Oligonucleotide purification solutions

According to the latest data, from January to November 2023,31 nucleic acid drug companies in China have been financed with a total amount of nearly RMB 4 billion yuan, among which many companies have raised more than RMB 100 million yuan. The nucleic acid drug field is hot. (Medical Maike statistics, list of 31 companies attached at the end of the article)

As the representative of nucleic acid drugs, small nucleic acid drugs have higher targeted drug formation, high specificity and long efficacy by virtue of their direct effect on disease genetic targets, showing great potential. This unique advantage also makes it a common focus in the R & D and investment fields.

After the synthesis process of small nucleic acid drugs, there will be a variety of impurities, among which short nucleotides (n-x) or long nucleotides (n+x) are very close to the properties of the target products, so it is difficult to remove them. How to remove these product-related impurities while ensuring a high yield is a very big challenge for the purification process.

In this article, we take a look at oligonucleotide purification solutions that can be used for commercial production.

01 Basic concepts of oligonucleotides

Oligonucleotide drug, also known as oligonucleotide drug, is a short-chain nucleic acid composed of more than ten to dozens of nucleotides in series, which can achieve the purpose of treating diseases by interfering with the expression of target genes.

Small nucleic acid drugs in a narrow sense refer to siRNA (small RNA interfering drugs); small nucleic acid drugs in a broad sense refer to ASO (antisense oligonucleotide drugs) and miRNA targeting (microRNA), saRNA (small activating RNA), and Aptamer (RNA aptamer) in addition to siRNA.

The current global drug development focus is mainly on siRNA and ASO.

02 Oligonucleotide production process

The production of small nucleic acid drugs usually adopts chemical synthesis method, through target sequence screening, oligonucleotide synthesis, fragmentation and deprotection, purification, isolation and other steps. The synthesis of oligonucleotides is the key, and phosphoramidite chemical solid phase synthesis is widely used in the market at present. Although chemical modification can improve the stability of drugs, it also brings problems such as increased impurity types and high purification difficulty, which brings hidden dangers to the quality and safety of final drugs.

During the solid phase synthesis of oligonucleotides, various impurities are produced, including oligomers (short nucleotide N-x/long nucleotide N+x), by-products of modification reaction, incomplete products of deprotection, oligonucleotides lacking purine bases and other degradation products. To obtain high purity, high quality oligonucleotides, chromatographic purification must be used to remove these impurities.

Because these impurities are very similar to the target product in charge, molecular size and hydrophobic properties, it is difficult to purify the oligonucleotide. It is necessary to use high-resolution chromatographic media and chromatographic processes to complete the separation of impurities from the target product.

03 Oligonucleotide purification process

The quality and production efficiency of oligonucleotides can be ensured by using appropriate high-resolution chromatographic media and chromatographic processes.

In terms of purification process, common oligonucleotide purification methods are reverse chromatography (RP) and anion exchange chromatography (AEX), among which anion exchange chromatography (AEX) is recommended for the following reasons: firstly, it avoids the use of high-cost eluent containing organic solvents, does not need to be equipped with explosion-proof equipment, and can also reduce the cost of waste liquid treatment; secondly, the principle is simple, easy to amplify, and can effectively bind with negatively charged oligonucleotides.

Usually, a certain concentration of salt is used to remove impurities without DMT protection, then the tritylation is carried out on the column by acid washing, and finally the target oligonucleotide is isolated and purified by different salt concentrations. Purified samples require desalination and concentration (precipitation or TFF).

04 Selection of chromatographic medium

PolyGel 15Q/30Q Anion Exchange Chromatography Purified Oligonucleotides PolyGel 15Q Product Details

PolyGelTM 15Q and PolyGelTM 30Q are based on polymer PS-DVB.(Polystyrene-divinylbenzene copolymer) as the matrix anion exchange chromatography medium, with small particle sizes of 15μm and 30μm, using special functional group modification methods, specially tailored for the purification of small nucleic acid drugs, with extremely high resolution, large operating space, high loading capacity, even under denaturation conditions (pH 12), can still purify oligonucleotide samples, effectively avoid single strand self-complementation or aggregation of GC rich oligonucleotides, Especially suitable for oligonucleotide purity.

PolyGel 15Q anion exchange chromatography medium was used to purify oligonucleotide. After one-step purification, the purity could reach more than 96%, and the recovery rate was 85%~90%, which completely met the quality requirements.

Note: In addition to PolyGel 15Q, PolyGel 30Q is also a preferred oligonucleotide purification filler, which can be selected according to the molecular size, sequence and charge of the sample to be purified.

At present, high-quality oligonucleotide production capacity and stable supply chain have been scarce in China. Separation and purification of oligonucleotide final products from similar impurities has always been one of the difficulties restricting high quality and batch production of oligonucleotides.

As a rising star in the field of purification in China, Kono Biotech has faced up to difficulties. Combined with independent innovative technology, it has conducted in-depth research, fully considered the balance before product purity and recovery rate. Through multiple experimental verification of different batches of samples and different scales, it can ensure that the sample yield is above 85%, and its purity is above 95%. It provides customers with efficient oligonucleotide purification solutions. Help customers solve problems in production preparation.

product features

Two small particle sizes of 15μm and 30μm can be selected, with high resolution, high loading capacity and large operation space to meet the diverse purification requirements of oligonucleotides.

With polymer PS-DVB as matrix, it has thermal stability and chemical stability, which can realize the application of various technologies;

Operating at high temperatures can speed up mass transfer and improve the resolution of long-chain oligonucleotides.

Under denaturation conditions such as high pH, purification is not affected, and aggregation of modified, self-complementary and GC-rich oligonucleotides is effectively avoided;

Excellent performance, easy to scale up, low cost, can meet the needs of different scales from laboratory to industrial production.

Appendix: Domestic Financing in Nucleic Acid Drug Field from January to November 2023