[ method guidelines ] Method Guidelines. *OST: Oligonucleotide Separation Technology. Contents I. PRINCIPLES OF OLIGONUCLEOTIDE SEPARATIONS

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1 Method Guidelines *OST: Oligonucleotide Separation Technology columns are based on Waters second generation of hybridsilica BEH Technology particles and can be effectively used for the lab scale purification and analysis of detritylated synthetic oligonucleotides using ionpair, reversedphase chromatography. This document provides useful method guidelines for the effective use of this column chemistry for this group of compounds. Contents I. PRINCIPLES OF OLIGONUCLEOTIDE SEPARATIONS II. III. IV. SAMPLE PREPARATION RECOMMENDED MOBILE PHASES RECOMMENDED INJECTOR WASH SOLVENT V. GENERAL CONSIDERATIONS IN DEVELOPING SEPARATIONS VI. ANALYSIS OF MODIFIED OLIGONUCLEOTIDES VII. PURIFICATION CONSIDERATIONS

2 I. P RINCIPLES OF OLIGONUCLEOTIDE SEPARATIONS Separations of detritylated synthetic oligonucleotides on an XBridge OST C 18 column are based on ionpair, reversedphase chromatographic principles (IPRPLC). As shown in Figure 1, the ionpairing additive in the mobile phase is adsorbed on a hydrophobic sorbent and provides for chargetocharge interactions with negative charges contained on the oligonucleotide backbone (e.g., phosphate groups). Figure 2: Separation of a 15 60mer Deoxythymidine Ladder on Figure 1: Proposed Mechanism of IPRPLC for Synthetic Oligonucleotide Separations TEA As a result, an efficient chargebased (lengthbased) oligonucleotide separation is achieved (Figure 2). Gradient elution using an acetonitrile or methanol eluent displaces both ionpairing agent and the oligonucleotides from the sorbent surface. Separation selectivity and resolution decreases with increasing oligonucleotide length (Figure 2) making the separation of long oligonucleotides challenging. Modified oligonucleotides such as phosphorothioates and 2O alkyl modified species are also more difficult to analyze. Special mobile phase may be required (see Section III, Recommended Mobile Phases). chain PO group on Oligo chain + Sample Injected: Approximately 100 pmoles of a detritylated 15 60mer oligonucleotide ladder diluted in 0.1 M TEAA Waters, 2.5 µm (2.1 x 50 mm) Mobile Phases: A: 0.1 M TEAA, B: Acetonitrile / 0.1M TEAA, 20/80, v/v 0.2 ml/min Column Temp.: Gradient delay: 0.45 ml Gradient: 40 to 62.5% B in 30 minutes (812.5% acetonitrile, 0.15% acetonitrile per minute) 260 nm, 5 scans per second Two commonly used ionpairing agents for oligonucleotide applications are triethyl ammonium and dimethylbutyl ammonium ions. The final ph of these mobile phases containing either of these ionpairing reagents is adjusted by the addition of Acetic Acid, or in some cases, Hexafluoroisopropanol (HFIP). These mobile phases are volatile making them suitable for LCMS applications. The ability to adequately resolve synthetic oligonucleotide mixtures by ionpair, reversedphase chromatography is significantly affected by the particle size of the material contained in an efficiently packed column (see Figure 3). Consequently, columns are efficiently packed with 2.5 micron material to maximize detritylated oligonucleotide component resolution. In order to improve oligonucleotide separation efficiency and speed, elevated separation temperature (e.g. ) is recommended. Elevated temperature will also reduce operating LC System back pressure. 2

