<h3>Why Anion Exchange Chromatography Is a Strong Choice for Large-Scale Oligonucleotide Purification</h3>Oligonucleotide therapeutics are becoming increasingly important in modern drug development. Short DNA- and RNA-based medicines are moving through clinical pipelines in growing numbers, and several have already received FDA approval. As this field expands, one question becomes increasingly important: how can these molecules be purified efficiently, reproducibly, and on a manufacturing scale?One of the most reliable answers is anion exchange chromatography (AEX).In earlier discussions, we looked at the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6846">fundamentals of oligonucleotide analysis and the core principles behind AEX separations</a>. Here, the focus shifts to preparative purification at larger scale and to the practical issues that arise when moving from laboratory development toward production. As an example, this article highlights the purification of an oligo(dT) sample on the liter scale, demonstrating how AEX can deliver both high purity and strong recovery.<h4>Why purification is so important</h4>Following chemical synthesis, crude oligonucleotide material contains a mixture of desired product, truncated sequences, and other synthesis-related impurities. For pharmaceutical use, especially in clinical and regulated settings, this crude material must be refined to a much higher purity level.Although multiple HPLC approaches are available for oligonucleotide purification, not every method is equally suitable for scale-up. Some techniques work well in analytical or small-scale formats but become less practical when transferred to larger production environments. What is needed is a purification strategy that combines high performance with straightforward scalability.<h4>What makes AEX attractive?</h4>Anion exchange chromatography has established itself as an effective purification method for oligonucleotides, especially when larger sample volumes are involved. Its main strengths include:<ul><li>High loading capacity, allowing large sample amounts to be processed efficiently</li><li>Strong separation performance, enabling high-purity product recovery</li><li>Robust scalability, from development work through to GMP-oriented production</li><li>More environmentally friendly operation, since aqueous buffer systems can be used instead of harsher organic solvents</li></ul>These features make AEX a practical option for laboratories and manufacturers seeking a reproducible purification platform that can grow with the needs of a project.<h4>Example: large-scale purification of oligo(dT)</h4>To illustrate the approach, an oligo(dT) sample was purified on the liter scale. Oligo(dT) is widely used in molecular biology, for example in mRNA isolation and as a primer in cDNA synthesis.Before chromatography, the sample required the usual preparation steps: cleavage from the solid support, deprotection with ammonia, and subsequent neutralization. The purification itself was performed on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1621">AZURA® Prep HPLC system</a>, which is designed for high-throughput preparative workflows and offers precise gradient formation, UV detection, and automated fraction collection.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_AZURA_preparative_HPLC_system_b989fe64fb.webp" alt="KNAUER: AZURA preparative HPLC system" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_AZURA_preparative_HPLC_system_b989fe64fb.webp 107w," sizes="100vw" width="653" height="954">The separation used a strong anion exchange resin with gradient elution based on a phosphate buffer and sodium bromide as the counterion. Sodium bromide was selected because it is less aggressive toward stainless steel components than chloride, helping protect instrument hardware during repeated operation. For optimal resolution, anion exchange particles in the 10–15 µm range are typically preferred.<h4>Performance in practice</h4>The results clearly demonstrate the suitability of AEX for oligonucleotide purification at scale.<ul><li>Purity before purification: 89%</li><li>Purity after AEX purification: 95%</li><li>Yield after purification: 90%</li></ul>These values show that the method can effectively improve product quality while maintaining a high recovery, which is a key requirement in preparative and manufacturing settings.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Chromatogram_of_oligonucleotide_purification_from_1_l_sample_a768bb4759.webp" alt="KNAUER: Chromatogram of oligonucleotide purification from 1 l sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Chromatogram_of_oligonucleotide_purification_from_1_l_sample_a768bb4759.webp 245w," sizes="100vw" width="1920" height="848"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Analytical_chromatogram_of_purified_and_pooled_oligo_d_T_after_AEX_0d6e5b31c5.webp" alt="KNAUER: Analytical chromatogram of purified and pooled oligo (dT) after AEX." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Analytical_chromatogram_of_purified_and_pooled_oligo_d_T_after_AEX_0d6e5b31c5.webp 240w," sizes="100vw" width="1085" height="705"><h4>Important limitations to keep in mind</h4>Like any chromatographic method, AEX also has its limitations and process considerations.<ul><li>Phosphodiester vs. phosphorothioate separations can be challenging, and resolution may decrease for longer or more heavily modified sequences.</li><li>Aggregation effects caused by secondary structures can reduce purification efficiency.</li><li>Because AEX is typically performed under aqueous conditions, there is also an increased need to monitor and control endotoxin-related process risks.</li></ul>These factors do not diminish the value of AEX, but they do underline the importance of careful method development and process control.<h4>Final thoughts</h4>For oligonucleotide-based therapeutics, purification is not just a supporting step — it is a critical part of the path from crude synthesis to clinical or commercial material. The purification strategy must be scalable, robust, and capable of consistently delivering high-quality product.Anion exchange chromatography meets many of these demands. It offers a compelling balance of purity, yield, scalability, and operational practicality, making it an important tool for both development and production environments.For teams preparing material for research, clinical studies, or GMP manufacturing, AEX on AZURA® Prep systems represents a strong and practical route toward reliable oligonucleotide purification.For additional details, the related application note “<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VBS_0082_Large_scale_purification_of_oligonucleotides_with_ion_exchange_chromatography_5e622f1914.pdf">Oligonucleotide Purification via Ion Exchange</a>” provides more information on the method.<blockquote><ul><li>For further information on this topic, please contact the author at <a href="mailto:krop@knauer.net">krop@knauer.net</a></li><li>If you are planning to build or optimize an oligonucleotide workflow, you can also contact sales@knauer.net</li></ul></blockquote>

