Oligonucleotide Purification Columns and Consumables

Oligonucleotide Purification Columns and Consumables

Discover how Waters oligonucleotide lab scale preparative columns and consumables can help you quickly achieve exceptional direct scale-up high recovery and throughput purifications. Built with industry-leading pH and temperature tolerance, each batch of sorbent is QC tested and selected using Waters oligonucleotides standards to ensure lot-to-lot performance and reproducibility.

Discover how Waters oligonucleotide lab scale preparative columns and consumables can help you quickly achieve exceptional direct scale-up high recovery and throughput purifications. Built with industry-leading pH and temperature tolerance, each batch of sorbent is QC tested and selected using Waters oligonucleotides standards to ensure lot-to-lot performance and reproducibility.

Learn how to achieve high performance oligonucleotide preparative chromatography by utilizing XBridge Oligonucleotide OBD Prep Columns and efficient method optimization considerations
Learn how to achieve high performance oligonucleotide preparative chromatography by utilizing XBridge Oligonucleotide OBD Prep Columns and efficient method optimization considerations

Overview

Purifying a full-length oligonucleotide product from impurities generated during synthesis is essential to ensure their functionality, safety, and efficacy in pharmaceutical and diagnostic applications. Due to their polyanionic nature, purifying oligonucleotides with high selectivity and resolution is challenging. Ion-pairing reversed-phase chromatography (IP-RP) has emerged as the dominant and preferred technique.

High pH/temperature stable and batch-tested columns with controlled levels of secondary interactions help quickly remove time-consuming bottlenecks. Waters XBridge Oligonucleotide BEH C18 Optimal Bed Density (OBD) Columns are the go-to-choice for preparative columns, delivering assured performance, consistent scalability, and high recovery purification of various oligos with complex chemical modifications.

  • Batch-tested and QC-certified oligonucleotide BEH C18 particles deliver predictable scale-up and consistent purification across a range of column diameters
  • BEH C18 hybrid silica particle technology delivers industry-leading pH and temperature stability with exceptional resolution and column life
  • MaxPeak Premier High Performance Surfaces (HPS) technology (available on 10 mm I.D. columns) provides an extra option to reduce non-specific adsorption for maximum sensitivity, reproducibility, and productivity
  • A range of high-quality, lot-traceable analytical standards facilitate system suitability testing, method development, and troubleshooting


Optimum Bed Density packing

Optimum Bed Density packing

For preparative columns with smaller aspect ratios, high pressure slurry packing with small particles often fails to achieve the desired bed density found in well-designed analytical columns. Excessive axial compression at the inlet can result in particle breakage, channeling, and reduced bed permeability. Thoughtfully designed OBD columns offer significant benefits, including longer column lifetime, improved efficiency, better peak shape, and lower back pressure.

MaxPeak High Performance Surfaces with 10 mm I.D. columns

MaxPeak High Performance Surfaces with 10 mm I.D. columns

Nucleic acids, including oligonucleotides are polyanionic (negatively charged) and as such readily adsorb metal oxide surfaces in stainless steel columns. Waters XBridge Premier Oligonucleotide BEH C18OBD Columns are built with hardware components modified to have an inert hybrid organic/inorganic silica-based surface chemistry that greatly reduces non-specific adsorption (NSA) of nucleic acids, thus enabling enhanced recovery and improved reproducibility for certain compounds that need to be isolated from a drug substance for further study. XBridge Premier Oligonucleotide BEH C18 OBD Columns also eliminate the need for column passivation/sample conditioning so you can start your fraction collection runs without delay. 

Oligonucleotide batch-tested and selected BEH C18 130 and 300 Å 2.5 and 5 µm particles

Oligonucleotide batch-tested and selected BEH C18 130 and 300 Å 2.5 and 5 µm particles

Waters batch-certified Bridged Ethyl Hybrid (BEH) technology for oligonucleotides ensures extra performance, reproducibility, and scale-up for purification. High temperature and high pH are prevalent strategies for minimizing secondary interactions in oligonucleotide separation and purification. BEH particle technology withstands harsh conditions such as high pH, temp, and additives that usually are required for oligonucleotide analysis.

Batch-tested and QC-certified XBridge Oligonucleotide OBD Prep Columns provide extra assurance for reproducibility and method transfer, and support emerging purification needs for standard and new chemically diverse siRNAs, ASOs, and oligonucleotide reagents. Each batch of Oligonucleotide BEH C18 sorbent is QC tested and selected using Waters MassPrep Oligonucleotide Standard and a stringent resolution and peak tailing assessment from a triethylammonium acetate IP-RP separation. These carefully examined sorbents are available in 2.5 and 5 µm particle sizes as well as 130 and 300 Å pore sizes to give oligonucleotide chemists a range of versatile options to purify a variety of synthesis products.

