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Synthesizing the molecule chemically will enable therapeutic oligonucleotides (TOs) to be produced. TOs can be used to modulate the expression of one or more genes. They can also be used to fix mutations/lesions in genes to reinstate normal function. Additionally, they can be used to target/guide gene editing nucleases to your regions of interest within the genome.
TOs are drug candidates used to treat diseases using virtually every mechanism that alters gene expression or edits the genome. ASOs induce degradation of mRNA or block its translation. siRNA hybridizes to mRNA and directs it for RNAi mediated degradation. These antisense oligonucleotide therapies are dependent on stability, uptake, targeting and minimal side effects.
| Therapeutic Class | Molecular Structure | Primary Mechanism |
| ASOs | Single-stranded DNA or RNA | RNase H-mediated cleavage or steric blocking |
| siRNAs | Double-stranded RNA | RNA interference pathway |
| gRNAs | Single-stranded RNA with scaffold | Nuclease targeting and DNA cleavage |
| mRNA | Single-stranded coding RNA | In vivo protein translation |
Table 1 Therapeutic Oligonucleotide Classes and Their Mechanisms
Classes of TOs include ASOs, short interfering RNAs, gRNAs, and mRNAs. Antisense oligonucleotides are often single-stranded oligonucleotides complementary to their target mRNA sequence. ASOs can induce degradation by cellular nucleases or sterically block translation. Short interfering RNAs are double-stranded oligonucleotides that use incorporated protein factors to induce cleavage of target transcripts through endogenous RISC activity. Guide RNAs direct customizable protein nucleases to bind and cleave genomic DNA at desired loci and are composed of a sequence-specific region and a constant handle region. Messenger RNAs are transcripts that direct the translation of proteins. Synthetic mRNAs can be used to induce immunity to pathogens (as mRNA vaccines), for enzyme replacement therapy or protein supplementation. For these oligonucleotides to have a desirable shelf life and activity within the body they may need chemical modifications such as backbone modification, sugar modification, or conjugation to the ends.
Mechanism of action of antisense oligonucleotides.1,5
Chemicals used for therapeutic purposes need to meet a higher quality standard than those used in experimental settings. Shorter oligonucleotides, deletions, or deprotected precursors may have off-target effects or cause unforeseen immunological responses. They may also take up binding sites that would otherwise be available to the active compound or be metabolized into toxic products in vivo. Guidelines from regulatory bodies therefore require extensive characterization of the active pharmaceutical ingredient (identity, purity, qualification of impurities), usually with acceptance criteria tighter than >95% of full-length species for certain classes of drugs. Therapeutic oligonucleotides must be produced following good manufacturing practice (GMP) guidelines on validated equipment/procedures in cleanrooms. Ultimately, the needs of therapeutics translate to higher oligonucleotide volumes and increasingly intricate modification profiles.
Synthesis scales are also very different when making oligonucleotides for therapeutic use versus research use. Scale-wise, it is common practice to synthesize milligram quantities of oligonucleotides for research purposes. Correspondingly, typical purity measurements involve a single analytical method that provides researchers with a general idea of the purity of the oligonucleotide. Ultra-pure oligonucleotides for therapeutic use are produced in kg quantities under good manufacturing practice regulations with fully validated synthetic methods and purification methodologies. Thus it is essential that all synthetic processes and methods are fully validated and can detect impurities below 1%. Additionally process controls must be put into place to ensure batch-to-batch consistency. This is also driven by the fact that these oligonucleotides will be used directly in patients and therefore identity, purity and potency need to be rigorously defined and maintained throughout the shelf life of the drug. Requirements are significantly more stringent for oligonucleotides containing chemical modifications such as phosphorothioates. In addition to controlling stereochemical outcome at the site of modification, these modifications can introduce impurities that must be carefully controlled beyond what is typical in academic synthesis labs.
