2 FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S FP-S Average: Rel.Std.Dev:.4%.79%.2%.98% Table 2 reveals RSD values for retention time and asymmetry of <.5 % and. %, respectively. We observe little, if any, column degradation over the course of this test. 6 High-Throughput Oligonucleotide Analysis on a Small Particle Pellicular Anion-Exchanger

7 Conclusions We confirm the DNAPac PA2 RS column family meets the following criteria: In 5 mm format: Greater throughput, and efficiency as well as resolution exceeding the standard 4 x 25 mm DNAPac PA2 (Figures and 2). In 25 mm format: Better throughput and much better resolution than the standard 4 x 25 mm DNAPac PA2 (Figure 3). Simple resolution of n from n- length ONs is readily demonstrated, even for fairly long sequences in less than minutes (cf. Figures 2A and 2B). Difficult ON separations, such as resolution of linkage and diastereoisomers, are demonstrated (Figures 4 and 5), even with formats only 5 mm in lengths (Figure 4). The small-particle column exhibits very good ruggedness, maintaining very small peak widths for over 35 injections. References. Thayer, J.R., V. Barreto, S. Rao, and C.A. Pohl. 25. Control of oligonucleotide retention on a ph-stabilized strong anion exchange column. Analytical Biochemistry 338: Thayer, J.R., S. Rao, N. Puri, C.A. Burnett, and M. Young. 27. Identification of aberrant 2-5 RNA linkage isomers by pellicular anion exchange chromatography. Analytical Biochemistry 36: Thayer. J.R., N. Puri, C. Burnett, M. Hail and S. Rao. 2. Identification of RNA linkage isomers by anion exchange purification with electrospray ionization mass spectrometry of automatically desalted phosphodiesterase-ii digests. Analytical Biochemistry 399: Thayer, J.R., Y. Wu, E. Hansen, M.D. Angelino, and S. Rao. 2. Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography. Journal of Chromatography A: 28: Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa Australia Austria Belgium Brazil Canada China (free call domestic) Denmark Europe-Other Finland France Germany India Italy Japan Korea Latin America Middle East Netherlands New Zealand Norway Thermo Fisher Scientific, Sunnyvale, CA USA is ISO 9:28 Certified. Russia/CIS Singapore Sweden Switzerland Taiwan UK/Ireland USA PN2949_E 5/4S