<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>How to Choose the Right Column for Oligonucleotide Analysis</h3><h4>Finding the Best Column for Your Oligonucleotides</h4>Selecting an appropriate chromatographic column for oligonucleotide analysis can be challenging. These molecules are long, highly charged, and often require specialized separation strategies. With multiple chromatographic techniques available, choosing the most suitable approach for your application is not always straightforward.However, understanding the characteristics of oligonucleotides and the analytical requirements of your workflow can significantly simplify the process.<h4>Why Are Oligonucleotides Difficult to Separate?</h4>Over the past several years, oligonucleotides have become increasingly important in molecular biology, biotechnology, and therapeutic research. Their growing role in regulated environments, including pharmaceutical development, means that precise and reliable analytical methods are essential.From a chromatographic perspective, oligonucleotides present several challenges.These molecules are typically highly polar and strongly charged, consisting of phosphodiester or phosphorothioate backbones. During synthesis, numerous closely related impurities can be generated. These include fragments that differ slightly in chain length (shortmers or longmers), as well as molecules with minor structural variations such as single-base substitutions or functional group modifications.In addition, oligonucleotide samples frequently contain residual reagents, salts, and by-products from synthesis. These components can overload chromatographic columns and negatively affect separation efficiency.For analytical laboratories, this means that a suitable column must deliver:<ul><li>High resolution between closely related structures</li><li>Symmetrical and well-defined peaks</li><li>Fast analysis times</li><li>Compatibility with mass spectrometry (MS) detection</li></ul>At the same time, the method must provide reliable reproducibility and minimize sample loss during elution or desalting.Understanding these analytical requirements is the first step toward selecting the most appropriate chromatographic solution.<h4>Which Chromatographic Approach Should You Choose?</h4>Separating oligonucleotides is inherently complex because many compounds differ only slightly in structure, sometimes by a single nucleotide or modification.Different chromatographic techniques are therefore used depending on the analytical objective.For purification of synthesized strands, preparative liquid chromatography or size exclusion chromatography (SEC) is typically employed.For analytical characterization, UHPLC coupled with mass spectrometry is widely used because it enables both qualitative and quantitative analysis of products and impurities.Before purification or analysis, samples may also require desalting, which can be achieved efficiently using dedicated SEC desalting cartridges to remove interfering salts.Even when the general purpose—purification or analysis—is known, it is still necessary to decide how detailed the separation needs to be and whether robustness or analytical depth should take priority.<blockquote>“Clearly defining your chromatographic goals is the first step toward selecting the right separation method.”Dr. Ulrike Krop, Head of Applications &amp; Academy, KNAUER</blockquote><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Figure_1_Decision_tree_Which_separation_technology_for_your_purification_task_5564a4be93.jpg" alt="KNAUER: Figure 1: Decision tree - Which separation technology for your purification task?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Figure_1_Decision_tree_Which_separation_technology_for_your_purification_task_5564a4be93.jpg 178w," sizes="100vw" width="1920" height="1683"><h4>Choosing a Method for Oligonucleotide Purification</h4>When the goal is purification, the amount of material to be processed plays a key role.If only small quantities of oligonucleotides need to be purified, columns with smaller internal diameters and lower flow rates are typically sufficient.For applications where high resolution is required, ion-pair reversed-phase chromatography (IP-RP) can be an effective option.Alternatively, laboratories that prioritize environmentally friendly eluents, cost efficiency, and high loading capacity may prefer anion exchange chromatography (AEX) for preparative separations.If the objective is mainly to separate smaller fragments or remove residual synthesis reagents, size exclusion chromatography (SEC) provides a simple and environmentally friendly solution.