What are Oligonucleotides?
Oligonucleotides are polymeric sequences of nucleotides - RNA, DNA and their analogs. Initially synthetic oligonucleotides were manufactured and used principally as reagents in Life Science research (e.g. PCR) and in DNA-based diagnostic kits. More recently, oligonucleotide (nucleic-acid-based) therapeutic drugs have emerged as another area of increasing importance.
The first oligonucleotide approved by the US FDA was Novartis Pharmaceutical’s Vitravene (formiversen) in 1998, although this was subsequently discontinued in 2004 due to lack of clinical need. Currently, there are only 8 FDA-approved oligonucleotide-based therapeutics on the market, however there are hundreds more in the clinical pipeline, from antisense oligonucleotides (AON) and small interfering RNA (siRNA) to full-length messenger RNA (mRNA) for gene therapy and vaccine applications
What are the manufacturing challenges?
For companies pursuing oligonucleotide-based therapies, the relative complexity of their synthesis can pose purification challenges. Oligonucleotides are short DNA or RNA molecules that are “grown” via the addition of nucleotide groups in a series of automated solid-phase synthesis cycles. A failure in this process can allow impurities to emerge. Phosphoramidites (amidites) comprised of the nucleic bases A, C, G, U or T are the starting materials and so if impurities are present in these bases these can be incorporated. Often, some chemical modifications are necessary to improve their pharmacokinetics and enhance their stability in-vivo. Incorporation of these modifications through the various chemical processes employed has the potential to give rise to impurities in the product.
Irregularities in the manufacturing process can lead to the formation of the following common impurities:
- N-1: Shortmers – oligonucleotides missing one or more nucleotides,
- N+1: Longmers – oligonucleotides that include more than the intended number of nucleotides,
- Lack of protecting groups (derivatives of existing functional groups that decrease reactivity and increase stability)
- Phosphodiester impurities (PO) in phosphorothioate oligonucleotides.
Oligonucleotide Regulatory View
Oligonucleotides are considered to be more similar to small molecules than to biologics since they are manufactured by solid phase synthesis. However, as mentioned by Mohan Sapru of the US FDA, there are no FDA regulations at the moment that specifically address the quality control expectations of the diverse oligonucleotide products (1).
Characterization of Oligonucleotides
The term “oligonucleotides” is broad and encompasses a range of chemical entities from low molecular weight siRNA (15-20 nucleotides) to mRNA and DNA expressing whole genes containing many thousands of nucleotides. In addition, chemical modifications and formulations necessary for the functionality of these therapeutics add a further layer of complexity and potential risk and increase the analytical challenge.
BioPharmaSpec provides specialized, high resolution mass spectrometric analysis of oligonucleotide products to confirm identity, and assess for impurities and degradants. Our scientists use their extensive structural characterization experience to apply many of the below techniques to your oligonucleotide as required to confirm the desired product from simple molecular weight determination through to detailed structural sequence and impurity analysis. The selection of appropriate analytical techniques depends on the oligonucleotide size, structure, chemical composition and modifications. Various chromatographic, electrophoretic and high performance liquid chromatography mass spectrometry techniques are used for data acquisition to support identification and purity assessments of DNA, RNA diagnostic oligonucleotides and oligotherapeutics:
- Molecular weight confirmation using ESI-MS, LC-MS and/or MALDI-TOF
- Detailed structural analysis using LC-MS
- Confirmation of sequence using LC- MS/MS and/or exonuclease treatment followed by MALDI-TOF MS analysis
- Impurity profiling and identification using LC-MS
- pI/charge variants using icIEF/IEX
References
1. M. Sapru, “CMC Regulatory Considerations for Oligonucleotide Drug Products: FDA Perspective,” (2017). https://pqri.org/wp-content/uploads/2017/02/3-SapruPQRI-FDA-Conference-Oligo-2017-Presentation.pdf
Are you developing an oligonucleotide product?
Please contact our scientists today to discuss the most efficient characterization strategy for your oligonucleotide product.