<h3>A Practical Guide to Oligonucleotide Production – From Concept to Final Product</h3>In 2020, an internal innovation at KNAUER significantly reshaped our perspective on nucleic acid technologies. The development of Impingement Jets Mixing (IJM) for lipid nanoparticle (LNP) generation—originally driven by the urgent need for mRNA-based COVID-19 vaccines—sparked a deeper engagement with DNA and RNA-based therapeutics. Although mRNA vaccines were the first application, it soon became clear that the same platform could support a broad range of oligonucleotides, including short DNA and RNA sequences.This realization naturally expanded our focus beyond formulation alone. Purification strategies, analytical control, and scalable manufacturing of oligonucleotides became equally compelling challenges. Today, simplifying and optimizing oligonucleotide production workflows is a central pillar of KNAUER’s technology roadmap.<h4>Technologies for LNP Manufacturing</h4><ul><li>NanoScaler</li><li>Customized NanoProducer</li></ul>These systems provide flexible solutions for research and production-scale LNP generation, supporting nucleic acid delivery applications.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Systems_for_LNP_production_Middle_Nano_Scaler_Right_Customized_Nano_Producer_1cf4727f38.jpg" alt="KNAUER: Systems for LNP production. Middle - NanoScaler, Right - Customized NanoProducer" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Systems_for_LNP_production_Middle_Nano_Scaler_Right_Customized_Nano_Producer_1cf4727f38.jpg 245w," sizes="100vw" width="1547" height="524"><h4>What Is Required to Manufacture Oligonucleotides?</h4>Whether producing oligonucleotides for internal use or as a CDMO, establishing a dedicated oligo production laboratory requires a sequence of well-defined steps. This introductory article outlines a mid-scale production workflow (from approximately 1 µmol up to several hundred mmol). Each operation is introduced here and will be explored in greater depth in future articles, with particular attention to purification and quality control. But before assessing purity, the oligonucleotide itself must first be synthesized.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Typical_workflow_for_oligonucleotide_production_a52b1aabd0.jpg" alt="KNAUER: Typical workflow for oligonucleotide production" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Typical_workflow_for_oligonucleotide_production_a52b1aabd0.jpg 245w," sizes="100vw" width="1593" height="510"><h4>Typical Oligonucleotide Production Workflow</h4><h5>Step 1: Solid-Phase Oligonucleotide Synthesis</h5>Oligonucleotide assembly begins on a solid support, most commonly controlled pore glass (CPG) or polystyrene resin. Individual nucleotides are sequentially added through a cyclic series of reactions:<ul><li>Detritylation – removal of temporary protecting groups</li><li>Coupling – attachment of the next nucleotide</li><li>Oxidation or thiolation – stabilization of the phosphate backbone</li><li>Capping – deactivation of unreacted chains</li></ul>These repetitive steps involve precise reagent delivery, washing, and activation, and are typically performed on automated synthesizers. KNAUER’s own oligonucleotide synthesizer is currently approaching completion.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Solid_phase_synthesis_5d16092ede.jpg" alt="KNAUER: Solid-phase synthesis" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Solid_phase_synthesis_5d16092ede.jpg 203w," sizes="100vw" width="809" height="622"><h5>Step 2: Cleavage and Deprotection</h5>Once synthesis is complete, the crude oligonucleotide must be released from the solid support. This is commonly achieved using ammonia-based reagents. Subsequent treatment with heated ammonium hydroxide removes remaining protecting groups. After filtration to remove the solid matrix, the solution is neutralized and prepared for initial analytical evaluation.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Flow_scheme_KNAUER_Synthesizer_a4212a41a2.jpg" alt="KNAUER: Flow scheme KNAUER Synthesizer​" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Flow_scheme_KNAUER_Synthesizer_a4212a41a2.jpg 245w," sizes="100vw" width="834" height="506"><h5>Step 3: Chromatographic Analysis and Purification</h5>Chromatography plays a central role in both the characterization and purification of oligonucleotides. Commonly used techniques include:<ul><li>Anion-Exchange Chromatography (AEX) Separation based on charge; cost-efficient but generates high salt loads requiring downstream removal.