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Figure_2_Decision_tree_What_are_your_priorities_for_your_analysis_2e26d9955e.jpg" alt="KNAUER: Figure 2: Decision tree – What are your priorities for your analysis?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Figure_2_Decision_tree_What_are_your_priorities_for_your_analysis_2e26d9955e.jpg 163w," sizes="100vw" width="1920" height="1840"><h4>Selecting an Analytical Strategy</h4>In analytical workflows, the decision often comes down to a balance between maximum resolution and operational robustness.High-throughput laboratories that routinely perform similar analyses and require stable long-term operation typically choose between IP-RP and AEX methods.Among these approaches, ion-pair reversed-phase chromatography has become widely established due to its excellent efficiency and resolution. In addition, IP-RP methods generally offer good compatibility with mass spectrometry, making them suitable for detailed impurity profiling.Columns specifically designed for oligonucleotide separations, such as Sepapure oliGO columns, are optimized for this application and provide reliable performance across a wide range of workflows.<h4>Methods for Advanced Research Applications</h4>In research and development settings, analytical flexibility and sensitivity may be more important than routine efficiency.For example, if the goal is to identify minor differences in side-chain modifications between two otherwise identical oligonucleotides, techniques such as HILIC (hydrophilic interaction chromatography) or mixed-mode chromatography can be particularly useful.These methods provide additional separation mechanisms and allow extensive method optimization. Depending on the chosen eluents, they can also remain compatible with mass spectrometric detection.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Benefits_and_Limitations_at_a_Glance_34c0cc3066.jpg" alt="KNAUER: Benefits and Limitations at a Glance" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Benefits_and_Limitations_at_a_Glance_34c0cc3066.jpg 138w," sizes="100vw" width="959" height="1082"><h4>Do You Need Bioinert Hardware?</h4>Bioinert chromatographic hardware is commonly associated with protein purification, where metal surfaces can interact with amino acid residues such as histidine or cysteine and alter protein structure.To prevent these interactions, chromatographic systems may use passivated surfaces, titanium components, or glass-lined flow paths. However, these solutions can significantly increase costs.For oligonucleotide separations, the benefits of bioinert hardware are less clear.<blockquote>“I have performed many analyses with different oligonucleotides and have not observed significant differences between bioinert and stainless steel hardware. Only in extremely fast UHPLC runs—around three minutes—might this become relevant.”Marvin Schaek, Product Manager Oligosynthesizer, KNAUER &amp; XNAPharma GmbH</blockquote>Since oligonucleotide structure is generally less sensitive to conformational changes than proteins, stainless steel hardware often performs adequately.However, in cases where phosphorothioate backbones are present, interactions with metal surfaces may occur. Some studies suggest that column passivation can improve peak shape in very fast UHPLC separations, although the impact in typical 10-minute methods appears minimal.<h4>Final Thoughts</h4>Anyone working with oligonucleotides will inevitably encounter the need for purification and high-quality chromatographic analysis.Fortunately, a variety of chromatographic techniques are available, allowing scientists to tailor their workflows to specific analytical goals. By carefully evaluating sample characteristics, desired resolution, and operational priorities, laboratories can select the most appropriate column and separation method.For IP-RP workflows ranging from UHPLC to semi-preparative scale, columns such as Sepapure oliGO provide a reliable and cost-effective option specifically designed for oligonucleotide applications.<blockquote>If you are planning to develop or improve your oligonucleotide workflow, feel free to <a href="sales@knauer.net">contact the KNAUER team</a> for further guidance.</blockquote>

