George Okafo, David Elder and Mike Webb

Analysis of Oligonucleotides and their Related Substances

George Okafo, David Elder and Mike Webb Hardback

Now available as an eBook from Amazon>>

Oligonucleotides are growing in importance in the pharmaceutical and biotechnology industries as therapeutic medicines. However, their structure and analysis are challenging and require a different approach to existing medicinal products. This book reviews the most up-to-date analytical strategies for synthetic oligonucleotides. It discusses the importance of characterising impurities in oligonucleotides, the different chemical classes and their synthetic origins. It also focuses on current analytical methodologies and strategies for characterising synthetic oligonucleotides using modern techniques in chromatography and spectroscopy.

This book will serve as an invaluable reference tool by giving guidance and tried-andtested approaches for characterising oligonucleotides.

Table of Contents


Chapter 1 Introduction to Oligonucleotides

George Okafo, David P. Elder and Mike Webb

1.1             What are Oligonucleotides?

1.2             Oligonucleotides as Drugs
1.3             The Discovery of the Cell Mechanism to Make Use of Double-Stranded Oligonucleotides
1.4             The Development of Oligonucleotides as Medicines
1.5             Oligonucleotide Suppliers
1.6             Quality by Design Applied to Oligonucleotide Manufacture
1.7             Regulatory Guidance
1.8             Advances in Analytical Methodology


Chapter 2 Oligonucleotide Impurities and their Origin

Hagen Cramer, Kevin J. Finn and Nanda D. Sinha

2.1             Introduction
2.2             Brief Historical Perspective of Oligonucleotide Synthesis
2.3             Raw Material Related Impurities
2.4             Process Related Impurities
2.5             Chemistry Specific Impurities  
2.6             Table of Impurities and Average Masses
2.7             Summary  
2.8             Outlook


Chapter 3             Separation of Oligonucleotides and Related Substances

Bernhard Noll and Ingo Roehl

3.1             Introduction
3.2             Chromatographic Analysis of Oligonucleotides
3.3             General Principles of Chromatographic Separation
3.4             Ion Exchange Chromatography  
3.5             Reverse Phase Chromatography  
3.6             Size Exclusion Chromatography


Chapter 4             Analytical Characterisation of Oligonucleotides by Mass Spectrometry

Patrick A. Limbach

4.1             Introduction  
4.2             MS Instrumentation
4.3             Oligonucleotides in the Gas Phase  
4.4             Method Development
4.5             Applications
4.6             Quantitative Analysis
4.7            Future Developments


Chapter 5             Analytical Characterisation of Oligonucleotides by NMR Spectroscopy

Elena Bichenkova

5.1             Introduction
5.2             Different Formats of NMR Used for Nucleic Acids  
5.3             Application of 1H NMR for Oligonucleotides
5.4             Application of 31P NMR Spectroscopy for Oligonucleotide Analogues
5.5             Diffusion Ordered Spectroscopy for Oligonucleotide Characterisation
5.6             Application of NMR for Structural Analysis of Oligonucleotides with Therapeutic or Diagnostic Potentials           
5.7             Future Perspectives for NMR Characterisation of Oligonucleotides


Chapter 6             Analytical Characterisation of Oligonucleotide using Thermal Melting Curves

George Okafo, David P. Elder and Mike Webb

6.1             Introduction
6.2             In-Silico Modelling Approaches  
6.3             Spectroscopic Methods for Determining Tm
6.4             Thermal Method for Determining Tm Values
6.5             Future Directions
6.6             Conclusions


Chapter 7             Oligonucleotide Stability and Degradation

Daren Levin

7.1             Introduction
7.2             Secondary Structure Considerations  
7.3             Types of Degradation Products
7.4             Considerations for the Analysis of Degradation Products
7.5             Storage of Oligonucleotides
7.6             siRNA Stability Case Study  
7.7             Summary

Author Biography

George Okafo studied chemistry and biochemistry at Imperial College of Science, Technology and Medicine, London and continued at Imperial College to undertake research for a PhD in chemical carcinogenesis. Dr Okafo then continued his research as a postdoctoral research fellow at the University of Toronto, Canada in the Institute of Medical Biophysics, where he studied the mechanism for nitrosamine-induced chemical carcinogenesis. Dr Okafo has more than 22 years’ experience in the pharmaceutical industry in legacy companies of GSK (SK&F and SB). His current role is a science director in the externalisation group (SCINOVO) in GSK R&D that provides expert drug development consultancy to GSK external drug discovery collaborators. Dr Okafo has published 45 papers, authored three book chapters focused on analytical and separation sciences (HPLC, GC, CZE, MEKC, LC-MS) and detection modes (UV, fluorescence), and is the co-owner of two patents on fluorescence detection. Dr Okafo’s recent publications have focused on analytical strategies for characterising synthetic oligonucleotides and, in 2010, he co-organised an international analytical symposium on characterising therapeutic oligonucleotides. George is a member of the Royal Society of Chemistry and an Associate of the Royal School of Chemistry, London. 


David Elder studied chemistry (BSc) and analytical chemistry (MSc) at Newcastle upon Tyne, before moving to Edinburgh University to undertake research for a PhD in crystallography. Dr Elder has 34 years’ experience in the pharmaceutical industry at a variety of different companies (Sterling, Syntex and GSK). For the last 19 years he has been employed by GSK. He is currently a director in the externalisation group (SCINOVO) in GSK R&D. Dr Elder is a member of the British Pharmacopoeia (Expert Advisory Group PCY: Pharmacy), a member of the Analytical Division Council (Royal Society of Chemistry, UK) and a council member of the Joint Pharmaceutical Analysis Group, UK. He is also a member of the PhRMA and EfPIA sub-groups on genotoxic impurities and was part of the PQRI group that assessed the control strategies for alkyl mesylates. He has published over 40 papers and given over 60 presentations at international fora on a variety of pharmaceutical topics. He has authored six book chapters focused on degradation, impurity identification/control (both genotoxic and standard impurities) and in vitro approaches to assess genotoxicity. David is a fellow of the Royal Society of Chemistry (FRAC), a chartered scientist (CSci) and chartered chemist (CChem). He is also a member of the Chartered Quality Institute (MCQI) and Chartered Quality Professional (CQP). 


Mike Webb was awarded his BSc in chemistry from the University of Essex. He went on to a masters degree in molecular spectroscopy at Kingston University and a PhD in molecular recognition at Imperial College of Science, Technology and Medicine in London. After a short career in academia, Dr Webb joined the pharmaceutical industry, where he has remained for 32 years. During the bulk of this time Dr Webb has worked in positions of increasing responsibility in analytical chemistry, specialising in spectroscopy and later separation sciences. Dr Webb has published a number of papers, presented at international meetings and has edited two books on the analysis of pharmaceuticals. In January 2010 Dr Webb took a short secondment into a small group looking at oligonucleotide delivery. In October 2010 he took up his current role as Vice-President of API Chemistry & Analysis UK. He is responsible for the synthetic design, scale-up and analytical control of active drug substances in development in the UK.    


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