Characterization and Impurity Analysis of Oligonucleotide Therapeutics

Ashleigh Wake

Ashleigh Wake is director of Biological Services, Intertek.

Pharmaceutical Technology, Pharmaceutical Technology-01-02-2017, Volume 41, Issue 1
Pages: 30-33

Analytical technologies play a key role in the characterization and quantitation of oligonucleotide therapeutics.

The potential and anticipation surrounding oligonucleotides as therapeutics has been apparent in the pharmaceutical industry for more than 30 years (1). Until recently, however, the number of success stories has been limited with the actual level of growth failing to meet these initial expectations.

As of September 2016, three antisense drugs have been approved for use in the United States. The development pipeline for these therapies appears to be strong, with detailing more than 140 active clinical programs for oligonucleotides in various stages of development.

This resurgence in oligonucleotide development can be attributed to a combination of factors including improved chemistries, a better understanding of the basic biology of oligonucleotides, more sophisticated delivery systems, and most importantly, increasing success in the clinic (2).

Undoubtedly to support these developments, advancement in analytical technology has also been a fundamental aspect, specifically to facilitate characterization and quantitation of the oligonucleotide of interest as well as any synthetic contaminants (3).

Oligonucleotides are generally produced through a synthetic solid-phase chemical synthesis in a manner that likens them directly to traditional small-molecule pharmaceuticals. Oligonucleotides, however, display a diversity in mode of action, which on a cellular level involves interactions more typical of a biological moiety (3). This lack of ready definition as either a large or small molecule has led to many challenges from a regulatory perspective in terms of providing guidance, and subsequently, as yet, neither FDA or EMA have issued official documentation with respect to expectations surrounding quality control of oligonucleotides.

Despite the lack of formal guidance, FDA has issued papers detailing current thinking in respect to quality control (4). These documents provide an overview of the data required to support product registration in respect to identity, purity, quality, and strength. The actual analytics involved represent a diverse and complex analytical program. Table I provides an overview of a typical characterization program.

Identity testing

Given the complex nature of the molecule, as with many of the quality control analytics, it is recommended that orthogonal approaches be used to verify the identity of the test material.

Oligonucleotide structure and sequence

Determination of the molecular weight and confirmation of the nucleotide sequence of an oligonucleotide are fundamental criteria for analysis in terms of confirmation of the identity of the molecule and thus a regulatory expectation. Several methods can be applied to gain this information. Historically, digestion approaches such as enzymatic methods (e.g., Sanger) or chemical methods (e.g., modified Maxam Gilbert) followed by mass spectrometry have been widely used. Methods involving digestion are often complex and relatively time-consuming and the likelihood of success is restricted, in some ways, to the analysis of short chain length species. Mass spectrometric approaches, alternatively, can often be hindered by the polar nature, low thermal stability, complexity, and large molecular weights of oligonucleotides (5), which can hinder the ability to obtain good spectra and thus make clear assignments on mass and sequence.

Advancements in high-resolution mass spectrometry and in particular, tandem methods (MSMS), have provided a viable alternative for the determination of both mass and sequence of oligonucleotides. When considering intact mass, normal resolution instrumentation can only be used to obtain the average molecular weight; high-resolution mass spectrometry has, however, facilitated the determination of accurate mass. This method is based on obtaining negative ion spectra of the oligonucleotide followed by deconvolution. The accuracy of these measurements is typically less than 5 ppm, and as such, the mass can be used as an aid to establishing the empirical formula of the molecule, which is in turn used to postulate or confirm structure (6).

Such high resolution readily allows discrimination of nucleosides differing by only 1 mass unit, such as Cytidine monophosphate (CMP) (monoisotopic mass 323.05185 Da) and Uridine monophosphate (UMP) (monoisotopic mass 324.03587 Da), including distinguishing between the 13C isotope of CMP and 12C isotope of UMP, which effectively have the equivalent mass at a lower resolution (324 Da).Quinn et al. (7) also detailed how tandem MS can be used to confirm the presence of truly isobaric nucleosides, such as Adenosine monophosphate (AMP) and Deoxyguanosine monophosphate (dGMP), both of empirical formula C10H14N5O7P and a monoisotopic mass of 347.06308 Da). In discrimination between species of this type, structural differences are relied upon for definitive identification. In the case of AMP and dGMP, for example, the position of the oxygen atom differs, which can be distinguished by MS analysis and thus allow these isobars to be distinguished.

The benefit of these advanced MS-based methods is further demonstrated when considering identification of the position of modified nucleosides, a feature that could not be established from the earlier digestion/chromatography approaches.

Chain length

Despite improvements in the automation and understanding of the chemistries involved in oligonucleotide synthesis, and despite the most ardent post synthesis clean-up, it is inevitable that there will be some heterogeneity with regards to chain distribution in the final material. Monitoring of this distribution presents a further fundamental aspect of quality control.

The most accepted methodologies for performing this assessment are capillary gel electrophoresis (CGE) and anion exchange-high-performance liquid chromatography (SAX-HPLC), given both methods’ inherent ability to separate truncated species. Each approach offers advantages over the other. CGE methods require little or no development to reach maximum performance and can generally be applied to larger oligonucleotides without loss of resolution over that can be prevalent with the HPLC approach. Alternatively, SAX-HPLC methods are generally more reproducible, the columns last longer, and the response of and amount of loading into the instrument are not affected by species of differing mass to charge ratios (8).


