Research and Development

Impact of Impurities in Amidites on
Quality of Oligonucleotides

Quality Control of Amidites
for Improved Control of Product Quality and Purity

Phosphoramidites (amidites), which comprise nucleic base, A, C, G, U or T, are the starting materials of oligonucleotides. Impurities present in an amidite may be incorporated into the oligonucleotide during synthesis and cause some oligonucleotide-related impurities. It is difficult to remove these impurities from the oligonucleotide even through the use of state-of-the-art purification technology;
therefore, quality control of amidites plays an important role in assuring the quality of oligonucleotides.

Critical Impurities in Amidites

Impurities in an amidite can be divided into three classes (see Figure below): Classes 1 and 2 are classified as noncritical, while Class 3 is classified as critical. Critical impurities are believed to be reactive and have the potential to be inserted in a growing chain of oligonucleotides during synthesis, ultimately affecting oligonucleotide quality.

Quality Control of Amidites´╝ÜImpurities in Amidites
Class 1 Non-reactive and non-critical
Class 2 Reactive but non-critical
Class 3 Reactive and critical image

Strict Impurity Control in Amidites

Even a slight amount (0.2%) of a Class 3 impurity present in an amidite theoretically causes 10% impurity in the oligonucleotide. Therefore, this class of critical impurity should be closely controlled and monitored using appropriate analytical tools such as HPLC. The following LC chart shows a model solution containing various types of Class 3 impurity that was analyzed using the HPLC method. This method is derived from our expertise and experience in the field of analytical and organic chemistry through R&D, production, and quality assurance of low-molecular-weight drugs.

For example, to closely control oligonucleotide quality, we developed more than 10 analytical methods for controlling impurities in amidites with more than 50 synthesized standard samples of impurities.

Impact of Impurities in Amidites on Quality of Oligonucleotide

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Quality Control of Amidites through Chemical Processes

To control the quality of amidites, we have developed chemical processes for amidites based on data from parameter studies that use our established analytical methods, along with expertise and experience in the area of organic synthesis and chemical production.

Ex) LC Chart Spiked with Amidite Impurities
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Develop another method if separation is poor!