Outstanding column lifetime

Outstanding column lifetime

XBridge and XBridge Premier Oligonucleotide OBD Prep Columns are packed with Waters BEH (Bridge Ethyl Hybrid) C18 hybrid silica particles and demonstrate remarkable column longevity under high pH and temperature conditions while maintaining outstanding separation performance. With careful care, Oligonucleotide BEH C18 Columns have been known to exceed 1,0000 injections without loss of efficiency or retention. 

Predictable loadability

Predictable loadability

In preparative purifications, loading capacity is influenced by multiple parameters. Column efficiency, pore size, particle size, and method parameters play significant roles. Larger pore size columns allow for better mass transfer and sharper peak shapes of larger analytes. With their reduced surface area, they can exhibit lower loading capacity. Waters XBridge Oligonucleotide OBD Prep Columns are available in different pore sizes and a wide range of column I.D.s for predictable scalability and improved loading capacity.

High purity fractions and higher yields

High purity fractions and higher yields

High purity and quality oligonucleotide purification is critical to minimize the safety and efficacy concerns related to unwanted impurities in toxicological studies. The high-efficiency 2.5 µm XBridge Oligonucleotide OBD Prep Columns facilitating isolation of early and co-eluting impurities while maintaining chromatography profile without compromising high recovery.


“Waters XBridge Oligonucleotide BEH 300 Å C18 5 µm OBD Columns showed definitive improvement in removing early eluting impurities.”

— Amy Howie, Director of Product Quality and Fulfillment, Oligo Factory, Inc.

— Amy Howie, Director of Product Quality and Fulfillment, Oligo Factory, Inc.

Oligo Factory is a leading CMO specializing in custom oligonucleotides for late-stage discovery, clinical phases, diagnostics, and RUO with a mission to provide high-quality, cost-effective oligonucleotides while providing flexible, individualized service for every customer. They are working with Waters Corporation to optimize the purification of sgRNA molecules critical to the research and clinical use of CRISPR gene editing. 


DNA molecule spiral

Solutions


Particle stability with MaxPeak HPS protection

Particle stability with MaxPeak HPS protection

Oligonucleotides phosphodiester groups can adsorb the metals used in the construction of standard columns. Use of low adsorption column hardware components can improve peak shape by minimizing chelation, leading to more reliable retention and more consistent chromatographic parameters. XBridge Premier Oligonucleotide BEH C18 OBD 130 Å and 300Å 5 µm Prep Columns are manufactured with MaxPeak HPS vapor deposited components to enhance peak shape and improve recovery are available in 10 mm I.D. and 50-, 100-, and 150-mm lengths.

  • Reversed-Phase Analysis
  • Oligonucleotides
  • Purification

Particle stability and performance for methods development

Particle stability and performance for methods development

Batch-tested and QC-certified XBridge Oligonucleotide OBD Prep Columns are available in a wide range of diameters and multiple lengths for lab scale purification options that are predictable and flexible for direct scale up. Sizes include:

  • Reversed-Phase Analysis
  • Oligonucleotides
  • Purification

High purity oligonucleotide and nucleic acid formulations for benchmarking performance.

High purity oligonucleotide and nucleic acid formulations for benchmarking performance.

Oligonucleotide/nucleic acid standards for benchmarking performance

Oligonucleotide/nucleic acid standards for benchmarking performance

Complementing our oligonucleotide reversed-phase columns, Waters provides a range of high-quality, lot-traceable analytical standards to facilitate system suitability testing, method development, and troubleshooting. These include our MassPREP Oligonucleotide Standard containing a 15–35mer oligodT ladder used to QC verify all Waters oligonucleotide branded columns, a 20mer ssDNA standard for benchmarking LC-MS/MS fragmentation and sequence analysis, a lipid-conjugated heavily modified antisense oligonucleotide (ASO), ssDNA, and dsDNA ladders, and more.

  • Reversed-Phase Analysis
  • Oligonucleotides
  • Purification

Protect and prolong analytical column lifetime.

Protect and prolong analytical column lifetime.

Prep column guard for extended column lifetimes

Prep column guard for extended column lifetimes

XBridge Oligonucleotide BEH C18 OBD Prep Columns are available with column guards. The column cartridges are packed with the same sorbent and can be used to prevent contamination and fouling of the prep columns resulting in longer column lifetime. Placing this guard assembly ahead of the prep column will provide longer column lifetimes and reduce purification costs.