Internucleoside linkages

Introducing modification to a nucleoside linkage has been a critical feature in the advancement of oligonucleotide therapeutics. Such alterations help to overcome the two main challenges affecting the efficacy of these molecules, specifically, delivery to the target in vivo and increasing bioavailability. An example of the effect of this engineering is that introduction of phosphorthioate linkages increases resistance to nucleases, but the incorporation of too many bonds can reduce the function of the species.

Modifications are, however, a necessity, and as such, powerful techniques that allow continued monitoring of the distributions are required. SAX-HPLC and nuclear magnetic resonance (NMR) spectroscopy, in particular 31P NMR, provide powerful data in this respect to monitoring linkage. SAX-HPLC is particularly useful where quantitation is required (i.e., discrimination of the amounts of phosphate diester [P=O] or phosphorothioate diester). 31P NMR can yield powerful data about the type of internucleoside linkers (phosphodiester P=O, phosphorothioate P=S, methyl phosphonate, phosphonate, or any other modified phosphate), the nucleobase, and oligo backbone composition. NMR also provides information on the ratios of various species such as that between the P=O and P=S; however, this technique is restricted to ratio and other techniques needed to give true amounts.

Melting temperature (Tm)

Melting temperature is often considered the most critical quality attribute of an oligonucleotide. This property relates to the temperature at which a double-stranded oligo denatures and separates into two single strands. The melt temperature, or Tm, is defined as the temperature at which 50% of the molecule is double stranded and 50% single stranded, also known as the molecule being classed as 50% annealed. Critically, Tm can be influenced by external factors, such as salt concentration, the presence of denaturants, and hybridization conditions. Altering the Tm by manipulating external or environmental factors is often used to increase solubility of a product or to enhance in-vivo stability of the material.

Many algorithms exist for determination of theoretical Tm. These theoretical values aid product development. For determination of actual Tm, however, NMR and circular dichroism (CD) provide the best methods for establishing the Tm of an oligonucleotide.

Impurities determination

In addition to confirmation of core structural and physiochemical features, continued monitoring of the purity and levels of product- and process-related impurities presents a fundamental attribute for oligonucleotides in continued quality control.

Product-related impurities include the following:

  • Addition sequences (n+1, n+2, etc.)

  • Deletion sequences (n n-1, n+2, etc.)

  • Phosphodiester analogs

  • Depurinated sequences

  • Partially deprotected sequences

  • Aggregated sequences.

When considering impurities involving addition or deletion of sequence, the methods of choice are SAX-HPLC or CGE, when considering chain length. For the other potential product-related species, a combination of chromatographic and spectroscopic methods are applied to cover all relevant components.

Aside from product-related impurities, residual species originating from the process require monitoring, and if necessary, specifications set. Such species include:

  • Organic volatile impurities (OVI) or residual solvents, typically quantified by gas chromatography (GC) with flame ionization detection (FID) or mass spectrometry (MS)

  • Inorganic molecules metals, inorganic salts, catalysts, cleavage reagents, and counterions typically quantified by inductively coupled plasma (ICP), MS, or OES.


In support of continued quality control of oligonucleotide therapeutics, a vast array of analytics is required to comprehensively control structural, physiochemical composition, as well as the purity and impurities of the test material. Looking forward, some of the challenges facing the resurgence in these therapies and the growing pipeline of oligonucleotides can be effectively addressed through application of these sophisticated analytical approaches and continued advancements in analytical technology.


1. A. Aartsma-Rus, Molecular Therapy 24 (2) 193-194 (2016).
2. R.L. Juliano, Nuclei Acids Research, published online April 15, 2016, doi: 10.1093/nar/gkw236
3. B2B Labs, Oligonucleotides: Opportunities, Pipeline and Challenges, accessed Dec. 12, 2016.
4. Rao V.B. Kambhampati, Points to Consider for the Submission of Chemistry, Manufacturing, and Controls (CMC) Information in Oligonucleotide-Based Therapeutic Drug Application, presentation at DIA Industry and Health Authority Conference on: Oligonucleotide-based Therapeutics (Bethesda, MD, April 2007).
5. M. Smith, Rapid Commun Mass Spectrom. 25 (4) 511-25 (2011).
6. R. Houghton, “Oligonucleotides: The Next Big Challenge for Analytical Science,” Chromatography Today, March 2011.
7. R. Quinn et al., Mass Spectrom. 48 (6) 703-712 (2013).
8. Analysis of Synthetic Deoxyoligonucleotides by Anion Exchange HPLC and Capillary Gel Electrophoresis, Waters Cooperation, poster at the 16th Annual Symposium of Column Liquid Chromatography (June 1992).

About the Author

Ashleigh Wake is director of Biological Services, Intertek.

Article Details

Pharmaceutical Technology
Vol. 41, No. 1
Pages: 30–33


When referring to this article, please cite it as A. Wake, “Characterization and Impurity Analysis of Oligonucleotide Therapeutics," Pharmaceutical Technology 41 (1) 2017.

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