Nucleic acid drugs, including small nucleic acids and mRNA drugs, represent a rapidly growing segment within the biopharmaceutical field. With continuous technological iterations, particularly innovations in nucleic acid chemical modifications and delivery technologies, several nucleic acid drugs have been successfully marketed, demonstrating remarkable efficacy across multiple therapeutic areas such as rare diseases, chronic diseases, oncology, and genetic disorders.
As a crucial category within nucleic acid drugs, oligonucleotide therapies involve the active design of drug sequences to precisely target and silence disease-related genes, offering a novel choice in therapeutic mechanisms. Compared to traditional drugs, small nucleic acid drugs possess significant advantages, including a broad therapeutic scope, shorter development cycles, higher success rates in development, and long-lasting therapeutic effects. This article will focus on introducing the core material selection and impurity analysis strategies in the production process of nucleic acid drugs.
01 Chemical Modifications
The Key to Enhancing the Efficacy of Nucleic Acid DrugsTo improve the stability, enhance the functionality, and reduce the side effects of nucleic acid drugs, chemical modifications of the core raw materials have become a critical approach. This involves various structural alterations to the nucleic acid molecules, such as:
DNA Series
Chemical modifications of DNA monomers hold significant importance in nucleic acid drug design, particularly in developing ASO drugs for gene regulation. Common modifications include 2'-deoxynucleotides (e.g., 2'-deoxythymidine) and 2'-fluoro-deoxynucleotides (e.g., 2'-fluorothymidine).
MOE Series
MOE modification enhances RNA stability and affinity by introducing a larger methoxyethyl group at the 2'-OH position. Applied in Antisense Oligonucleotides (ASO) drugs, such as Spinraza (nusinersen), used for treating Spinal Muscular Atrophy (SMA).
2'F/OMe Series
2'-OMe and 2'-F modifications are now used in almost all advanced siRNA designs. These two modifications reduce the vulnerability of the 2'-OH while maintaining the pre-organized A-type helix of the guide strand, which is necessary for successful RISC recognition and loading. They effectively increase RNA thermal stability and resistance to nuclease degradation, while reducing immunostimulation. An example is the siRNA drug Vutrisiran, used for treating hereditary transthyretin-mediated amyloidosis with polyneuropathy.
PMO Series
PMO monomers are a class of synthetic oligonucleotides with high affinity and stability, where the traditional ribose ring is replaced by a morpholino ring. PMO modifications enhance their resistance to nucleases, granting them a long half-life and good tissue penetration in therapeutic applications.
PS (Phosphorothioate Backbone) Modification
Because exonucleases can efficiently degrade RNA and DNA without relying on 2'-OH recognition, phosphorothioate (PS) modification is typically applied to the ends of siRNAs to address this issue. This is currently the primary strategy for terminal stabilization. In fact, simply adding two PS-modified backbone linkages at the 5' end can increase exonuclease stability by several orders of magnitude. For instance, a key difference between Alnylam's STC (Stabilized Trigger Complex) and ESC (Enhanced Stabilization Complex) platforms lies in the number of 5'-end PS modifications.
In PS modification, the phosphorus atom acts as a chiral center, resulting in two diastereomers, Rp and Sp. Currently, three trivalent GalNAc (N-acetylgalactosamine)-RNAi conjugate drugs have been approved: GIVLAARI®, OXLUMO®, and LEQVIO®. These three RNAi drugs are all mixtures of 64 diastereomers, generated by 6 randomly chiral PS modifications.
It is noteworthy that different stereoisomers (R and S configurations) of chiral drugs may exhibit significant differences in biological activity, efficacy, and safety. The ratio of R and S configurations has a major impact on the activity and efficacy of small nucleic acid drugs. Therefore, identifying stereoisomers represents an important direction for progress in oligonucleotide drug development.
02 Impurity Control Strategy
Impurity control at every stage of the small nucleic acid drug production process is crucial. Currently, the main impurities in small nucleic acids include: (1) Impurities with the same structure and sequence as the parent molecule; (2) Impurities containing only naturally occurring structural elements found in nucleic acids; (3) Sequence variants of the parent oligonucleotide; (4) Impurities containing structural elements not present in the parent oligonucleotide or naturally occurring nucleic acids.
- Terminal deletion (n-x)
- Unconjugated oligonucleotide (n-L)
- Single-strand impurities (in siRNA)
- Hydrolysis products (P=S -> P=O)
- 2',5'-Linked sugars in RNA
- Internal deletion (n-x)
- Dimer (n+x)
- Deletion (base fragment loss)
- Branched structure (n+x)
- Backbone modification
- Base modification
- Base oxidation
- Chain cleavage (depurination/depyrimidination)
- Transformation (n+Me)
- Acrylonitrile adduct (CNET)
- Residual protecting groups
- CAPALDI ET AL. NUCLEIC ACID THERAPEUTICS Volume 27, Number 6, 2017
- Guideline on the Development and Manufacture of 5 Oligonucleotides (Draft), July 2024
Each step in solid-phase synthesis has targeted impurity control strategies. These stringent impurity control measures ensure the high efficacy and safety of small nucleic acid drugs, providing reliable assurance for clinical applications.
- (1) Clearing reagents, solvents, and reaction by-products after each synthesis step ensures the purity of the solid support, thereby preventing the accumulation of insoluble impurities.
- (2) The cleavage and deprotection steps aim to remove the solid support and its associated insoluble impurities, further enhancing product purity.
- (3) The crude product ultrafiltration step further purifies the product by removing low-molecular-weight impurities, reagents, and reaction by-products.
- (4) The chromatographic purification step focuses on removing impurities from the oligonucleotide process, as well as impurities with different charge states and hydrophobicity, ensuring the quality of the final product.
- (5) Finally, the final ultrafiltration/diafiltration (UF/DF) step removes HPLC solvents and buffer salts to obtain the high-purity final product.
03 Conclusion
The rapid development of the nucleic acid drug market is an inevitable result of advancements in medical technology and growing clinical needs. With the launch of more innovative drugs and increasing market acceptance, small nucleic acid drugs are expected to become a major pillar in the pharmaceutical field in the coming years, offering more treatment options and health benefits to patients worldwide!