Analysis of Oligonucleotides
`and their Related
`Substances
`
`CHEMGENES EXHIBIT 2024
`Shanghai Hongene v. ChemGenes
`Case IPR2023-00490
`
`ChemGenes Exhibit 2024 - Page i
`
`

`

`THE CHROMSOC SEPARATION SCIENCE SERIES
`
`This series of high-level reference works provides a comprehensive look at key
`subjects in the field of separation science. The aim is to describe cutting-edge topics
`covering all aspects and applications of this important discipline. Each book is a vital
`technical resource for scientists and researchers in academia and industry.
`
`Published titles
`
`Monolithic Chromatography and its Modern Applications
`Edited by Perry G. Wang
`
`Analytical Characterisation and Separation of Oligonucleotides and their Impurities
`Edited by George Okafo, David Elder and Mike Webb
`
`Endorsed by
`
`The Chromatographic Society
`
`ChemGenes Exhibit 2024 - Page ii
`
`

`

`Analysis of Oligonucleotides
`and their Related
`Substances
`
`Edited by George Okafo, David Elder
`and Mike Webb
`
`ChemGenes Exhibit 2024 - Page iii
`
`

`

`Published in 2013 by ILM Publications
`Oak Court Business Centre, Sandridge Park,
`Porters Wood, St Albans, Hertfordshire,
`AL3 6PH, UK
`
`www.labmate-online.com/books
`
`Copyright # 2013 ILM Publications
`
`ILM Publications is a trading division of International Labmate Limited
`
`All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system
`or transmitted in any form or by any means, electronic, mechanical, photocopying, recording,
`scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988
`or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham
`Court Road, London, W1T 4LP, UK, without the permission in writing of the publisher.
`Requests to the publisher should be addressed to ILM Publications, Oak Court Business Centre,
`Sandridge Park, Porters Wood, St Albans, Hertfordshire, AL3 6PH, UK, or emailed to
`info@ilmpublications.com.
`
`Product or corporate names may be trademarks or registered trademarks but, for reasons of style
`and consistency, the TM and 1 symbols have not been used. Product or corporate names are
`used only for identification and explanation without intent to infringe. The publisher is not
`associated with any product or vendor mentioned in this book.
`
`This book contains information obtained from authentic and highly regarded sources. Reprinted
`material is quoted with permission, and sources are indicated. A wide variety of references are
`listed. Reasonable efforts have been made to publish reliable data and information, but the
`author and the publisher cannot assume responsibility for the validity of all materials or for the
`consequences of their use.
`
`British Library Cataloguing in Publication Data
`
`A catalogue record for this book is available from the British Library
`
`ISBN 978-1-906799-14-4
`
`Typeset by Keytec Typesetting Ltd, Dorset, UK
`
`Printed in Great Britain by Biddles, part of the MPG Books Group, Bodmin and King’s Lynn
`
`ChemGenes Exhibit 2024 - Page iv
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`

`

`Table of Contents
`
`List of Acronyms
`
`The Editors
`
`The Contributors
`
`Foreword
`
`Preface
`
`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
`References
`
`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
`
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`ix
`
`xv
`
`xvii
`
`xxi
`
`xxiii
`
`1
`
`1
`3
`
`5
`5
`11
`
`13
`14
`17
`18
`
`21
`
`21
`
`22
`29
`48
`80
`87
`87
`89
`
`

`

`vi
`
`Analysis of Oligonucleotides and their Related Substances
`
`Chapter 3
`
`Chapter 4
`
`Chapter 5
`
`Acknowledgements
`References
`
`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
`References
`
`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
`References
`
`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
`Future Perspectives for NMR Characterisation of
`Oligonucleotides
`Acknowledgements
`References
`
`5.7
`
`89
`89
`
`101
`
`101
`103
`110
`114
`127
`141
`152
`
`157
`
`157
`158
`169
`172
`177
`190
`191
`192
`
`201
`
`201
`205
`219
`
`229
`
`232
`
`232
`
`236
`240
`240
`
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`