Oligos Made Easy - Part 6: Column Navigator

<h3>A Dedicated Solution for Oligonucleotide Analysis</h3>Oligonucleotides have become key players in modern pharmaceutical and biological research, particularly in the development of highly targeted and personalized therapies. As increasing numbers of oligonucleotide-based drugs enter clinical trials or receive regulatory approval, analytical laboratories face growing demands for both throughput and data quality. These molecules present unique chromatographic challenges due to their long, highly polar structures and subtle variations in nucleobase sequence or functionalization.<h3>Sepapure oliGO – Designed for Oligonucleotide Separations</h3>Selecting the optimal chromatographic approach for oligonucleotide analysis can be complex. Many laboratories rely on ion-pair reversed-phase (IP-RP) chromatography because of its speed, robustness, and compatibility with mass spectrometry. However, commonly used ion-pair reagents can degrade conventional silica-based stationary phases.<a target="_blank" rel="noopener noreferrer" href="https://www.knauer.net/columns-oligonucleotide-columns">Sepapure oliGO</a> addresses this limitation through a hybrid silica matrix combined with specialized surface end-capping, ensuring resistance to aggressive IP-RP reagents. Its functionalized surface enables both strong reversed-phase interactions and controlled ionic contributions, effectively creating a mixed-mode separation mechanism. This design results in improved durability compared with standard mixed-mode columns, making Sepapure oliGO a robust choice for routine oligonucleotide workflows.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_Sepapure_oli_GO_columns_with_laser_etched_column_information_23f545be81.jpg" alt="KNAUER: Figure 1 - Sepapure oliGO columns with laser-etched column information." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_Sepapure_oli_GO_columns_with_laser_etched_column_information_23f545be81.jpg 223w," sizes="100vw" width="1186" height="828"><h3>Method Transfer and Comparative Performance</h3>In collaboration with BianoGMP GmbH, KNAUER’s application team evaluated Sepapure oliGO against a widely used reference column. The study compared oligonucleotides of varying lengths and purities, including a fully thiolated 43-mer DNA. Particular attention was paid to potential interactions between thiolated oligonucleotides and stainless-steel hardware, which have been reported to cause peak tailing.The results demonstrated that direct method transfer between the reference column and Sepapure oliGO was generally feasible without significant adjustment. Chromatographic resolution remained comparable, while retention times were slightly shorter on Sepapure oliGO. Furthermore, the anticipated benefits of expensive bioinert hardware were not confirmed under the tested conditions, suggesting that such effects may primarily influence very fast (U)HPLC separations.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_Overlayed_chromatogram_of_crude_product_of_a_43mer_thiolate_oligonucleotide_7ae9613efd.jpg" alt="KNAUER: Figure 2: Overlayed chromatogram of crude product of a 43mer thiolate oligonucleotide. Light blue - reference column and dark blue – Sepapure oliGO." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_Overlayed_chromatogram_of_crude_product_of_a_43mer_thiolate_oligonucleotide_7ae9613efd.jpg 245w," sizes="100vw" width="1223" height="453"><h3>Low Backpressure for Reliable Operation</h3>Sepapure oliGO can be applied effectively in both HPLC and (U)HPLC workflows. Its low operating backpressure minimizes risks associated with clogging, excessive system wear, and performance deterioration over time. Typical IP-RP separations using 3 µm particles generated pressures around 200 bar, enabling laboratories without ultra-high-pressure systems to perform high-quality oligonucleotide analysis.<h3>Best Practices for Column Performance</h3>To maintain optimal performance, users should observe recommended operating conditions, including a broad pH range of 1.5–10 and temperatures up to 60 °C. Employing a guard column can prevent coarse synthesis by-products from reaching the analytical column. If pressure increases are observed, backflushing at low flow rates can restore performance.When the system is idle, thorough flushing to remove salts is essential. For storage, the column should be kept in a 1:1 mixture of methanol–water or acetonitrile–water. Gradient transitions are recommended when switching between standard RP eluents and IP-RP reagents. Regeneration procedures developed for RP columns are also suitable for Sepapure oliGO.<h3>Beyond Analytical Separations</h3>For large-scale purification, ion-exchange chromatography (AEX) may offer advantages. Scalable preparative systems are available to support purification workflows, ranging from compact laboratory setups to pilot-scale platforms with flow rates up to 800 mL/min. Bioinert configurations can be integrated where required, allowing laboratories to tailor purification solutions to their specific oligonucleotide applications.<blockquote><ul><li>If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.</li></ul></blockquote>

Oligos Made Easy - Part 8: Sepapure oliGO

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Oligos Made Easy - Part 8: Sepapure oliGO

Oligos Made Easy - Part 6: Column Navigator

Oligos Made Easy - Part 1: Overview

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Under Pressure: Mastering Backpressure in HPLC

PFAS in pharmaceuticals