3 Figure 3: Effectiveness of Waters BEH Technology HybridSilica C 18 Particle Size on Deoxythymidine Ladder Separations Figure 4: Impact of Ionpairing System on Separation of a 1030mer Heterooligonucleotide Ladder Sample Injected: Mobile Phases: Column Temp.: Gradient delay: Gradient: Approximately 100 pmoles of detritylated 15 60mer crude oligonucleotide ladder diluted in 0.1 M TEAA Waters BEH HybridSilica C 18 particles (2.1 x 50 mm) A: 0.1 M TEAA, B: Acetonitrile / 0.1M TEAA, 20/80, v/v 0.2 ml/min 0.45 ml 40 to 62.5% B in 30 minutes (812.5% acetonitrile, 0.15% acetonitrile per minute) 260 nm, 5 scans per second Sample: 3 exonuclease Mobile phases: Column Temp.: Gradient delay: 20 mer: TCC CTA GCG TTG AAT TGT CC 25 mer: TCC CTA GCG TTG AAT TGT CCC TTA G 30 mer: TCC CTA GCG TTG AAT TGT CCC TTA GCG GGT Ladder was prepared by hydrolyzing detritylated 20, 25, and 30mer oligonucleotides with a Waters, 2.5 µm (4.6 x 50 mm) Upper chromatogram: 0.1 M TEAA with acetonitrile gradient; Lower chromatogram: 16.3 mm TEA 400 mm HFIP with methanol gradient 1.0 ml/min 0.45 ml 260 nm, 5 scans per second In addition to ionpairing, a hydrophobic reversedphase mechanism also takes place in the oligonucleotide separation. The residual interaction of nucleobases has an impact on overall retention and separation selectivity, especially when using Triethylammonium Acetate (TEAA) ionpairing mobile phases. Separation of N and N1mers may be either enhanced or suppressed by the sequence contribution. More potent ionpairing systems such as Triethylammonium ion with Hexafluoroisopropanol counter ion provide for more regular chargebased separations (Figure 4). II. SAMPLE PREPARATION 1. Dissolve the detritylated synthetic oligonucleotide sample in Mobile Phase A (e.g., 0.1 M TEAA). For example, a µmole scale synthesis can be prepared in 0.1 ml of 0.1 M TEAA. Proportionately larger or smaller volumes of 0.1M TEAA are required when dissolving samples from different scale syntheses. Due to the nature of gradient separations, relatively large volumes of sample (in low organic strength eluent) can be injected and concentrated onto the head of the column before beginning the gradient elution program. 2. Samples must be completely in solution and free of particulates before injecting onto the column. Remove all particles from the sample (Controlled Pore Glass Synthesis Support, etc.), which may block the inlet column frit, increase the operating pressure, and shorten the column life time. Sample contamination with high concentration of salts and/or detergents may also interfere with analysis. 3

4 3. To remove particulates the sample may be filtered with a 0.2 μm membrane. Be sure that the selected membrane is compatible and does not dissolve with the selected Mobile Phase diluent. Contact the membrane manufacturer with solvent compatibility questions. An alternative method of particulate removal involves centrifugation for 20 minutes at 8,000 rpm, followed by the transfer of the supernatant liquid to an appropriate vial. III. RECOMMENDED MOBILE PHASES The most common ionpair mobile phase for synthetic oligonucleotide separations is based on Triethylammonium Acetate (TEAA). This mobile phase can be prepared by titrating Glacial Acetic Acid aqueous solution with Triethylamine (TEA). Note: To maximize column life, it is ESSENTIAL that all prepared OST Mobile Phases be filtered through a solvent compatible, 0.45 µm membrane and contained in bottles that are clean and particulate free. TEAA 1L of 0.1 M TEAA may be prepared as follows: 1) Perform work in a hood. 2) Add 5.6 ml of glacial Acetic Acid into 950 ml of water and mix well. 3) Slowly add ml of TEA. 4) The ph should be adjusted to ph 7 +/ 0.5 by careful addition of Acetic Acid. 5) Adjust final volume to 1 L with water. Alternatively, premixed TEAA can be used [(e.g., Sigma 1 M TEAA (part no )]. Mix 100 ml with 900 ml of water to prepare 1 L of 0.1 M TEAA mobile phase. Alternative ionpairing reagents are recommended for improved separation of phosphorothioates or when performing LCMS analyses. An ionpairing mobile phase based on Triethylamine (TEA) and Hexafluoroisopropanol (HFIP) as the buffering acid produces an efficient eluent system for improved separations involving these application types. As indicated below, two ionpairing systems are useful. For routine detritylated oligonucleotide applications, aqueous buffer consisting of 8.6 mm TEA and 100 mm HFIP is effective. For applications such as those involving the separation of Grich oligonucleotides, it is advisable to use aqueous buffer consisting of 15 mm TEA and 400 mm HFIP (ph 7.9). TEAHFIP System 1 1L of 8.6 mm TEA / 100 mm HFIP is prepared as follows: 1) Perform work in a hood 2) Add 10.4 ml of HFIP (16.8 g) into g of water and mix well. 3) Slowly add 1.2 ml of TEA. 4) The ph is approximately 8.3 +/ 0.1. TEAHFIP System 2 1 L of 15 mm TEA / 400 mm HFIP is prepared as follows: 1) Perform work in a hood 2) Add ml (67.17 g) of HFIP into g of water and mix well. 3) Slowly add 2.08 ml (1.52 g) of TEA. 4) The ph of final buffer is approximately 7.9 +/ 0.1. IV. RECOMMENDED INJECTOR WASH SOLVENTS Between analyses, the HPLC system injector seals should be washed. A 90% Water / 10% Acetonitrile injector wash solvent is recommended. V. GENERAL CONSIDERATION IN DEVELOPING SEPARATIONS Separation of detritylated synthetic oligonucleotides by ionpair, reversedphase chromatography uses very shallow gradients. With both TEAA and TEAHFIP ionpairing systems, a rate of strong eluent change between % Acetonitrile (or Methanol) per minute is recommended. However, the formation of shallow gradients can place performance demands on LC pumps and mixers that can compromise the quality of the separation. Consequently, it is strongly advised that Mobile Phase B formulation contain a premix blend of aqueous and organic solvents (e.g., Mobile Phase A= 0.1 M TEAA and Mobile Phase B = Acetonitrile / 0.1M TEAA, 20/80, v/v) to minimize potentially inadequate solvent mixing that can compromise component resolution. 4

5 As illustrated in Figures 5 through 7, these analyses were performed with the following mobile phases: Mobile Phase A: 0.1 M TEAA Mobile Phase B: Acetonitrile (ACN) containing 0.1 M TEAA, 20:80 (v:v) The 0.1% ACN / min gradient change from an initial 5 to 10% Acetonitrile concentration over 50 minutes was programmed as specified in Table 1: Table 1 Time % A % B Actual Acetonitrile (ACN) Concentration 0 min % 50 min % Example: For the initial 5% Acetonitrile concentration: Initial %B = desired ACN % / Volume Fraction of ACN in Mobile Phase B. So, initial %B = 5% / 0.2 = 25% For the final 10% Acetonitrile concentration: Final %B = desired ACN % / Volume Fraction of ACN in Mobile Phase B. So, final %B = 10% / 0.2 = 50% With TEAA mobile phases, the unmodified oligonucleotides elute within a 710 % ACN gradient window. However, C and G rich oligonucleotide sequences are generally less retained (i.e., elute within a 58% ACN gradient window) than A and T rich sequences (i.e., elute within a 811% ACN gradient span). When using a shallow gradient, the total length of analysis for an unknown sample sequence may be excessive. Use of a fast scouting gradient with a 1% ACN per minute change is recommended in such cases. Information gathered from this scouting analysis can then be used to create a more appropriate and time efficient set of gradient conditions for the particular sample. Gradient slope has a direct impact on the achievable oligonucleotide component resolution (along with the type of ionpairing agent, sequence, and oligonucleotide modification). Steeper gradients (e.g., 1% ACN change per minute on a 4.6 x 50mm column at a 1.0 ml/min flow) are recommended for labeled oligonucleotides or for short, 515 mer sequences. Separation of longer sequences are typically performed using more shallow gradient slopes (e.g. 0.15% ACN change