Key Phosphoramidite Considerations for Oligonucleotide Manufacturing

Oligos can be synthesized in two ways: chemically and biosynthetically. Chemical synthesis, which is the industry-wide standard for oligo synthesis and the focus of this article, occurs through chemical reactions using phosphoramidites as the building blocks. Biosynthesis occurs via enzymatic reactions and is an emerging commercial technology for oligonucleotide synthesis.

Phosphoramidite method of oligonucleotide manufacturing

The phosphoramidite method is the dominant chemical approach used for oligonucleotide manufacturing because it enables rapid, precise, and scalable synthesis of DNA and RNA sequences. In this solid-phase synthesis process, the first nucleoside is attached to an insoluble support, and additional nucleoside phosphoramidites are added sequentially through automated synthesis cycles. Each cycle includes four core steps: deblocking to expose the growing chain’s 5′-OH, coupling of an activated nucleoside phosphoramidite, capping of unreacted sites to help prevent sequence errors, and oxidation or sulfurization to stabilize the backbone linkage (Figure 1). By repeating these cycles, highly defined oligonucleotide sequences can be assembled one base at a time with high efficiency. The phosphoramidite platform also supports incorporation of therapeutic modifications—such as phosphorothioate backbones, 2′-O-methyl groups, and locked nucleic acids (LNAs) which improve nuclease resistance, binding affinity, and pharmacological properties. After synthesis, the oligonucleotide is cleaved from the solid support, deprotected, purified, and quality tested, making phosphoramidite chemistry the foundation of modern oligonucleotide therapeutics, primers, probes, and synthetic biology applications.

DNA and RNA oligos can be used in various diagnostic applications for genetic diseases, infectious diseases, pharmacogenomics, and more. Oligos, which are commonly used as primers and probes in these diagnostic techniques, bind specific target sequences—primarily nucleic acids but sometimes proteins and small molecules (as in the case of aptamers)—for target detection. These diagnostic assays commonly use oligo primers and/or probes in techniques such as PCR, microarrays, sequencing, and in situ hybridization.

Another application of oligos is in therapeutics, where short, synthetic oligonucleotides modulate gene expression or function through antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), or guide RNAs (gRNAs) used in CRISPR gene editing. Therapies using these approaches have been approved or are currently going through clinical trials to treat diseases such as muscular dystrophy, polyneuropathy, and hypercholesterolemia.

DNA or RNA oligo manufacturing begins with designing an oligo of a specific nucleic acid sequence (with modifications as needed), to chemically synthesize, purify, and analyze. This is followed by formulation and production for its specific application. Automated oligo synthesizers complete sequential chemical reactions using phosphoramidites to produce the nucleotide chains of synthetic oligos. Typically, oligo synthesizers add phosphoramidites as the building blocks from the 3′ to 5′ direction, the opposite of enzymatic synthesis. One advantage of synthesizing oligonucleotides chemically is that modifications or labeling of the building blocks necessary for final oligo function can be more easily introduced than through enzymatic methods.

Phosphoramidites used for therapeutic applications should be considered with some attributes different from diagnostic usage in mind. First and foremost for therapeutics, the raw materials must be produced under robust quality systems with appropriate quality management and documentation with extensive testing for consistency, purity, traceability, and stability, to help meet requirements set by regulatory bodies. In addition, manufacturing must be scalable, especially when a therapeutic is ready to go into clinical trials and eventually manufacturing.

Because of their therapeutic uses, phosphoramidites are often modified to increase nuclease resistance and thermal stability while lowering their immunogenicity. Such modifications include 2′-O-methyl (2′-OMe), 2′-O-methoxy-ethoxy (2′-MOE), and 2′-fluoro. For delivery to target organs, conjugating moieties such as N-acetylgalactosamine (GalNAc) to the oligos for uptake (e.g., in the liver, by hepatocytes) is a common approach.

In addition to these modifications, controlling impurities that can be generated during phosphoramidites synthesis is critical to the development of your therapeutic. Impurities are defined by their reactivity in oligonucleotide synthesis or by the ability to be removed during the manufacturing process. They can be categorized as:

a. Critical impurities

• Potentially are incorporated into oligonucleotides
• Contain amidite functionality and acid-labile protecting group

b. Noncritical impurities

• Cannot be incorporated into oligonucleotides
• Could also be reactive but are removed in processing

A top concern for developers of oligo therapeutics would be impurities that are reactive and critical. Nonreactive and noncritical impurities as well as reactive but noncritical impurities are less of a concern. The presence of reactive and critical impurities can be limited through manufacturing processes implemented by phosphoramidite suppliers, such as rigorous control of raw material specifications, in-process manufacturing controls and testing, and final release testing.

Certain aspects of phosphoramidites should be considered when they are used in the manufacturing of oligos for diagnostic purposes. At minimum, your phosphoramidite supplier should be able to accommodate small, manageable starting synthesis scales, with the eventual ability to scale to the industrial scale required for many diagnostic kits. Options for base modifications, linkers, and dye labeling should also be available. Table 1 describes some modifications that are common for phosphoramidites as used in diagnostic applications.

Table 1. Phosphoramidite modifications of oligos for diagnostic applications.

For a more visual explanation of this topic, please access our phosphoramidite infographic