Choosing the Right SEC Column for Oligonucleotide Separation

Application Note Biopharma/Pharma Authors Andy Coffey, Anne Blackwell, and Charu Kumar Agilent Technologies, Inc. Abstract The use of analytical size exclusion chromatography (SEC) for the separation of oligonucleotides from other components based on hydrodynamic radius is complicated by the range of sizes that can be present, as well as fundamental differences in the conformation between DNA and RNA. This makes selecting the correct pore size SEC column more challenging. Using oligonucleotide ladders, often developed for electrophoresis calibration, makes understanding the pore structure of SEC columns easier, but it remains essential to perform thorough method optimization to overcome challenges with some molecules. This application note provides guidance in choosing the appropriate pore size columns for oligonucleotide separations. Choosing Appropriate Pore Size Columns for Size Exclusion Chromatography of Oligonucleotides 2 Introduction SEC has become the method of choice for aggregate analysis of biotherapeutic molecules. Unlike proteins, which generally have a compact structure, oligonucleotides are much larger. They may be single- or double-stranded, and they may have a complex structure containing loops and hairpins. These structures may also vary depending on the conditions used for analysis. This study used SEC of oligonucleotide standards available in a wide range of sizes. These sizes corresponded to differing dimensions in solution that covered the entire resolving range of the columns used in the study. This allowed a direct comparison of the chromatographic properties of each column, such as exclusion limit (rather than pore size) based on the number of nucleotides. Although oligonucleotides are highly hydrophilic, they can exhibit secondary interactions through hydrogen bonding or from ionic interactions. Also, variations that introduce hydrophobic modifications such as labels can also cause secondary interactions. It is therefore advisable to screen different mobile phase conditions to ensure robust methodology. Experimental Reagents and chemicals All reagents were HPLC grade or higher. Instrumentation Data acquisition was performed on an Agilent 1260 Infinity II bio-inert LC system using Agilent OpenLab CDS software. Sample preparation DNA Ladder standards (part numbers S7025 and D0428) were purchased from Merck Sigma-Aldrich, dissolved in mobile phase, and stored frozen until needed. RiboRuler HR was purchased from Thermo Fisher and diluted 1:10 in mobile phase before use. Mobile phase preparation Three different mobile phase solutions were investigated. These were prepared by dissolving the necessary compounds in Milli-Q water, then filtering through a 0.22 µm membrane filter. The eluents were: – 150 mM sodium phosphate, pH 7.0 – 2X PBS (approximately 20 mM phosphate + 280 mM NaCl), pH 7.4 – 50 mM potassium phosphate, 200 mM potassium chloride, pH 7.0 Parameter Value Column See Table 2 Mobile Phases – 150 mM sodium phosphate, pH 7.0; or – 2X PBS (approximately 20 mM phosphate + 280 mM NaCl), pH 7.4; or – 50 mM potassium phosphate, 200 mM potassium chloride, pH 7.0 Flow Rate 0.35 or 0.175 mL/min Column Temperature 30 °C Injection Volume 5 µL Total Run Time 30 minutes per injection Detection UV, 260 nm Table 1. HPLC method conditions. Column Description A Agilent AdvanceBio SEC 500 Å, 4.6 × 300 mm, 2.7 µm B Agilent AdvanceBio SEC 1000 Å, 4.6 × 300 mm, 2.7 µm C Waters GTxResolve Premier SEC 1,000 Å, 4.6 × 300 mm, 3 µm Table 2. Columns tested. 