The Purification of Oligonucleotides

As a new generation of gene medicine, oligonucleotides comprise 15-50 nucleotides, which can directly regulate gene expression and thus play a role in treating diseases. Oligonucleotides are usually used as primers for DNA sequencing, and their synthesis speed is fast, but the products are usually mixed with by-products of chemical reactions. Therefore, further purification of products is usually required to remove non-target substances in the chemical reaction, and the purification requires complicated procedures. High-performance liquid chromatography (HPLC) is the earliest chromatographic analysis way for the purification of drugs, containing reversed-phase chromatography (RP-HPLC) and anion-exchange chromatography (IE-HPLC).

Key Technologies

At CD BioGlyco, we leverage dual HPLC purification technologies:

• Reversed-phase HPLC (RP-HPLC) using C18 columns to exploit hydrophobic interactions with DMT groups for sequences under 100 bases.
• Ion-exchange HPLC (IE-HPLC) separating by charge differences without DMT dependency, ideal for polar oligonucleotides up to 40 bases.

We optimize mobile phases with ion-pairing reagents and ensure high-resolution purification for PCR, gene editing, and drug development applications.

HPLC-Purified: Where Every Base Counts.

CD BioGlyco provides mature HPLC-based oligonucleotide purification services, including purification of unmodified sequences as well as sequences with complex modifications, for example, linkers, spacers, and modified bases. C18 or ion-exchange chromatographic columns are used as tools for HPLC, which are mostly suitable for the purification of sequences with less than 100 bases. HPLC is a column chromatography, and usually, different types can be selected for purification according to the characteristics of the sequence.

RP-HPLC

Separation mechanism

Separation of target sequences from short sequences based on relative hydrophobicity.

• Retain
  Target sequences contain a dimethoxytrityl (DMT) unit at the 5' OH of the last base. When the hydrophobic 5'-DMT of the desired sequence After the column, hydrophobic interactions can be established with the alkyl chains on the surface of the stationary phase support, which can be highly retained by RP-HPLC.
• Elute
  Elute the desired sequence by cleaving the 5'-DMT.

Tips

• Due to the capacity and separation performance of RP-HPLC columns, it is often used as the method of choice for large-scale synthesis.
• As the length of the sequence increases, the hydrophobicity will decrease, while affecting the purity and yield of the product.
• Standard RP-HPLC can achieve >85% sequence purity.

IE-HPLC

Separation mechanism

Separation of target sequences from short sequences based on relative charge differences.

• Retain
  After the mixed sequence enters the chromatographic column, it forms a charge interaction with the polycationic stationary phase resin.
• Elute
  Short sequences are washed away through the ionic strength of the mobile phase increases. The desired sequence is then eluted at a higher ionic strength.

Tips

• Oligonucleotide drugs are highly polar, and the charged group (phosphate) in the backbone has a strong negative charge. Their retention is weak under conventional reversed-phase chromatography conditions, so it is necessary to add ion-pairing reagents to the mobile phase.
• IE-HPLC purification is possible in the absence of 5'-DMT on the sequence. The method has the characteristics of good separation selectivity, short analysis time, less sample consumption, and less solvent consumption.
• IE-HPLC is suitable for the purification of small amounts of sequences, usually no more than 40 bases.

Fig.1 HPLC-based oligonucleotide fragment purification. (CD BioGlyco)

Workflow

Our HPLC-based purification workflow is a meticulous, multi-step process designed to achieve the highest possible purity and yield.

• Sample Preparation

Following solid-phase synthesis, the crude oligonucleotide is cleaved from the solid support and deprotected. The crude product, which may still contain the 5'-terminal DMTr group for purification purposes, is dissolved in an appropriate buffer.

• Chromatographic Separation

The crude oligonucleotide solution is loaded onto a high-resolution HPLC column. A controlled mobile phase gradient is run, causing the shorter, non-tritylated failure sequences and other impurities to elute first, followed by the desired full-length product. Unwanted tritylated peaks usually elute later in the chromatogram.

• Fraction Collection

Based on the chromatogram, fractions containing the full-length oligonucleotide are carefully collected. We use real-time monitoring and advanced software to identify and isolate the product peak with high precision, often collecting the product between 50% of the peak maximum on the upside and 50% on the downside to maximize purity.

• Post-Purification Processing

The pooled fractions containing the purified oligonucleotide are then subjected to a post-purification step. The final product is desalted, concentrated, and lyophilized to a stable, solid form.

Publication Data

Applications

• Molecular biology: High-purity oligonucleotide fragments can be obtained for PCR amplification and sequencing reactions.
• Genetic engineering: Purified and synthesized sequence fragments can be applied to genome editing and protein expression regulation.
• Drug development: Purified drug candidates help to determine the purity, stability, and activity of drug candidates to support further research and evaluation of drug development.

Advantages

• CD BioGlyco provides efficient oligonucleotide fragment purification services that quickly separate target compounds and remove by-products.
• We have established strict quality management system production standards to ensure reliable experimental results.
• Our purification service has high resolution and will obtain high-purity products (over 85%).

Frequently Asked Questions

Associated Services

Our HPLC-based oligonucleotide purification delivers sequence-specific isolation of full-length oligonucleotide fragments by resolving critical impurities, essential for applications demanding molecular precision. To extend this analytical rigor to glycomics and glycoprotein analysis, we offer specialized Enzymatic Release Services that cleave and isolate key glycoconjugates from purified biomolecules:

• Enzymatic Release of N-Glycan
• Enzymatic Release of Fucose
• Enzymatic Release of Galactose
• Enzymatic Release of N-Acetylgalactosamine
• Enzymatic Release of O-Glycosylated Protein
• Enzymatic Release of Core-1 O-Glycan
• Enzymatic Release of Sialic Acid

CD BioGlyco offers mature HPLC purification services for your oligonucleotide fragment and delivers high-purity products. When using a single HPLC purification, sequences longer than 70 bases in length or sequences with more than 2 modifications are not guaranteed to be 85% pure. For these sequences, consider a two-step purification to ensure high purity. Other purification techniques used for oligonucleotides are also provided, including Desalination-based Oligonucleotide Fragment Purification, Cartridge (OPC)-based Oligonucleotide Fragment Purification, and PAGE-based Oligonucleotide Fragment Purification. Please feel free to contact us for detailed information if you are interested in oligonucleotide fragment purification services.