`

`Table of Contents
`
`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
`References
`
`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
`Storage of Oligonucleotides
`7.5
`siRNA Stability Case Study
`7.6
`Summary
`7.7
`Acknowledgements
`References
`
`Index
`
`vii
`
`243
`
`243
`244
`248
`259
`263
`264
`266
`
`271
`
`271
`272
`274
`
`282
`286
`287
`293
`293
`294
`
`297
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`

`

`List of Acronyms
`
`1X PBS
`2-AP
`2D
`3-HPA
`
`A
`AA
`Ac
`ACN
`ADTT
`AEX
`API
`ATT
`
`BAP
`BDMAB
`BEH
`BNA
`BSE
`Bz
`
`C
`C&D
`CD
`cDNA
`CDTA
`CE
`CGE
`CID
`CNET
`COSY
`CPG
`CQAs
`
`phosphate buffered saline with the same molarity as standard saline
`2-aminopurine
`2-dimensional
`3-hydroxypicolinic acid
`
`adenosine
`atomic absorption spectroscopy
`acetyl
`acetonitrile
`1,2-dithiazole-5-thione (xanthane hydride)
`anion exchange chromatography
`active pharmaceutical ingredient
`2-amino thiothymine
`
`bacterial alkaline phosphatase
`butyldimethylammonium bicarbonate
`ethylene bridged hybrid
`bicyclic nucleic acid
`bovine spongiform encephalopathy
`benzoyl
`
`cytidine
`cleavage and deprotection
`circular dichromism
`complementary DNA
`trans-1,2-diaminocyclohexane-N,N,N9,N9-tetraacetic acid
`cyanoethyl
`capillary gel electrophoresis
`collision-induced dissociation
`N3-cyanoethyl-thymidine
`correlation spectroscopy
`controlled pore glass
`critical quality attributes
`
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`x
`
`CSP
`
`dA
`Da
`DABP
`dC
`DCA
`DCI
`DCM
`DDTT
`
`DE
`DEA
`dG
`DLS
`DMAP
`DMT
`DNA
`DOSY
`DQF-COSY
`DSC
`dsRNA
`DTD
`
`EDITH
`EDTA
`ESI-MS
`
`FDA
`FID
`FLP
`FLP(PO)
`
`FTICR
`FT-NMR
`FWHM
`
`G
`Gapmer
`
`GC
`
`HA
`HAA
`HFIP
`
`Analysis of Oligonucleotides and their Related Substances
`
`calf spleen phosphodiesterase
`
`deoxy adenosine
`Dalton, unit of atomic mass
`3,4-diaminobenzophenone
`deoxy cytidine
`dichloroacetic acid
`4,5-dicyanoimidazole
`dichloromethane
`3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-
`thione
`delayed extraction
`diethylamine
`deoxy guanosine
`dynamic light scattering
`4-N,N-dimethylamino pyridine
`dimethoxytrityl
`deoxyribonucleic acid
`diffusion-ordered spectroscopy
`double quantum filtered-COSY
`differential scanning calorimetry
`double-stranded RNA
`dimethylthiuram disulfide
`
`3-ethoxy-1,2,4-dithiazoline-5-one
`ethylenediaminetetraacetic acid
`electrospray ionisation mass spectrometry
`
`US Food and Drug Administration
`free induction decay
`full-length product
`phosphorothioate oligonucleotide having a single phosphodiester
`linkage
`Fourier transform ion cyclotron resonance
`Fourier transform NMR spectroscopy technique
`full width at half maximum
`
`guanosine
`class of oligonucleotide having a stretch of deoxynucleoside bases
`flanked by LNA, 29OMe 29-F or MOE at either end
`gas chromatography
`
`hexylamine
`hexylammonium acetate
`1,1,1,3,3,3,-hexafluoro-2-propanol
`
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`List of Acronyms
`
`xi
`
`HFIP/TEA
`HMBC
`HPLC
`HPLC-MS
`HSQC
`