Oligos Made Easy - Part 7: Large Scale AEX

News from LabRulezLCMS Library - Week 12, 2026

The leaflet outlines the role and handling of analytical equipment and systems in the process of a laboratory dealing with the analysis of chemical elements and molecules. It is an introduction to topics being considered by the Eurachem Analytical Equipment and Systems Qualification Working Group.&nbsp;Various steps are required to draw a conclusion concerning the object being examined (sampling target), see Fig. 1. The core part, the analytical measurement, is based on an analytical principle. The measurement is based on a physical or chemical interaction, e.g. between light of a certain wavelength and the sample, which leads to a displayed measured value. This measurement is carried out using analytical equipment. This equipment must be fit for its intended use. Complex analytical equipment with extensive data processing is referred to as a system. Analytical equipment is used to perform the analytical method. The method must be validated to demonstrate that it is fit for purpose.&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Eurachem_Fig_1_Analytical_principle_method_and_procedure_1_b751d25236.jpg" alt="Eurachem: Fig. 1 Analytical principle, method and procedure [1]" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Eurachem_Fig_1_Analytical_principle_method_and_procedure_1_b751d25236.jpg 245w," sizes="100vw" width="841" height="439"><h4>The pyramid of quality&nbsp;</h4>The pyramid of quality (Fig. 2) covers the analytical method. In order to produce a reproducible, reportable result that is suitable for the intended purpose, the equipment (system) (1st level) and analytical method (2nd level) must be well defined and under control, qualified or validated, respectively.&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Eurachem_Fig_2_Pyramid_of_quality_979312ac6b.jpg" alt="Eurachem: Fig. 2 Pyramid of quality" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Eurachem_Fig_2_Pyramid_of_quality_979312ac6b.jpg 163w," sizes="100vw" width="708" height="677">The equipment must have similar performance when carrying out the analyses as when developing and validating the method. Particularly in the case of methods based on libraries or training datasets, the long-term stability of the equipment must be ensured. In the case of transfer of the method to other equipment, both sets of equipment must have similar performance.&nbsp;The performance of the equipment and the behaviour of the method are monitored by quality control measures. This is the purpose of the internal or short term (3rd level) and inter-laboratory or long term (4th level) quality assurance. When selecting the quality assurance elements, the findings from levels 1 and 2 are taken into account.&nbsp;<h4>Is "calibration" sufficient?&nbsp;</h4>All relevant quality assurance systems, e.g. those meeting the requirements of ISO/IEC 17025 [2] and related standards, require equipment and systems to be traceably calibrated. But is that enough to prove the fitness for the intended use of the analytical equipment?&nbsp;According to the definition in VIM [3] calibration is the operation that, under specified conditions, establishes a relation between the quantity values with measurement uncertainties provided by measurement standards and corresponding indications with associated measurement uncertainties. It is implicitly assumed that the influencing quantities (e.g. ambient conditions, temperature, flow rates, intensities of signal sources, etc.) are constant and do not exhibit any short or long-term fluctuations or drifts. This assumes that no changes occur due to influencing quantities.