per minute on a 4.6 x 50mm column at a 1.0 ml/min flow). The organic solvent concentration at initial sample loading conditions has to be well chosen. If the initial organic solvent strength is too high, then some desired oligonucleotide sequences may be unretained. In the other extreme, when the gradient starts with too low an organic concentration, the analysis is excessively long without the benefit of enhanced component resolution. A suitable gradient separation method can be approximated from the oligonucleotide base (C, G, A, and T) composition. The initial gradient is typically adjusted while keeping the gradient slope constant. Table 2: Suggested Gradient Conditions for NonStandard Detritylated Synthetic Oligonucleotide Sequences Gradient 1 [Standard oligonucleotides (1)] Gradient 2 [High GC content or short oligonucleotides (2)] Gradient 3 [High AT content or long oligonucleotides (3)] Initial % ACN 7.00% 5.25% 7.50% Final % ACN 10.75% 9.00% 12.50% Gradient Length(4) 15 min 15min 20min 1: Standard oligonucleotides: 10 30mers 2: Short oligonucleotides: Less than 10mer 3: Long oligonucleotides: 30 60mers 4: Assuming use of a 2.1 x 50mm column at a flow of 0.2 ml/min and a separation temperature of. The retention of single and dual dyelabeled oligonucleotides is dictated by the nature of label. For example, the retention of 25 mer oligonucleotide increases according to the type of label attached as follows: no label<6fam< <TAMRA<TET<HEX<Cy3. VI. ANALYSIS OF MODIFIED OLIGONUCLEOTIDES columns are suitable for analysis of unmodified as well as modified detritylated oligodeoxyribonucleotides and oligoribonucleotides. Phosphorothioate and 2 O alkyl modified oligonucleotides can also be analyzed with IPRPHPLC method. However, these full length oligonucleotide products are usually more difficult to resolve from their shorter length failure sequences. The recommended ionpair system for phosphorothioate oligonucleotide analysis is TEAHFIP (see Recommended mobile phases). An example of a 25mer phosphorothioate oligonucleotide analysis is shown in Figure 5. 5

6 Figure 5: Analysis of a Digested 25mer Phosphorothioate Oligonucleotide Figure 6: Analysis of a 20mer Oligodeoxythymidine Crude Synthesis Mixture Sample: Mobile phase: Column Temp.: Gradient delay: Gradient: Detritylated 25mer phosphorothioate oligonucleotide mix (CTC TCG CAC CCA TCT CTC TCC TTC T) digested with 3 exonuclease, 2.5 µm (2.1 x 50 mm) A: 15 mm TEA with 400 mm HFIP B: methanol 0.2 ml/min 0 ml 15 to 20% B in 20 minutes (0.25% methanol per minute) 260 nm, 2 scans per second Sample: Mobile phase: Column Temp: Gradient Delay: Gradient: ~600 pmol of a detritylated 20mer, ~18 pmol of 19mer, ~4.5 pmol of 17mer was injected on column., 2.5 µm (2.1 x 50mm) A: 0.1 M TEAA, B: Acetonitrile / 0.1M TEAA, 20/80 (v/v) 0.2 ml/min 0 ml 35 to 50% B in 30 minutes (710% acetonitrile) 260 nm, 2 scans per second Peptide nucleic acids (PNA) can also be analyzed using columns. The ionpairing system recommended for analysis of PNA is similar to those used for peptide analysis (0.1% Trifluoroacetic Acid or Formic Acid). UV detection of eluted oligonucleotide peaks is often performed at 260 nm. Injection of 50 pmol of detritylated oligonucleotide sample on a 2.1 x 50mm column yields relatively abundant peaks. Limits of quantitation (LOQ) vary with the type of oligonucleotide, LC system and detector; LOQ generated on Waters 2996 PDA detector equipped with micro UV cell is approximately 1 pmol (2.1 x 50mm ). The Limit of detection (LOD) estimate is shown in Figure 6. VII. PURIFICATION CONSIDERATIONS columns are designed for laboratory scale oligonucleotide purifications and analyses. Sufficient amount of isolated material suitable for molecular biology and other experiments can be prepared