Scale-up is another issue. A research chemical might be prepared on the scale of milligrams. For human treatment purposes, several kilograms may be needed. There is a challenge associated with scaling up synthesis so that the clinical batches have comparable quality to the research batches. All reaction conditions used in the solid phase chemical synthesis need to be proven to give good coupling efficiency and deprotection rates independent of the size of the reactor. Moreover, the purification process needs to work efficiently with larger volumes of crude material. The reproducibility from batch-to-batch is crucial for therapeutic oligonucleotides. Certain levels of truncated species, backbone modification and incorrect stereochemistry may be well tolerated in research materials. However, these impurities may cause unwanted side effects or diminished activity of therapeutics. In-process control parameters must be monitored and controlled so that batch meets its specification. Any batch not meeting its specification must be held until the issue is resolved.
Synthesis of therapeutic oligonucleotides is highly regulated compared to those used for research. Regulatory guidelines ensure that synthetic oligonucleotides intended for human use are consistent with good manufacturing practice and have defined chemistry, manufacturing and controls. For example, regulatory agencies require greater characterization of the active pharmaceutical ingredient (API). Sequence verification must be performed on the API, along with testing for possible impurities and their levels. Stability studies must also be conducted to ensure integrity of the product under various storage conditions. As well, oligonucleotides for therapeutic use must meet higher purity standards to avoid immunogenicity and off-target binding than those for research purposes. Limits are put in place for residual solvents, heavy metals and other potential biological contaminants. Manufacturing must take place in clean rooms by qualified personnel using validated equipment. Traceability is important, so detailed documentation is required from starting materials all the way to finished product release. These regulations increase both cost and time associated with therapeutic oligonucleotide production.
Factors to consider when designing therapeutic oligonucleotides include many that are common to basic oligonucleotide design with the additional requirement that desired activity be coupled with favorable toxicity. Therapeutics are distinguished from research oligonucleotides by a set of characteristics that allow the molecule to persist intact in vivo and access their target sequences. This set of requirements often necessitates compromises between affinity and other desired attributes in order to yield viable drug candidates. Attributes considered during therapeutic oligonucleotide design include sequence selection, chemical modifications, and secondary structure. Sequence selection algorithms can be employed to increase drug-like properties such as target accessibility, cell permeability, and pharmacokinetics. Chemical modifications provide levers during design to tune properties such as increasing stability, limiting immune stimulation and tuning thermodynamic properties by changing the nucleotide recipe.
Optimization identifies accessible target sites in mRNA or genomic DNA to bind to without interfering secondary structure. In antisense and small interfering RNA design, this means finding accessible areas of the transcriptome with little secondary structure that would otherwise hide the target site from the drug. Also, the duplex energy between the therapeutic oligonucleotide and target should not be too high as to render the complex inert to dissociation (failed release into cell) or degradation (when degradation is desired). Computational identification of chemical modifications that increase binding affinity and resistance to nucleases such as locked nucleic acids (LNAs) or constrained ethyl (cEt) can be placed within the sequence to maintain high target affinity while limiting self-targeting. Length of oligonucleotide and percentage of G-C pairs are also adjusted to attain an optimal melting temperature with reduced likelihood of self-targeting.
Off-target cross-talk with undesired targets is another safety aspect to consider. Antagonistic oligonucleotides are capable of forming hybrid complexes with transcripts other than the target sequence provided there is homology. Off-target binding can lead to silencing of genes required for normal cell functions or can induce toxic effects. Transcriptome-wide in silico analysis can be used to filter out sequences that have high homology to other genes. Sequences with potential perfect seed matches are of particular interest. Activation of the innate immune system is another safety concern mediated by sequence specific elements that can bind and activate pattern recognition receptors such as toll-like receptors or cytosolic receptors. Modified nucleotides such as substitution of uridine with pseudouridine and/or 5-methylcytidine, as well as modification of the backbone itself can change immunogenicity profiles. In this way the oligonucleotide can avoid recognition by the immune system yet retain its pharmacologic properties. Backbone chemistries can also be chosen to avoid activating cellular processes that recognize foreign nucleic acids decreasing the likelihood of undesirable immunogenicity.