  • Reversed-Phase Analysis
  • Oligonucleotides
  • Purification

Applications

Oligonucleotides phosphodiester groups can adsorb the metals found in column bodies and frits. Using low adsorption, MaxPeak High Performance Surface column hardware can improve peak shape by minimizing chelation, leading to more reliable retention and more consistent chromatographic parameters. XBridge Premier Oligonucleotide BEH C18 OBD Prep Columns are manufactured with MaxPeak HPS vapor deposited components to enhance peak shape and improve recovery.

Oligonucleotides phosphodiester groups can adsorb the metals found in column bodies and frits. Using low adsorption, MaxPeak High Performance Surface column hardware can improve peak shape by minimizing chelation, leading to more reliable retention and more consistent chromatographic parameters. XBridge Premier Oligonucleotide BEH C18 OBD Prep Columns are manufactured with MaxPeak HPS vapor deposited components to enhance peak shape and improve recovery.


Application Notes

Application Notes

One challenge with lab scale oligo purification is the isolation of impurities that can be similar to the target molecules. Small particle size particles are usually not prevalent for lab-scale purification due to the high pressure they can generate. Waters batch-tested XBridge Oligonucleotide BEH C18 OBD 2.5 µm Prep Columns provide a new level of assurance for low pressure and efficient isolation of complex CRISPR sgRNA, siRNA, and oligo-conjugates. Optimized purification parameters with focused gradient mode and HFIP-free mobile phase support highly efficient, high-throughput, hazardless, and cost-effective purification.

One challenge with lab scale oligo purification is the isolation of impurities that can be similar to the target molecules. Small particle size particles are usually not prevalent for lab-scale purification due to the high pressure they can generate. Waters batch-tested XBridge Oligonucleotide BEH C18 OBD 2.5 µm Prep Columns provide a new level of assurance for low pressure and efficient isolation of complex CRISPR sgRNA, siRNA, and oligo-conjugates. Optimized purification parameters with focused gradient mode and HFIP-free mobile phase support highly efficient, high-throughput, hazardless, and cost-effective purification.


Application Notes

Application Notes

CRISPR technology plays a critical role in gene editing modification. The technology utilizes single guide RNA (sgRNA) hence the significance of sgRNA purity for improved therapeutic accuracy and precision. The traditional on-market available 8 to 20 µm HPLC columns may not meet the need for drug safety and accuracy due to lack of required resolving power for isolating the impurities from target sequences. 

The new batch-tested XBridge Oligonucleotide BEH C18, 300 Å, 5 µm Column improves separation efficiency and retention mechanism for larger nucleic acids such as sgRNA and related applications. The smaller particle sizes and optimized pore dimensions with 300 Å are applied to achieve new single pass, purity levels that are required for high-precision CRISPR applications.

CRISPR technology plays a critical role in gene editing modification. The technology utilizes single guide RNA (sgRNA) hence the significance of sgRNA purity for improved therapeutic accuracy and precision. The traditional on-market available 8 to 20 µm HPLC columns may not meet the need for drug safety and accuracy due to lack of required resolving power for isolating the impurities from target sequences. 

The new batch-tested XBridge Oligonucleotide BEH C18, 300 Å, 5 µm Column improves separation efficiency and retention mechanism for larger nucleic acids such as sgRNA and related applications. The smaller particle sizes and optimized pore dimensions with 300 Å are applied to achieve new single pass, purity levels that are required for high-precision CRISPR applications.



Application Notes

Application Notes


The data speaks for itself

The data speaks for itself
Fraction purity determinations for a 25 mg purification run using a novel XBridge Oligonucleotide BEH C18 130 Å 2.5 µm OBD Prep Column.
(A) Comparison of the chromatograms obtained for hydrocortisone phosphate using MaxPeak Premier (black trace) and standard (gray trace) ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 2.1 x 50 mm Columns. The mass load was 2 ng. Acetonitrile gradient separations were carried out with a 10 mM ammonium formate (pH 3.0) aqueous mobile phase, a flow rate of 0.5 mL/min and a temperature of 30 °C. The peaks were detected using a PDA detector, with the 246 nm channel displayed. (B) Log-log plot of peak area vs mass load for hydrocortisone phosphate separated using MaxPeak Premier (black symbols) and standard (gray symbols) columns, with mass load from 2–200 ng.

Resources


  • White Paper

Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces

Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces

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Learn more about Oligonucleotide Purification Columns and Consumable Solutions.

Learn more about Oligonucleotide Purification Columns and Consumable Solutions.

Learn how to achieve high performance oligonucleotide preparative chromatography by utilizing XBridge Oligonucleotide OBD Prep Columns and efficient method optimization considerations
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