Oligonucleotide therapeutic analysis for identity, purity, quality, strength, structure characterisation, physicochemical properties and bioanalysis supporting antisense & other oligo product development
Oligonucleotide therapeutic analysis is an important component of drug development, registration and quality control. Robust and accurate analytical characterisation is required to confirm identity, determine safety, efficacy, purity, quality, and strength and also to identify and quantify contaminants. Oligo-based therapeutics such as oligo-based drugs (RNA, antisense oligonucleotides (DNA-like) or aptamer) are gaining increasing interest with a strong development pipeline evident.
Given the sheer complexity and diversity in the types of oligonucleotides in clinical development, including differences in length, chemical modifications, conjugation and other features, a range of diverse analytical approaches are required.
Our capabilities include Good Laboratory Practice (GLP) bioanalysis and GLP or Good Manufacturing Practice (cGMP) characterisation services for the different classes of oligo-based drugs. Intertek has supported oligo medicine drug development from the inception of therapeutic oligo products. Our analytical scientists support product development, from quality control testing of amidite starting materials and early stage product characterization through to GMP batch release testing for either single strand or duplex products. 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 Characterization
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 for assurance of 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 a 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 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.
Oligonucleotide Stability Studies
With comprehensive oligo testing services that are integrated with ICH stability storage and testing laboratories, we can help you to evaluate the stability of your oligo product through accelerated and long-term stability studies. Prior to these, we can develop (and validate) methods to identify and quantify any degradation products, as some categories of oligos are prone to degradation (e.g by depurination, depyrimidination).
Ensuring a safe and efficacious product, Intertek are experienced in GLP or GCP bioanalysis supporting toxicokinetic (TK) and pharmacokinetic (PK) studies for preclinical and clinical development of oligonucleotide products. Our GLP laboratories typically apply a range of bioanalytical techniques including ELISA, mass spectrometry approaches (LC-MS/MS, and UPLC-MS/MS) and capillary gel electrophoresis (CGE).
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.
Ashleigh Wake, Laboratory Director for Intertek Pharmaceutical Services, Manchester, UK features in this months Chemistry Today. Access the article here