Chemical modifications are essential to converting unstable RNA into a drug that can persist within the body and reach therapeutically relevant levels in tissues of interest. Unmodified RNA is susceptible to degradation by ribonucleases that are found throughout the body and is rapidly removed from the bloodstream by renal filtration. Modifications can help to address these issues by increasing stability, improving cellular uptake, and aiding in targeting of therapeutics to areas of interest. Many types of modifications can be employed including: modifications to the ribose at the 2'-position, backbone modifications that replace the phosphodiester linkage, and conjugation of ligands or hydrophilic polymers to the ends of the RNA. These can be used in various positions of the RNA sequence and must be chosen such that they do not impede the intended activity of the RNA (i.e. promoting degradation of the transcript through antisense mechanisms, inducing RNAi, or increasing translation) while improving the pharmacological characteristics.
Structures of AON chemical modifications and the GalNAc conjugate.2,5
Modifications to the sugar moiety at the 2'-position represent the most commonly used approach. 2'-O-methyl and 2'-O-methoxyethyl groups enhance nuclease stability by obstructing enzymatic cleavage of the phosphodiester linkage and increase duplex melting temperatures. 2'-Fluoro substitutions greatly improve metabolic stability and affinity while being more closely linked to toxicity issues. Backbone modifications such as phosphorothioate backbones have been demonstrated to significantly improve resistance to exo- and endonucleases with maintenance of activity through recruitment of proteins to elicit functionality. Chirality also affects how phosphorothioate-modified RNAs behave in the body. Stereoselective synthesis of phosphorothioate RNA is now possible and enantiomerically pure RNA backbones can be synthesized.
Conjugation of RNA therapeutics to macromolecules is one strategy to promote cellular uptake. Conjugating GalNAc to siRNA therapeutics enables efficient hepatocyte specific delivery by receptor mediated endocytosis. GalNAc targets asialoglycoprotein receptors which are overexpressed on hepatocytes. Using this method, treatments can still be remarkably effective, even in small amounts. Many different diseases which were previously inaccessible to RNA therapeutics can now be targeted by GalNac-conjugated therapeutics.
| Modification Category | Specific Chemistry | Primary Function | Typical Application |
| Sugar modifications | 2'-O-methyl, 2'-fluoro | Nuclease resistance, affinity enhancement | Antisense, siRNA |
| Backbone modifications | Phosphorothioate | Metabolic stability, protein recruitment | Broad therapeutic classes |
| Targeting conjugates | GalNAc multimers | Hepatocyte-specific delivery | Liver-targeted therapeutics |
| Pharmacokinetic modifiers | Polyethylene glycol | Extended circulation, reduced clearance | Systemic |
Table 2 Chemical Modifications in RNA Therapeutics
Production of clinical scale therapeutic oligonucleotides is challenging. Optimization of process chemistry parameters increases in complexity with longer sequences, incorporation of chemical modifications, and scale of manufacture. Current mainstream oligonucleotide production via solid-phase phosphoramidite chemistry becomes inefficient for the highly modified and longer sequences used in contemporary RNA therapeutics. When dealing with such sequences challenges with lowering synthetic yield, increasing impurities, lack of process control, etc., become more pronounced. Bulk synthesis also presents many unforeseen hazards such as scale-up issues that affect the upper limits of reactors and purification, and validating analytical methods. Rigorous process development, analytics testing, and quality control are required to successfully produce clinical oligos.