</li><li>Ion-Pair Reversed-Phase Chromatography (IP-RP) Offers excellent resolution and typically simplifies downstream processing due to volatile buffers, though at higher operating costs.</li><li>Hydrophilic Interaction Liquid Chromatography (HILIC) An increasingly popular analytical tool, particularly suitable for short or chemically modified oligos, without ion-pairing reagents. Its primary strength lies in analysis rather than large-scale purification.</li></ul><h5>Step 3a: In-Process Quality Control</h5>Throughout production, oligonucleotides must be monitored for purity, sequence fidelity, and structural modifications. Liquid chromatography remains the primary analytical technique for these assessments, commonly using IP-RP, AEX, or HILIC methods. This is an area where KNAUER’s analytical expertise and HPLC platforms play a key role.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Representative_KNAUER_analytical_HPLC_system_and_Chromatogram_of_Oligonucleotide_Analysis_26f4619c5b.jpg" alt="KNAUER: Representative KNAUER analytical HPLC system and Chromatogram of Oligonucleotide Analysis" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Representative_KNAUER_analytical_HPLC_system_and_Chromatogram_of_Oligonucleotide_Analysis_26f4619c5b.jpg 245w," sizes="100vw" width="1342" height="674"><h5>Step 3b: Preparative Chromatographic Purification</h5>Following analytical assessment, purification removes synthesis-related impurities such as truncated sequences or residual protecting groups. AEX and IP-RP are the most frequently applied methods. Process development teams often evaluate both approaches at small scale before selecting the optimal strategy. Notably, both modes can be implemented on the same preparative system, such as the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=41&amp;manufacturerItems=503&amp;search=lab">AZURA® Lab </a>or <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=41&amp;manufacturerItems=503&amp;search=pilot">Pilot Prep HPLC platforms.</a><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Representative_KNAUER_preparative_LC_System_and_Chromatogram_of_Oligonucleotide_Purification_19415a7614.jpg" alt="KNAUER: Representative KNAUER preparative LC System and Chromatogram of Oligonucleotide Purification" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Representative_KNAUER_preparative_LC_System_and_Chromatogram_of_Oligonucleotide_Purification_19415a7614.jpg 245w," sizes="100vw" width="1335" height="673"><h5>Step 4: Desalting or Solvent Exchange via Tangential Flow Filtration (TFF)</h5>After chromatographic purification, oligonucleotides remain in matrices containing salts or organic solvents. Tangential flow filtration enables gentle and scalable buffer exchange. Low-molecular-weight components pass through the membrane, while oligonucleotides are retained. This step prepares the product for formulation or further processing. KNAUER is currently developing a modular TFF solution tailored to this application.<h5>Step 5: Endotoxin Reduction</h5>For therapeutic applications, endotoxin levels must be rigorously controlled. Lipopolysaccharides from bacterial sources can be efficiently reduced using an additional TFF step. By selecting appropriate membranes, multiple diafiltration cycles can lower endotoxin concentrations by several orders of magnitude, supporting regulatory compliance depending on dose and administration route.<h5>Step 6: Lyophilization</h5>For preclinical and early-stage batches, freeze-drying is the standard finishing step. The oligonucleotide solution is frozen below –40 °C and subjected to vacuum sublimation, producing a dry, stable product that can be stored at ambient temperature and rapidly reconstituted when needed.<h4>Connecting the Workflow with KNAUER Technology</h4>KNAUER provides a cohesive portfolio supporting oligonucleotide production from synthesis to final formulation. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=37&amp;manufacturerItems=503&amp;search=azura">AZURA® (U)HPLC systems</a> and dedicated columns such as Sepapure® oliGO enable robust analytical control and efficient purification. Preparative LC platforms are optimized for oligo workflows, while new solutions—including an in-house oligonucleotide synthesizer and a modular TFF system—are being developed to streamline desalting and endotoxin removal.Together, these technologies allow laboratories to build scalable, integrated oligonucleotide production environments without relying on fragmented, multi-vendor solutions.

