Oligonucleotide-based therapeutics have become one of the most promising drug classes in modern medicine. Their ability to target disease at the genetic level is unmatched, but their chemistry brings a new set of analytical challenges. Every modification in sequence, length, or backbone can influence activity, safety, and stability. Reliable methods to characterize these molecules are therefore essential for both developers and regulators to ensure their quality and consistency throughout the product lifecycle.
To address this need, Kymos Italy has developed OligoXplorer, a research and development project designed to create a comprehensive analytical platform for the CMC characterization of oligonucleotide-based therapeutics.
The project has been carried out in collaboration with the University of Camerino (UNICAM) – School of Biosciences and Veterinary Medicine and co-funded by Regione Marche under its innovation program (PR Marche FESR 2021/2027).
Beyond its scientific scope, OligoXplorer marks an important milestone for Kymos Group: it consolidates our experience in TIDES and expands our portfolio with the CMC testing for oligonucleotides.
Understanding the Analytical Challenge
Oligonucleotides sit between small molecules and biologics, too large for traditional chemical methods, too small for most bioassays. Their large size, high polarity, and tendency to form secondary structures make them difficult to separate, quantify, and sequence. As said before, minor changes in sequence length or their chemistry can affect potency, specificity, and stability.
For this reason, regulatory agencies expect comprehensive CMC packages built around orthogonal analytical methods that confirm sequence identity, quantify content, and characterize impurities; all under validated, reproducible conditions.
OligoXplorer was conceived precisely to address these challenges: to explore, develop, and validate methods capable of supporting every stage of oligonucleotide characterization, from early development to GMP release testing.
Three Main Analytical Areas
The project focused on three main analytical areas: sequencing, assay, and related substances. Each covering a critical quality attribute (CQA) in oligonucleotide control.
Sequencing: Confirming Molecular Identity
Determining that an oligonucleotide carries the right sequence is the first step toward ensuring safety and efficacy.
Our team in Italy has established and refined LC-MS and HR-MS workflows capable of confirming sequence identity and revealing subtle variants such as shortmers, longmers, or chemical modifications.
Chromatographic fractionation helped isolate these variants before MS analysis, improving resolution and reliability. The outcome is a set of methods that provide clear, high-quality identity data suitable for routine CMC testing use.
Assay: Measuring Content and Strength
Quantifying oligonucleotides is equally challenging, and accurately doing so is vital for potency and dose reproducibility. Their physicochemical properties can affect recovery and response, especially during routine testing.
To overcome this, Kymos Italy has developed and validated LC-UV and LC-MS methods for precise quantitation.
Method validation covers linearity, precision, and robustness, while chromatographic optimization helps separate the main peak from degradation or impurity signals. The result is a validated, fit-for-purpose assay method ready for use in batch release and stability programs in GMP environments.
Related Substances: Ensuring Purity and Product Integrity
Even small impurities can affect therapeutic performance. During synthesis, oligonucleotides can produce a range of truncated or elongated species (n–1, n+1), or generate chemical impurities through oxidation or depurination. Identifying and quantifying these is essential for product safety and regulatory compliance.
Through OligoXplorer, our Italian team has established LC-UV and LC-MS impurity-profiling workflows to detect and quantify these species with high sensitivity. Comparative studies across batches confirmed their suitability for process control and stability assessment.
Together, these developments provide a complete picture of product purity and integrity.
Complementary Analytical Capabilities
Alongside the core analytical areas developed through OligoXplorer, Kymos Group can cover other tests that are often required for oligonucleotide materials and formulations. Some are carried out directly in-house, while others are performed through trusted partners with proven experience in the field.
Our teams can determine lipid content in LNP formulations by HPLC-CAD, a key parameter to evaluate encapsulation efficiency and formulation consistency. We also provide testing for residual solvents, sodium acetate content, and elemental impurities, ensuring full compliance with current ICH and EMA quality standards.
Physical characterization is equally important. Using Dynamic Light Scattering (DLS), we can measure particle size distribution, aggregation state, and polydispersity, helping to assess the stability and homogeneity of the product.
By combining these complementary studies with our CMC analytics, Kymos Group offers a complete picture of oligonucleotide quality.
From Research to Implementation
The methods developed under OligoXplorer are not just prototypes; they are already implemented at Kymos Italy and available as validated analytical services for oligonucleotide developers as a coherent analytical platform for oligonucleotide CMC. Each method has been developed, optimized, and validated using representative synthetic oligos and can now be adapted to specific client projects.
This includes identity confirmation, assay quantitation, and impurity profiling, all performed under GMP-compliant conditions and supported by our experienced analytical teams.
With this, Kymos Group becomes one of the few European CROs to offer integrated CMC and bioanalytical support for oligonucleotide therapeutics, combining the strengths of our sites in Italy and Germany.
Looking Ahead
With OligoXplorer, Kymos Italy now operates as a center of excellence for oligonucleotide CMC analytics within the Group. The experience gained through this project has allowed our teams to consolidate expertise in mass spectrometry, chromatographic method development, and impurity profiling applied to oligonucleotides, capabilities that are now fully available to support client projects.
For partners developing oligonucleotide-based drugs, these capabilities mean faster, more reliable CMC support, from early method development to regulatory submission.
Ultimately, this initiative strengthens Kymos Group’s mission: to combine scientific excellence, regulatory compliance, and innovation in analytical testing, helping our partners bring safer, more effective oligonucleotide-based medicines to patients.
