Oligonucleotide Characterisation and Analysis

Oligonucleotide characterisation and analysis is an important component of drug development, registration, and quality control. A robust and accurate analytical characterisation is needed to confirm identity, determine safety, efficacy, purity, quality, and strength, as well as to identify and quantify contaminants of oligonucleotide therapeutics.

Given the sheer complexity and diversity in the types of oligonucleotides (e.g. RNA, antisense oligonucleotides (DNA-like) or aptamer) in clinical development, including differences in length, chemical modifications, conjugation and other features, a range of diverse analytical approaches are required.

Among our capabilities are Good Manufacturing Practice (cGMP) characterisation services for the different classes of oligo-based drugs. We determine the identity, structure, biologic potency, physicochemical properties, and purity and impurities and perform method development and validation where required to meet your registration or quality control needs.

Oligonucleotide Therapeutic Physicochemical Characteristics
Our teams use mass spectrometry (MS) methods to determine the molecular weight of the oligo, typically using electrospray ionisation (ESI). High resolution accurate-mass spectrometry (Orbitrap, QToF) allows accurate mass spec data (HRAM) to be generated to support characterisation requirements. We also provide a full set of physicochemical property testing, including optical rotation, pH, pKa, and moisture content.

Oligonucleotide Identity, Structure and Characterisation
We apply orthogonal approaches to verify the identity of oligonucleotides including molecular weight and molecular sequencing, as an assurance of identity of an oligonucleotide.

Molecular weight and molecular sequencing are used to verify the identity of an oligonucleotide. To confirm or determine the sequence of the oligos, we use enzymatic or chemical digestion approaches followed by mass spectrometry, including high-resolution accurate-mass spectrometry (Orbitrap, QToF).

Our expertise in 31P nuclear magnetic resonance (NMR) spectroscopy can yield powerful data about the type of internucleoside linkers (phosphodiester, phosphorothioate, methyl phosphonate, phosphonate, or any other modified phosphate), the nucleobase, and oligo backbone composition. Monitoring modifications to nucleoside linkages via NMR spectroscopy, in particular 31P NMR can deliver powerful data on the type and ratio of different types of internucleoside linkers.

Higher order structures can be evaluated by electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD) spectroscopy, and thermal denaturation measurements. Melting temperature (Tm) is a critical quality attribute of an oligonucleotide, and we monitor this using NMR and circular dichroism (CD).

Due to their impact on the structure, it is important to determine the presence of counterions, particularly for divalent ions (e.g. Mn, Ca, Cu, Zn, Mg, Fe, etc). We use inductively coupled plasma spectroscopy (ICP-MS or ICP-OES) to achieve elemental information and trace metal levels.

Purity and Impurities Analysis for Oligonucleotide Therapeutics
To understand the purity of the oligonucleotide, we provide drug substance and drug product assays and can build a well-understood impurity profile by applying a range of chromatography (e.g. UPLC, HPLC, LC-MS, CGE) and spectroscopy methods (UV absorbance spectrum, extinction coefficient, FTIR, ¹³C and ¹H NMR). 

Determining the levels of product-related impurities such as addition sequences (n+1, n+2, etc.), deletion sequences (n-1, n-2,etc.), we apply HPLC with UV and MS detection approaches. 

For other product-related species such as phosphodiester analogs, depurinated sequences, partially deprotected sequences, and aggregated sequences, we apply a combination of chromatographic (e.g HPLC) and spectroscopic (e.g ³¹P NMR) methods. Our scientists also deliver highly detailed MS characterization of product related impurities using QToF, MALDI-TOF MS, ESI-MS, LC-MS, LC-MS/MS with expert data interpretation to determine the levels of early and late eluting impurities. 

Determination of process residuals (residual solvents, trace metals) and other reagents (e.g. activators, deprotection agents, trace levels of EDTA) that may be present in the sample are also achieved through a combination of chromatographic and spectroscopic methods.  

Supporting Oligonucleotide Therapeutic Development
Our scientists have supported oligo medicine drug development since the inception of this class of therapeutics. Through our dedicated suite of characterisation techniques, we are focused on helping you to meet industry requirements for regulatory compliant (GMP or GLP) characterisation and quality control for oligonucleotide-based drugs. Bringing quality and safety to life, our Total Quality Assurance expertise is delivered consistently to help you ensure you meet and exceed your quality, safety, and efficacy requirements.