Understanding and analyzing oligonucleotides is critical for ensuring the identity, purity, quality, and structural integrity of these therapeutics. At BioPharmaSpec, we offer expert-driven, fully outsourced oligonucleotide characterization services designed specifically to meet the challenges of oligonucleotide analytical development.
Our specialized techniques, including high-resolution mass spectrometric analysis, provide structural elucidation and impurity profiling to support your oligonucleotide development from early-stage research through regulatory submission.
We provide a broad suite of analytical solutions for oligonucleotide therapeutics, including:
- Intact oligonucleotide analysis (Molecular Mass Determination)
- Sequence confirmation and identity testing
- Counterion analysis
- Spectral characterization (31P, HSQC and DOSY NMR, CD, FT-IR)
- Thermal stability (melting temperature, DSC analysis)
- Identification as duplex (for siRNA duplexes)
- Structure elucidation for Antibody-Oligonucleotide Conjugates (AOCs)
- Product and process-related impurities
- Modified linkages
- Stereoisomers
- Insertion, deletion and truncated sequences
- Base modifications
- Cross-linked structures
- Residual single stranded oligonucleotides (in double stranded products)
- Aggregation
- Process-related: residual solvents, elemental impurities etc.
What is an Oligonucleotide?
Oligonucleotides are short polymeric sequences of nucleotides, encompassing RNA, DNA, and their analogs. Initially, synthetic oligonucleotides were primarily used as reagents in life science research, such as DNA-based diagnostic kits. More recently, they have become increasingly important as therapeutic agents, forming the basis of nucleic-acid-based drug development.
The term "oligonucleotide" refers to a diverse range of molecules consisting of short, single- or double- stranded DNA or RNA. These synthetic nucleic acid polymers have a broad application to a wide scope of disease states. The chemical modifications and formulations required for therapeutic efficacy add further complexity to their characterization, making detailed structural and physicochemical analysis essential for ensuring product integrity and regulatory compliance.
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Why Choose Biopharmaspec?
- Expert Scientists: Highly trained specialists with deep analytical expertise in oligonucleotide characterization.
- Cost-Effective Outsourcing: Avoid the significant costs of purchasing, maintaining, and staffing complex instrumentation while gaining access to state-of-the-art capabilities.
- Comprehensive Support: From intact mass determination to detailed impurity profiling and counterion analysis, we provide a full spectrum of analytical services.
- Regulatory Alignment: Our methods align with ICH Q6B and global regulatory expectations, ensuring compliance and facilitating streamlined approvals.
Common Challenges in Oligonucleotide Manufacturing
The synthesis of oligonucleotides is a highly complex, multi-step process that requires precision to ensure product integrity. These short DNA or RNA molecules are assembled through automated solid-phase synthesis cycles, where nucleotide groups are sequentially added. A failure in this process can allow impurities and structural inconsistencies to emerge, impacting the safety and efficacy of the final product.
Phosphoramidites (amidites) - the building blocks of oligonucleotides - are derived from the nucleic bases A, C, G, U, and T. If impurities are present in these starting materials, they can be incorporated into the final oligonucleotide sequence. Additionally, chemical modifications are often introduced to enhance pharmacokinetics, in vivo stability and targeted delivery, but these modifications can also introduce further impurities, making rigorous analytical characterization essential.
Common manufacturing-related impurities include:
- Shortmers (N-1) & Longmers (N+1): Oligonucleotides missing or containing extra nucleotides due to incomplete or overextended synthesis cycles.
- Protecting Group Removal Issues: Residual functional groups that were not successfully cleaved, affecting reactivity and stability.
- Phosphodiester Impurities: Unintended phosphodiester bonds in phosphorothioate oligonucleotides, which can alter therapeutic performance.
- Base Modifications & Cross-Linked Structures: Chemical modifications that introduce unintended structural variations.
At BioPharmaSpec, our high-resolution mass spectrometry, chromatographic and electrophoretic-based approaches detect and quantify these impurities, helping our clients determine mitigation strategies to ensure batch-to-batch consistency and regulatory compliance in oligonucleotide development.
Oligonucleotide Characterization
Mass spectrometry (MS) is a key analytical tool for confirming the identity, purity, and structural integrity of oligonucleotides. BioPharmaSpec applies high-resolution MS techniques, including LC-MS (MS), MALDI-TOF MS, and HRMS, to assess sequence fidelity, detect modifications, and characterize impurities with precision.
Our scientists use their extensive experience in structural characterization to select the most appropriate analytical approaches based on the size, structure, chemical composition, and modifications of the oligonucleotide. From molecular weight determination to detailed sequence confirmation and impurity profiling, we ensure comprehensive analysis using chromatographic, electrophoretic, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) techniques.
For Higher Order Structure, a battery of orthogonal techniques, including CD, FT-IR, Microfluidic Modulation Spectroscopy (MMS) and NMR, can be deployed. In addition, aggregation can be investigated using SEC-MALS and AUC.
With specialist expertise and state-of-the-art instrumentation, you can trust BioPharmaSpec to deliver high-confidence data to support regulatory submissions, manufacturing consistency, and product development.
| Molecular mass | LC-MS, MALDI-TOF MS, ESI-TOF MS |
| Sequence and identity | LC-MS/MS, LC-MS, IP-HPLC-TOF-MS |
| Identity of potential counter ions | ICP-OES |
| Extinction coefficient | UV |
| Spectral characterization | 31P, HSQC and DOSY NMR, Circular Dichroism (CD), FT-IR |
| Melting temperature | UV |
| Identification as duplex (siRNA duplexes) | Non-denaturing IPRP HPLC UV |
| Thermodynamic transitions | Differential scanning calorimetry (DSC) |
| Tertiary structure, as appropriate (e.g. Antibody Oligonucleotide Conjugates (AOC)) | Near UV Circular Dichroism (CD) |
| Product and process related impurities | Suitable analytical methods with appropriate limits of detection (LOD) and limits of quantitation (LOQ) to detect the likely impurities. |
Supporting Your Oligonucleotide Development
At BioPharmaSpec, we provide a comprehensive portfolio of analytical testing to support informed decision-making throughout your oligonucleotide development.
Get in touch today to discuss your oligonucleotide characterization needs and streamline your path to regulatory approval.