Previous BioPharmaSpec blogs have explored the importance of characterizing the primary structure of chemically synthesized oligonucleotides and how this can be achieved using mass spectrometric techniques. However, just as with the characterization of proteins and peptides, it is a regulatory requirement from both the EMA and FDA that the higher order structure (HOS) of oligonucleotides is investigated. This is covered in both general and specific guidance documents (1,2). As with other aspects of biopolymer drug characterization, the concept of orthogonality is critical for HOS investigations. To meet this expectation, it is important to use multiple techniques.
A variety of techniques are used to analyze oligonucleotide HOS. These include methods that focus on secondary and tertiary structure, such as Circular Dichroism (CD), Fourier Transform Infrared Spectroscopy (FT-IR), Microfluidic Modulation Spectroscopy (MMS), and Nuclear Magnetic Resonance (NMR). Additionally, techniques like Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) and Analytical Ultracentrifugation (AUC) investigate aggregation properties.
Size Exclusion Chromatography paired with Multi-Angle Laser-Light Scattering (SEC-MALS) is commonly used as an orthogonal tool to AUC, as it provides both separation and detection of closely related species, such as product-related impurities, including aggregates. Although MALS detection may not provide the mass accuracy of High-Resolution Mass Spectrometry (HRMS), it is still able to determine the average oligonucleotide mass, independent of elution position. Additionally, it provides mass distribution and polydispersity data, making it particularly valuable in characterizing aggregates. SEC-MALS is applicable to both single- and double-stranded species and can be used for both crude and purified materials, especially in cases involving structural heterogeneity and oligonucleotide conjugates with a propensity for aggregation.
Analytical Ultracentrifugation (AUC) is considered an orthogonal approach to SEC-MALS. AUC is widely used for biopolymer analysis, offering characterization such as size, shape, assembly, stoichiometry, and binding properties. One major advantage of AUC is its independence from phase interactions and its broad range of mass applicability (kDa-gDa). However, AUC generally lacks the resolution of SEC and other separation techniques for the separation of closely related components. While AUC has seen wide application in the cell and gene therapy (C>) area, its use as an analytical tool for oligonucleotides remains limited. However, this may change as conjugation and encapsulation become popular delivery system mechanisms.
Microfluidic Modulation Spectroscopy (MMS) is a recent development in infrared-based analysis. It allows for real-time background subtraction by alternately analysing the sample and buffer, providing valuable data for HOS investigations. The system can also generate IR-based HOS data through a thermal ramp cycle, allowing investigation into structural changes with temperature and gain insights into molecular robustness. Data generation during the thermal ramp cycle is particularly beneficial for analyzing double-stranded oligonucleotides, as it can provide information on denaturation.
Circular Dichroism (CD) is an analytical technique that exploits the interaction of chiral molecules with circularly polarized electromagnetic energy. It has become an essential tool for studying therapeutic biomolecules, including oligonucleotides. In the far-UV region, CD signals are produced by the optical absorption of the nucleotide backbone, making them sensitive to changes in oligonucleotide secondary and higher-order structures. CD provides the ability to detect structural differences due to manufacturing process variations and is useful for product comparability assessments. Furthermore, characteristics such as melting temperature and solution-phase inter/intramolecular interactions can also be examined using this technique.
Fourier Transform Infrared Spectroscopy (FT-IR) is frequently applied orthogonally with CD and, more recently, MMS, to study the higher order structures of oligonucleotides. This technique capitalizes on the unique vibrational signatures resulting from specific base pairing and backbone linkages. FT-IR spectra provide insights into several structural attributes, including conformers, binding affinities, base and phosphate modifications, and changes observed during denaturation and stability studies.
Nuclear Magnetic Resonance (NMR) is recognized by regulatory authorities as a valuable technique for obtaining structural data in oligonucleotide structural studies. NMR provides detailed insights into different structural aspects by probing magnetic atomic nuclei. It can be performed either on single nuclei, such as 1H and 31P, or extended to assess interactions of two magnetic nuclei in two-dimensional (2D) NMR analyses, such as 1H-13C NMR. The resulting spectra reflect the precise chemical environments of the magnetic nuclei, capturing not only the linear sequence of the molecule but also its higher order structure and through-space interactions.
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
Higher-order structure is crucial for the stability and effectiveness of many oligonucleotides, making it an essential quality attribute that must be experimentally defined as part of a therapeutic development strategy. Given the complexity and structural evolution of this therapeutic class, the importance of orthogonal methods for HOS investigation cannot be overstated. The wide range of available instrumentation allows for the integration of orthogonal approaches into experimental design, mitigating biases from any single analytical technique. The use of advanced techniques such as MMS will help meet the increasing demand for sensitive, robust, and rapid analytics, providing researchers with enhanced tools to probe the higher order structures of these multifaceted therapeutics.
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