Oligos Made Easy - Part 1: Overview

<h3>Charged for Action: The Hidden Secrets of AEX Oligonucleotide Analysis</h3>Oligonucleotides—short, well-defined fragments of DNA or RNA—have become indispensable tools in modern life sciences. They underpin cutting-edge applications in molecular diagnostics, gene therapy, and pharmaceutical development. Before oligonucleotides can be confidently used in research or clinical settings, however, their quality must be rigorously assessed. Purity, sequence integrity, length distribution, and chemical modifications all play a decisive role in their performance.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_2_45eee54fa0.jpg" alt="KNAUER: Oligos Made Easy - Part 2: AEX" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_2_45eee54fa0.jpg 245w," sizes="100vw" width="1534" height="711">Among the analytical techniques available today, anion exchange chromatography (AEX) stands out as a robust and highly informative method for characterizing oligonucleotides. By exploiting their intrinsic negative charge, AEX provides detailed insight into product composition and impurity profiles that are difficult to obtain by other approaches.<blockquote><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6808">Part 1 of the Oligos series: A Practical Guide to Oligonucleotide Production – From Concept to Final Product</a></blockquote><h4>How Scientists Read Oligonucleotides Using Charge</h4>At the core of AEX lies a simple but powerful principle: oligonucleotides carry a negatively charged phosphate backbone. This charge increases in a predictable way with strand length and, to a lesser extent, sequence composition. Anion exchange chromatography uses this property to separate oligonucleotides based on differences in charge strength and interaction with a positively charged stationary phase.As a result, AEX has become one of the most widely used analytical tools for evaluating oligonucleotide quality. It allows scientists to distinguish full-length products from truncated species, degradation products, or closely related variants—often with single-nucleotide resolution.<h4>Why Ion Exchange Works: Binding, Retention, and Release</h4>Ion exchange chromatography (IEX) relies on electrostatic interactions between analytes and a charged stationary phase. In the case of oligonucleotides, anion exchange is used, where negatively charged molecules interact with positively charged functional groups on the resin—most commonly quaternary amines.The system consists of two main components:<ul><li>Stationary phase: A positively charged resin that binds anionic analytes.</li><li>Mobile phase: An aqueous buffer system, typically combined with a salt gradient, that controls both pH and elution strength.</li></ul>Retention in AEX is governed primarily by charge. Molecules with weaker overall charge interact less strongly with the stationary phase and elute earlier, while more highly charged oligonucleotides bind more tightly and require stronger elution conditions.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_1_IEX_overview_including_elution_order_dependency_on_analyte_charge_3419ad3075.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 1 IEX overview including elution order dependency on analyte charge." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_1_IEX_overview_including_elution_order_dependency_on_analyte_charge_3419ad3075.jpg 245w," sizes="100vw" width="1583" height="654"><h4>The Five-Step Workflow of Ion Exchange Chromatography</h4>AEX separations generally follow a well-established sequence of steps:<ol><li>Column equilibration The column is conditioned with the starting buffer until the baseline and pH are stable. This ensures that all charged sites on the resin are ready for interaction.</li><li>Sample loading Samples, prepared in the starting buffer, are introduced under conditions that promote binding of the target oligonucleotides while minimizing retention of unwanted impurities.</li><li>Washing The column is washed with the starting buffer to remove uncharged or weakly interacting components. The baseline stabilizes as these species are flushed away.</li><li>Elution Bound oligonucleotides are released using a salt or pH gradient. Increasing ionic strength introduces competing ions that displace the analytes from the resin. Weakly bound species elute first; strongly charged oligonucleotides elute later.</li><li>Regeneration and re-equilibration A high-salt buffer removes any remaining bound material. The column is then returned to starting conditions in preparation for the next analysis.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_2_Five_steps_of_IEX_procedure_in_blue_outlined_using_an_example_chromatogram_in_red_38dd70b1b1.