The biopharmaceutical drug market has traditionally focused on protein and peptide-based drugs but in recent years there has been a noticeable upturn in the development and use of oligonucleotide-based drugs. Currently there are seventeen FDA approved oligonucleotide drugs that have been brought to the market, with hundreds more in the clinical pipeline and in pre-clinical development. Approved oligonucleotide therapeutics treat a variety of indications, although none presently are approved for the treatment of any type of cancer. However, there are several subcategories of oligonucleotides including antisense oligonucleotides, small interfering RNAs (siRNA), microRNA (miRNA), and aptamers, that show promise as therapeutics against various cancers. Oligonucleotides also cross over into the advancing area of gene therapy (see our recent blog “Analysis of Adeno-Associated Virus (AAV) Capsid Proteins”).
Oligonucleotide derived medicines require detailed structural analysis in the same way proteinaceous drugs do. So, is there any instrumentation or methodology that is particularly suited to structural characterization of oligonucleotides? The answer happily is yes, in the form of mass spectrometry. Mass spectrometry has been applied to the analysis of oligonucleotides for decades, with advancing analytical technologies rising to the challenge of increasingly complex molecules.
Single-stranded DNA oligonucleotides were traditionally analyzed using a combination of MALDI-TOF MS experiments. The average mass molecular weight of the intact native oligo in its phosphorothioate and/or phophodiester form(s) could be determined. The oligonucleotide could also be treated with two different phosphodiesterases, one cleaving from the 5’ end and one cleaving from the 3’ end, to allow ladder sequencing of the base residues by MALDI-TOF MS. This sequencing method is limited by the effectiveness of the phosphodiesterases on modified base residues and RNA based oligonucleotides.
These shortcomings can, in many cases, be overcome by application of high resolution ESI-MS and MS/MS procedures using mass spectrometers such as the Q-TOF geometry type of instruments. Modern high-resolution MS instruments allow more precise and confident molecular weight assignments than is possible with MALDI-TOF type mass spectrometers and nucleotide sequences can be confirmed and/or determined in much the same way as peptides are routinely sequenced by MS/MS, regardless of base modifications or backbone linkages. In other words, sufficient energy can be imparted to the oligonucleotides in the source of the mass spectrometer that meaningful fragment ions can be generated through cleavage at the phosphate backbone. With each nucleotide sugar having a unique mass (as is the case with most amino acids) the fragment ions generated allow sequence information to be determined through measuring the mass differences of the observed fragment ions. With ESI-MS in combination with UPLC, methods can be established to detect and aid in the identification oligonucleotide impurities such as N-1 “shortmers” or N+1 “longmers.”
The idea of drug targeting is now commonplace and chemistries have been, and continue to be, developed to successfully bind small molecule drugs to antibody delivery systems. Of course, if small chemicals can be successfully conjugated to antibodies, such as in Antibody Drug Conjugates (ADCs) then why not oligonucleotides? This type of forward thinking has led to the development of antibody-oligonucleotide conjugates (AOCs) where different oligonucleotide products have been successfully conjugated to antibodies which recognize different protein targets such as the HIV gp160 protein, CD71, CD44 and the epidermal growth factor receptor. These types of systems, whilst still in their infancy, provide an excellent mechanism for targeting oligonucleotides to where they are required. Again, analytics must be used to demonstrate that the correct product has been produced and here again mass spectrometry with its direct applicability to the molecule in its entirety as well as being able to be applied to the protein and nucleotide components separately, is ideal for the study of this type of product.
There are currently no ICH or FDA regulatory guidelines specifically for oligonucleotide products and they are generally assessed on a case-by-case basis by the authorities. With its general applicability to biopolymer analysis, mass spectrometry is a key tool in the structural investigations of oligonucleotides and the components of oligonucleotide containing drugs and can provide significant structural insights.
The development of oligonucleotide-based drug products is an area of ever-expanding research and development. Novel chemistries and ideas are being brought into the field and this requires the application of modern analytical technologies such as advanced mass spectrometric procedures, in conjunction with DNA focused chemical processes, to aid in the necessary characterization of these complex molecules.
Please contact our scientists to discuss appropriate characterization strategies for your oligonucleotide product.