Navigating the Analytical Complexity of Oligonucleotide Therapeutics

As polymers of nucleotides that can be either DNA or RNA, oligonucleotides encompass a wide range of sizes and products, from small interfering RNAs (siRNA) and microRNA (miRNA) products, to plasmid DNA (pDNA) and messenger RNA (mRNA) vaccines. Oligonucleotides are valuable in that they have a high level of specific targeting that conventional drugs lack.

Small oligonucleotide APIs, such as siRNAs and miRNAs, are considered complex small molecules, whereas pDNA and mRNA APIs can contain thousands of nucleosides. However, all of these formats present analytical challenges. Here, I’ll provide a brief overview of the techniques that should be considered. The biggest difficulty for developers is the sheer number of techniques required.

We’ll start with siRNA and miRNA. Although these are relatively small oligonucleotides, separating process impurities and degradation products from the active molecules still requires multiple HPLC purity methods. A denaturing ion-pairing reversed-phase HPLC (IR-RP HPLC) method is required to assess the purity of the individual sense and anti-sense strands of siRNA, while a non-denaturing method is needed for purity assessment of the intact duplex. IR-RP HPLC methods using traditional triethylamine acetate ion-pairing mobile phase systems have historically been used, but the mobile phase system is not compatible with mass spectrometry. These mobile phase systems prohibit the structural characterization necessary to fully understand the process impurities and degradation pathways of the molecules.

More recently, IP-RP HPLC methods using hexafluoroisopropanol with triethylamine or other amine containing additives have been developed that are compatible with mass spectrometry and can aid in structural characterization of the products. IP-RP and AEX methods can also be used to separate analytes based on charge to ensure that all impurities have been accounted for in at least one method.

Moving on to pDNA products, the analytical work becomes even more complicated. Tests are required to ensure the identity, integrity, and purity of mRNA products and the pDNAs used in their generation.

Prior to generation of mRNA drug substances, the pDNA must first be isolated. pDNAs containing the gene of interest are expressed and purified from host cells. Supercoiling of the pDNA is critical to expression of the mRNA and isolation of pDNA in a form that can be used for expression of mRNA represents a significant challenge in itself.

The purified pDNA is then used in the in vitro transcription reaction to generate the nascent mRNA molecule. After transcription, a 5’ cap and 3’ poly(A) tail are added. Since mRNA must enter cells to be translated into the target protein, special delivery systems are required to transport the mRNA across the cell membrane. The delivery system typically involves a lipid nanoparticle (LNP), the presence of which adds to the complexity of the final drug product and increases the difficulty of analytical methods.

pDNA requires identification testing using sequencing and restriction mapping. pDNA concentration is determined using UV spectroscopy and absorbance at 260 nm, while purity is assessed using capillary electrophoresis or HPLC analysis. Residual host cell DNA is quantified using quantitative PCR. Residual host cell proteins are quantified using ELISA and residual kanamycin can be quantified using LC/MS. Safety tests such as endotoxin, bioburden, and sterility must also be performed.

The list of tests for mRNA drug substances is even longer than for pDNA, and includes sequence confirmation by PCR, mRNA concentration by qPCR, digital PCR or UV spectroscopy, and mRNA intactness by electrophoresis. Techniques include:

• IP-RP with LC-MS/MS detection for 5’ capping efficiency.
• Electrophoresis or HPLC for 3’ poly(A) tail length.
• ELISA for double-stranded RNA and residual T7 RNA polymerase.
• Size exclusion chromatography for aggregation analysis.
• HPLC for fragments of product.
• qPCR for residual DNA template.
• LC-MS for free, non-incorporated nucleosides.

The ability of mRNA to express the target protein should also be evaluated using either a cell-based assay or in vitro translation with ELISA detection. For mRNA finished drug products in LNP, the LNP must be tested too. Lipid content can be tested using RP-HLPC with CAD detection; RNA encapsulation efficiency using fluorescence-based assays; and LNP size and polydispersity using dynamic light scattering.

There’s a lot to take in – and this is just a brief overview of the complex analytical requirements of oligonucleotides! Experience and access to the right, high-quality instrumentation really helps to overcome the challenges. Many companies won’t have the right kit in house, but there’s plenty of expert analytical service companies out there. Just make sure any partners you choose have the right expertise to handle oligonucleotides.

In the future, we will likely see even more oligonucleotides entering company pipelines as the industry continues to explore the indications that these therapeutics can treat. Persevering with the development and analytical challenges will be worth it as long as it brings valuable new therapies and vaccines to patients.