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 2 Five steps of IEX procedure (in blue), outlined using an example chromatogram (in red)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_2_Five_steps_of_IEX_procedure_in_blue_outlined_using_an_example_chromatogram_in_red_38dd70b1b1.jpg 245w," sizes="100vw" width="1583" height="674"><h4>Why AEX Is Especially Effective for Oligonucleotides</h4>The repeating phosphate backbone of oligonucleotides gives rise to a strong and uniform anionic character across a wide pH range (typically pH 7–9). This makes oligonucleotides exceptionally well suited for anion exchange separations.Key factors influencing AEX behavior include:<ul><li>Charge density: Longer oligonucleotides carry more negative charges and therefore exhibit stronger retention.</li><li>Resin chemistry: Strong and weak anion exchangers differ in functional groups and pH stability, influencing selectivity and robustness.</li><li>Buffer composition: The choice of buffering ions and salt gradients directly affects elution strength and resolution.</li></ul>Salt gradients—commonly based on chloride ions—compete with the oligonucleotide backbone for binding sites on the resin. By carefully tuning gradient slope and composition, analysts can achieve highly reproducible and finely resolved separations.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_3_Ionic_interactions_and_p_H_influence_on_Anion_Exchange_Chromatography_AEX_612e231ba4.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 3 Ionic interactions and pH influence on Anion Exchange Chromatography (AEX)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_3_Ionic_interactions_and_p_H_influence_on_Anion_Exchange_Chromatography_AEX_612e231ba4.jpg 245w," sizes="100vw" width="1547" height="593"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_4_AEX_buffer_selection_and_gradient_strategy_chromatogram_with_red_buffer_gradient_curve_53fd1bb11b.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 4 AEX buffer selection and gradient strategy (chromatogram with red buffer gradient curve)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_4_AEX_buffer_selection_and_gradient_strategy_chromatogram_with_red_buffer_gradient_curve_53fd1bb11b.jpg 245w," sizes="100vw" width="1600" height="327"><h4>AEX as the Gold Standard for Oligonucleotide Separation</h4>Anion exchange chromatography has become the method of choice for both analytical and preparative oligonucleotide workflows. Its advantages include:<ul><li>High resolving power: Closely related impurities, including n–1 or n–2 species, can often be separated and quantified.</li><li>Predictable retention behavior: In many cases, retention time correlates linearly with oligonucleotide length, simplifying identification.</li><li>Compatibility with modified oligonucleotides: Even chemically modified sequences retain their anionic nature, allowing AEX to resolve both products and related impurities.</li><li>Excellent control via gradient elution: Salt gradients provide precise tuning of selectivity and resolution.</li></ul>Typically, shorter or degraded oligonucleotides elute at lower ionic strength, while full-length products with higher charge density appear later in the chromatogram.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_5_AEX_Advantages_and_Challenges_7835521e34.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 5 AEX Advantages and Challenges." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_5_AEX_Advantages_and_Challenges_7835521e34.jpg 245w," sizes="100vw" width="1535" height="707"><h4>Final Perspective: Mastering AEX with Confidence</h4>Anion exchange chromatography may appear complex at first, but with thoughtful optimization of pH, buffer composition, and gradient conditions, it becomes a highly precise and reliable tool for oligonucleotide analysis. When properly applied, AEX delivers sharp peaks, excellent resolution, and robust performance—even for challenging sequences and modifications.For laboratories developing or refining oligonucleotide workflows, AEX offers both analytical depth and operational confidence.<blockquote>If you would like to discuss AEX method development or optimization for oligonucleotides, feel free to contact us at sales@knauer.net. More insights into practical oligonucleotide analysis will follow in our “Oligos Made Easy” series.For technical questions or in-depth discussion, the author can be reached at kindler@knauer.net</blockquote>

Oligos Made Easy - Part 2: AEX

Article

Product

Events

Webinars

Application

Academy

Video

Poster

Scientists

Plastics

Environment

Food

Water

Health

Presentation

Scientific article

Interview

Toxicology

Popularization

Science and research

Software

Covid-19

Career

Laboratories

Various

Zaměření

Type of News

Focus

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.

KNAUER

Analytical Strategies for Peptide & Protein Therapeutics

Advance Your SEC Knowledge Scientific Hands-on Workshop

36th MassSpec Forum Vienna 2026

Accurate mass fundamentals - Interpreting and applying your data

Process Analytical Techniques: Online HPLC for Real Time Monitoring and Control of Bioprocesses

Introducing the Orbitrap Excedion Pro Hybrid MS