50 bp DNA Ladder 1 kb DNA Ladder 50 bp 500 bp 100 bp 1,000 bp 150 bp 1,500 bp 200 bp 2,000 bp 250 bp 2,500 bp 300 bp 3,000 bp 350 bp 4,000 bp 400 bp 5,000 bp 450 bp 6,000 bp 500 bp 8,000 bp 600 bp 10,000 bp 700 bp 800 bp 900 bp 1,000 bp 2,000 bp 3,000 bp Table 3. DNA standards used. 3 Results and discussion The first experiment was to determine if using a slower flow rate would improve chromatographic performance as very large molecules diffuse more slowly. Figure 1 shows the separation of a 50 bp DNA ladder performed at 0.175 and 0.35 mL/min. The latter is the flow rate normally used with 4.6 mm id SEC columns. The main difference was the peak height – at the lower flow rate, the peaks spent longer in the detector flow cell, resulting in a greater response. Other factors such as peak width and resolution appeared to be very similar. However, when testing larger oligonucleotides using the 1 kb DNA ladder (Figure 2), the peak response was similar, confirming that larger molecules have slower diffusion times. Consequently, the resolution of the largest molecules was improved, and subsequent experiments were performed using the lower flow rate. Figure 3 shows the separation of the 1 kb DNA ladder using the Agilent AdvanceBio SEC 1000 Å, 2.7 µm column with three different mobile phase solutions as described in Table 1. The total buffer/salt concentration was either 150 or 300 mM, ensuring that nonspecific secondary interactions were minimized. The molecules eluted at almost the same retention time (RT) with very similar peak shape and resolution. The 2X PBS mobile phase conditions were used for creating the calibration data plots shown in Figure 4. Individual retention times are listed in Table 4 and were determined by integration of chromatograms shown in Figure 5. Figure 1. Separation of a 50 bp DNA ladder on an Agilent AdvanceBio SEC 1000 Å (column B), mobile phase 2X PBS. 123456789 10 11 12 13 14 15 Retention time (min) –0.5 0.0 0.5 1.0 1.5 2.0 Response (mAU) 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Retention time (min) –0.5 0.0 0.5 1.0 1.5 2.0 Response (mAU) 0.35 mL/min 0.175 mL/min Figure 2. Separation of a 1 kb DNA ladder on an Agilent AdvanceBio SEC 1000 Å (column B), mobile phase 2X PBS. 123456789 10 11 12 13 14 Retention time (min) 0 0.5 1.0 1.5 2.0 Response (mAU) 123456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Retention time (min) 0 0.5 1.0 1.5 2.0 Response (mAU) 0.35 mL/min 0.175 mL/min ×101 ×101 Figure 3. Separation of a 1 kb DNA ladder on an Agilent AdvanceBio SEC 1000 Å (column B) showing the effect of mobile phase. 12345678 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Retention time (min) 150 mM sodium phosphate, pH 7.0 2X PBS, pH 7.4 50 mM KPO + 200 mM KCl, pH 7.0 4 dsDNA (bp) RT (min) Column A Column B Column C 3,000 9.10 9.81 2,000 10.21 11.24 1,000 12.02 900 9.81 12.48 12.54 800 9.97 12.92 12.75 700 10.27 13.56 13.17 600 10.67 14.26 13.62 500 11.19 15.05 14.16 450 11.57 15.57 14.50 400 11.98 16.02 14.85 350 12.59 16.72 15.38 300 13.26 17.44 15.91 250 14.11 18.18 16.53 200 15.10 18.90 17.19 150 16.42 19.84 18.04 100 18.25 20.94 19.17 50 20.17 22.05 20.30 Table 4. Retention times of DNA ladder peaks. Figure 4. Oligonucleotide calibration curve comparison. 10 100 1,000 10,000 8 10 12 14 16 18 20 22 24 26 Nucleotides (bp) Retention time (min) Column A, AdvanceBio SEC 500 Å Column B, AdvanceBio SEC 1000 Å Column C Figure 5 . Chromatograms of a 50 bp DNA ladder using (A) column A, Agilent AdvanceBio SEC 500 Å; (B) column B, AdvanceBio SEC 1000 Å; and (C) column C, with mobile phase 2X PBS, pH 7.4. -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Response (mAU) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Retention time (min) 50 bp 100 bp 150 bp 200 bp 250 bp 300 bp 350 bp 400 bp 500 bp 450 bp 600 bp 700 bp 800, 900 bp 1,000, 2,000 bp ×101 A -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Response (mAU) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Retention time (min) 50 bp 100 bp 150 bp 200 bp 250 bp 300 bp 350 bp 400 500 bp bp450 bp 600 bp 700 bp 800 bp 900 bp 1,000 bp 2,000 bp 3,000 bp B 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Response (mAU) 1 2345678 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Retention time (min) 50 bp 100 bp 150 bp 200 bp 250 bp 300 bp 350 bp 400 bp 500 bp 450 bp 600 bp 700 bp 800, 900, 1,000 bp 2,000 bp ×101 C 5 Figure 6 shows chromatograms of the 1 kb DNA ladder standard run on both 1,000 Å pore size columns, illustrating the difference in pore volume (from exclusion point to total permeation point). In contrast, Figure 7 shows the chromatogram from an RNA ladder. RNA is most commonly single-stranded and the size is expressed in terms of number of nucleotides (nt) instead of base pairs (bp) used when describing DNA. One might expect a single strand of RNA containing 2,000 nucleotides to have the same size as a double-stranded DNA (dsDNA) molecule containing 2,000 base pairs. However, the double helix nature of the dsDNA appears to restrict the conformation that the molecule is able to adopt. Single-stranded RNA appears to be much more flexible and able to adopt a smaller size in solution than DNA, and therefore elutes later. This behavior has been noted previously.1 It is also important to take into consideration the likely need to either increase the denaturing conditions (for instance by increasing the temperature, or to include a chaotrope in the mobile phase) to improve peak shape for RNA molecule separations. Figure 6. Chromatograms of a 1 kb DNA Ladder for 1000 Å columns B and C (mobile phase 2X PBS, pH 7.4) showing the difference in pore volume. ×101 ×101 A B 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Retention time (min) 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Response (mAU) Response (mAU) Column B Agilent AdvanceBio SEC 1000 Å Exclusion point Permeation point 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Column C 20% less pore volume Figure 7. RiboRuler HR RNA Ladder on column A, Agilent AdvanceBio SEC 500 Å. 12345678 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Retention time (min) 0 1 2 3 4 5 6 7 8 9 Response (mAU) 200 nt 500 nt 1,500 nt 2,000 nt 3,000 nt 4,000 nt 6,000 nt 1,000 nt www.agilent.com DE-004830 This information is subject to change without notice. © Agilent Technologies, Inc. 2025 Printed in the USA, March 12, 2025 5994-8198EN Conclusion The columns used in this study are the latest Agilent AdvanceBio SEC columns in 500 and 1000 Å pore sizes, which provide exceptional pore volume for maximizing the useful range of oligonucleotides that can be separated. It is difficult to compare the resolving range of globular proteins to the resolving range of oligonucleotides, but a useful indication comes from the pore size of the column. For example, 500 Å pore size columns are suitable for DNA up to approximately 500 to 1,000 bp while 1000 Å pore size columns are suitable for DNA up to approximately 2,000 to 3,000 bp (Figure 8). Reference 1. Schneider, S.; Rieck, F. SEC-MALS for mRNA Characterization with the Agilent 1260 Infinity II Multi-Angle Light Scattering Detector, Agilent Technologies application note, publication number 5994-7745EN, 2024. Figure 8. Expected DNA and RNA resolving ranges of molecules for different pore size columns using Agilent AdvanceBio columns. AdvanceBio SEC 1000 Å AdvanceBio SEC 500 Å AdvanceBio SEC 300 Å 10 100 1,000 10,000 DNA size (bp) DNA resolving range AdvanceBio SEC 1000 Å AdvanceBio SEC 500 Å AdvanceBio SEC 300 Å 10 100 1,000 10,000 RNA size (nt) RNA resolving range