in a single injection. For example, a 4.6 X 50 mm XBridge OST C 18 column can suitably purify approximately nmoles of sample in a single injection. It is important to understand that column overloading results in a peak broadening and that some earlier eluting impurities may coelute with the component of interest. With a proper heartcutting technique, a good purity of the target oligonucleotide can be obtained without significant yield sacrifice (Figure 7). Chromatographers frequently develop a separation on the analytical scale before moving to preparative work. The steps required to optimize the analytical separation involve: 1) Selecting the appropriate column packing material and mobile phase. 2) Determining the optimal flow rate, gradient during and separation temperature. 3) Determining the amount of material that can be satisfactorily loaded and separated on the analytical scale column. 6

7 Once the separation has been optimized, one begins preparing for the preparative separation. The steps to successfully scale a separation from an analytical to a preparative column, containing the same packing material composition, are detailed below Step A: Calculate the flow rate for use on the Preparative column. Preparative Column Flow Rate = Analytical Column Flow Rate x (Diameter of Prep Column) 2 (Diameter of Anal Column) 2 Step B: To get similar chromatography, the gradient elution profile should be created on both columns using the same number of column volumes. When the analytical and preparative columns are of the same length, as is recommended for this application, then the gradient duration should be the same. Note: This assumes use of the same flow rate linear velocity for both runs as calculated above. For preparative runs, it is also important to note that an initial gradient delay is required to allow the entire sample to load onto the head of the column prior to beginning chromatography. * Custom Column ** Values are only approximate and vary depending on detritylated oligonucleotide length, base composition, and heartcutting fraction collection method used Figure 7 shows the separation of 90 nmoles of a detritylated 30 mer deoxythymidine crude reaction mixture on a 4.6 x 50mm XBridge OST C 18 column. The collection interval is suggested by the lines. Due to partial column overloading, the N 1, N 2... impurities are partially displaced and elute earlier than expected. With the proper hearthcutting technique, 9598% purity is typically achieved for 1535 mer oligonucleotides at this purification scale. Figure 7: Purification of a Detritylated 30mer Deoxythymidine Sample Step C: The last calculation involves determining how much sample can be loaded on the preparative column. This calculation compares the relative volumes of the two columns assuming that both columns are the same length as recommended for this application. Preparative Column Sample Load = Previously Determined Analytical Column Sample Load x (Diameter of Prep Column) 2 (Diameter of Anal Column) 2 Sample: Mobile phase: Column Temp.: Gradient delay: Gradient: Crude detritylated 30mer oligothymidine, 200 nmole dissolved in 100 µl of mobile phase A, 45 µl was injected on column, 2.5 µm (4.6 x 50mm) A: 0.1M TEAA with 400 mm HFIP B: Acetonitrile/0.1M TEAA, 20/80 (v/v) 1.0 ml/min 0 ml (compensated) 35 to 65% B in 24 minutes (713% ACN, 0.25% ACN per minute) 260 nm, 2 scans per second Table 3: Column Selection Guide for Detritylated Oligonucleotide Purification Column (mm) Approx Mass Load (µmoles)** Flow Rate (ml/min) 2.1 x x x x 50* x 50* x 50* Table 4: Ordering Information Description Particle Size Pore Size Dimension Part No. 2.5 μm 135Å 2.1 x 50 mm μm 135Å 4.6 x 50 mm μm 135Å 10.0 x 50 mm Custom

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