Chemical synthesis is less efficient as oligonucleotide length increases and shorter oligonucleotides have higher synthetic yields than longer oligonucleotides. Given this, if we assume typical DNA synthesis conditions, we should only expect to have ~60% full length material for a 100-mer oligonucleotide. Therapeutic oligonucleotides are frequently much longer (200 nt+), therefore even with 99% coupling efficiency the full length product will be negligible. Also, depending on the modification, chemical synthesis of modified oligonucleotides can present unique challenges. Steric bulk from additions such as fluorophores, backbone modification, or appending a group to the termini can hinder phosphoramidite addition. Lengthy secondary structures can prevent further elongation of the oligonucleotide. Modified chemistries can also slow down the coupling reaction or be incompatible with existing reagents. All of these effects require individualized synthesis protocols, longer coupling times, and creative purification techniques to obtain product from the reaction mixture that contains a significant amount of truncated sequences.
Transitioning from gram-scale to multi-kilogram-scale synthesis of therapeutic oligonucleotides demands rigorous process control to ensure consistent quality. Scaling up often requires modifications to fixed-bed reactors to handle larger volumes, with mixing and mass transfer considerations becoming more complex; insufficient agitation can result in channeling effects and suboptimal reaction efficiency. Heat management is another critical aspect during scale-up because the exothermic nature of coupling reactions can lead to excessive temperatures that degrade reactants or cause unwanted side reactions if not properly controlled. Furthermore, variability in the quality of raw materials from different suppliers may impact reaction efficiency, thus thorough supplier qualification and incoming quality checks become essential. Validation of scale-up processes involves confirming that key variables such as coupling duration, concentrations, and cleaning steps are scalable and documented changes to these processes may necessitate additional regulatory submissions.
The purification and testing requirements of drug oligonucleotides are much more rigorous than those of research oligonucleotides. Complex mixtures produced by chemical synthesis of oligonucleotides contain deletion products, truncated oligomers, and stereoisomers. As clinical-grade oligonucleotides must have essentially no side-effects, it is critical to remove these synthetic impurities. Characterization and purification methods must separate species that often only have a single chemical difference. Removal of these synthetic byproducts requires the development of orthogonal chromatographic purification methods. Adequate testing must confirm the identity of the molecule, both structurally and chemically. Quality control data will need to demonstrate the ability to produce drug oligonucleotides at a consistent level from batch to batch.
For therapeutic applications of oligonucleotides, purifications exceeding those afforded by desalting or precipitation methods typical of research grade oligonucleotides are required. In addition to the desired full-length product, synthesis results in failure molecules including n-1 and n+1 truncation sequences. Backbone modifications result in related species including chemical modifications of the backbone and stereoisomers that differ only by the orientation of the sulfur atom in phosphorothioate internucleotide linkages. Therefore, most therapeutic oligonucleotides are purified using high-resolution ion-pair reversed-phase chromatography first pioneered by Love et al. Lipophilic ion-pair reagents increase the overall hydrophobicity of the oligonucleotide such that it can be separated on organic reversed-phase columns. Ion-pair reversed-phase separations can resolve single nucleotide or stereochemical differences in the impurities. As an alternative, charge differences between full length products and deletion sequences can be resolved using ion-exchange chromatography. This selectivity can be highly useful and complementary to ion-pair reversed-phase separations for backbone chemistries with highly charged backbones. These methods can be optimized to resolve the various deletion sequences and other closely eluting impurities to baseline while preserving the modification integrity. Therapeutic oligonucleotides are purified to very high levels of purity in order to reduce immunogenicity and provide consistent biological activity.
Typically extensive characterization is performed to ensure that the purified oligonucleotide has the intended sequence and meets predefined purity criteria before being considered for clinical use. Identity can be confirmed through mass spectrometry, which can determine accurate molecular weight information to confirm the predicted full length molecular weight of the oligonucleotide sequence and ascertain the presence of any desired modifications such as backbone modifications/conjugations or end group conjugations. Impurities can also be characterized by LC/MS. Mass spectrometry can be used to help confirm the presence of undesired byproducts such as sequence impurities, deletion products, and oxidation products. Quantitative purity can be assessed by resolving the full length product from degradation products and synthetic byproducts (usually truncated strands) using HPLC or capillary electrophoresis. Components are separated based on hydrophobicity (reverse-phase chromatography) or charge-to-size ratio. All analytical methods should be validated to meet acceptance criteria for specificity, sensitivity, and accuracy according to current good manufacturing practices (CGMP) prior to use in a therapeutic manufacturing process.