&nbsp;Nevertheless this is not the case in everyday analytical work. The influence of fluctuations or changes in the influencing quantities is evaluated through robustness investigations during method development and validation. The magnitude of the fluctuations or deviations of the equipment is specified and verified during equipment qualification.&nbsp;<h4>The Fitness for Intended Use&nbsp;</h4>The life cycle (Fig. 3) of new equipment to be purchased by the laboratory begins with the definition of the requirements and the intended use of the equipment.&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Eurachem_Fig_3_Equipment_or_system_lifecycle_2919ea802d.jpg" alt="Eurachem: Fig. 3 Equipment or system lifecycle" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Eurachem_Fig_3_Equipment_or_system_lifecycle_2919ea802d.jpg 186w," sizes="100vw" width="887" height="742">The User Requirement Specifications (URS) define the selection criteria and the operational ranges of the equipment parameters (influence variables), e.g. flows, temperatures, analysis volumes, etc. Considerations to support data integrity and traceability are also included in the URS. For more complex systems, the interfaces and compatibility of the components are essential.&nbsp;All considerations made in this phase have an impact on the entire life cycle, i.e. right through to decommissioning. The stability and variability of the influence variables are monitored over the entire service life.&nbsp;In most cases, data and metadata should remain accessible and analysable beyond the end of the equipment's life. The laboratory must use its resources as sparingly and sustainably as possible. This includes the expected service life and the costs incurred. Data integrity considerations (ALCOA+ = Attributable, Legible, Contemporaneous, Original, Accurate, + Complete, Consistent, Enduring, Available) play a role during operation and at the end of life. These include paper records and electronic records.&nbsp;In the pharmaceutical industry, qualification of equipment and thus ensuring that analytical equipment and systems are suitable for the intended use, is mandatory, and the respective processes are often clearly defined. Specifications are also comprehensively defined for various equipment classes.&nbsp;The ISO standards, in particular ISO 9001 [4], ISO 10012 [5], ISO/IEC 17025 [2], ISO 17034 [6] or ISO/IEC 17043 [7], cover a very large area and a wide range of sectors. For specific sectors, an interpretation of the standards is required. Eurachem guides cover requirements for analytical chemistry and related disciplines. The Analytical Equipment and Systems Qualification Working Group is therefore developing guidance on establishing the fitness for intended use of analytical equipment and systems aimed at these disciplines.

Fitness for the intended use of analytical equipment and systems

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Eurachem is a European network promoting international traceability of chemical measurements and good analytical practice. It provides expert guidance, supports method validation, uncertainty evaluation, proficiency testing, and facilitates discussion on technical and policy issues in analytical quality.

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Based in Berlin, KNAUER is a medium-sized, owner-managed company that has been serving the sciences since 1962. We develop and manufacture scientific instruments of superior quality for liquid chromatography, including: Analytical HPLC/UHPLC, Preparative HPLC, Fast protein liquid chromatography (FPLC), Multi-column chromatography/Simulated moving bed (SMB) chromatography. High pressure dosing, in-line detection systems, and osmometry are additional business areas.

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Fitness for the intended use of analytical equipment and systems

News from LabRulezLCMS Library - Week 12, 2026

Oligos Made Easy - Part 7: Large Scale AEX