Oligonucleotide GMP manufacturing is tightly regulated and requires much more than a research-scale oligonucleotide synthesis facility. The GMP standard demands controlled rooms of defined cleanliness, validated equipment and methods, stringent documentation practices, trained staff and validated processes to ensure identity, purity and potency of therapeutic oligonucleotides. Processes must be shown to consistently produce batches meeting all product specifications and have controls in place to monitor the critical process parameters. Complete traceability of all materials used in the production process must be tightly controlled from receipt of starting materials through shipping of finished goods. Validated methods for testing each attribute of identity, purity and potency are required. Materials may need to meet predefined acceptance criteria. GMP regulations and requirements can grow more complex as programs progress through clinical development into commercial production. Agencies like the FDA and EMA will review quality systems through regulatory submissions and facility inspections.
Records are the basis for GMP compliance. Critical aspects of the oligonucleotide manufacturing process should be controlled, monitored and reproducible as documented in SOPs. Batch records need to include specifics about raw material lot numbers, equipment, processing parameters, in-process test results and any deviations. Process validation needs to prove that the process is able to consistently produce product meeting specifications. Therefore it needs to be well documented, including development studies, batches used for qualification and continued process verification. Traceability allows for tracing all components from suppliers through synthesis and purification steps up to final release. Problems can thus be easily traced back and if necessary rapidly contained. All these records allow for preparing regulatory submissions and discussions with the Health Authorities about the quality of the product and compliance of the manufacturing process.
The expectations of the regulatory agencies will change as the product advances from preclinical development through clinical trials and into commercialization. During the early stages of IND submission, the chemistry, manufacturing, and controls need to be described sufficient to ensure that the material to be administered to patients is well characterized and safe. At this stage, the agency will allow some flexibility in the definition of the manufacturing process and the refinement of product specifications. As clinical development progresses, the agency expects a better defined manufacturing process, narrower specifications justified with increased stability data and clinical experience, and validated analytical methods. Commercial manufacture will require validated processes, defined specifications with justification, and a stability program adequate to support the proposed shelf life. The sponsor should communicate frequently with the regulatory agencies to ensure that the CMC plan is meeting the expectations of the agency. Keep in mind that there is limited specific guidance for the manufacturing of synthetic oligonucleotides when compared to many small molecule drugs.
| Quality System Element | Implementation Requirement | Regulatory Purpose |
| Documentation | Comprehensive batch records and SOPs | Process control and traceability |
| Process validation | Demonstrated consistent performance | Reproducible product quality |
| Analytical validation | Verified test method performance | Reliable quality assessment |
| Change control | Formal evaluation of modifications | Maintained validated state |
| Stability program | Long-term and accelerated studies | Established shelf life and storage |
| Regulatory engagement | Proactive authority communication | Alignment with |
Table 3 GMP Requirements for Oligonucleotide Manufacturing
Therapeutic oligonucleotide synthesis requires significantly higher technical control than standard research-grade production. Sequence length, backbone modifications, conjugation strategies, and regulatory requirements all influence manufacturing complexity. Our capabilities in oligonucleotide synthesis for gene therapy and RNA therapeutics include:
By integrating controlled synthesis chemistry, purification expertise, and structured quality systems, we support oligonucleotide programs progressing from discovery to clinical evaluation.
Gene therapy and RNA therapeutic programs often face challenges related to sequence complexity, modification density, scalability, and regulatory compliance. Early alignment between molecular design and manufacturing strategy can reduce development risk and timeline delays. If your program involves:
a technical consultation can help clarify synthesis feasibility, purification requirements, and quality expectations at your current development stage. Contact our team to discuss your oligonucleotide synthesis strategy for gene therapy or RNA therapeutics and determine the appropriate manufacturing pathway for your program.
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