`hexafluoroisopropanol with triethylamine
`heteronuclear multiple-bond correlation spectroscopy
`high-performance liquid chromatography
`HPLC mass spectrometry
`heteronuclear single-quantum correlation spectroscopy
`
`iBu
`IC
`ICH
`ICP-MS
`ICP-OES
`IDMS
`IEX
`IM-MS
`IP
`IP-RP
`ITC
`
`LAL
`LC
`LCAA
`LC-MS
`
`LIT
`LNA
`L-TOF
`
`MALDI-TOF
`MD
`MMT
`MOE
`
`mRNA
`MS
`MSn
`n 1
`n 1(PS)
`
`ND
`Nd:YAG
`NMI
`NMR
`NN
`NOE
`
`isobutyryl
`ion chromatography
`International Conference on Harmonization
`inductively coupled plasma mass spectrometry
`ICP optical emission spectroscopy
`isotope dilution mass spectrometry
`ion exchange chromatography
`ion mobility-mass spectrometry
`ion-pairing
`ion-pairing reversed phase
`isothermal titration calorimetry
`
`Limulus amoebocyte lysate
`liquid chromatography
`long chain alkyl amine
`combination of chromatographic techniques with mass spectro-
`metric detection
`linear ion trap
`locked nucleic acid
`linear time-of-flight
`
`matrix-assisted laser desorption/ionisation – time of flight
`molecular dynamics
`monomethoxytrityl
`oligonucleotide having O-methoxyethyl substitution at the
`29-position
`messenger RNA
`mass spectrometry
`tandem mass spectrometry
`
`impurity characterised by FLP minus a single base
`impurity characterised as FLP minus the 39-terminal base having a
`39-terminal phosphorothioate monoester
`not defined/not determined
`neodymium-doped yttrium aluminium garnet
`N-methylimidazole
`nuclear magnetic resonance (spectroscopy)
`nearest neighbour
`nuclear Overhauser effect
`
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`xii
`
`Analysis of Oligonucleotides and their Related Substances
`
`NOESY
`
`nuclear Overhauser effect spectroscopy
`
`PADS
`PAGE
`PBS
`PCR
`PEG
`POS
`PS
`PTFE
`
`QbD
`QIT
`QqQ
`
`rMD
`RNA
`ROESY
`RP
`RTIL
`
`S/N
`SAX
`SEC
`siRNA
`SNP
`SPE
`SRM
`SVP
`
`T
`TBDMS
`TCA
`TEA
`TEA: 3HF
`TEAA
`TEAB
`THAP
`THF
`Tm
`TMP
`TOCSY
`TOF
`TSP
`
`phenylacetyl disulfide
`polyacrylamide gel electrophoresis
`phosphate buffered saline
`polymerase chain reaction
`polyethylene glycol
`polyorg sulfa
`polystyrene
`polytetrafluorethylene
`
`quality by design
`quadrupole ion trap
`triple quadrupole
`
`restrained molecular dynamics
`ribonucleic acid
`1H-1H rotational frame NOESY
`reversed phase
`room-temperature ionic liquid
`
`signal-to-noise ratio
`strong anion exchange
`size exclusion chromatography
`short interfering ribonucleic acid
`single nucleotide polymorphism
`solid-phase extraction
`selected reaction monitoring
`snake venom phosphodiesterase
`
`thymidine
`tert-butyldimethylsilyl
`trichloroacetic acid
`triethylammonium
`triethylamine-trihydrogen fluoride
`triethylammonium acetate
`triethylammonium bicarbonate
`2,4,6-trihydroxyacetophenone
`tetrahydrofuran
`melting temperature
`trimethyl phosphate
`total coherance transfer spectroscopy
`time-of-flight
`3-trimethylsilyl-propionate
`
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`

`

`List of Acronyms
`
`xiii
`
`U
`UPLC
`UV
`UVRR
`
`VT
`
`uridine
`ultra-high-pressure liquid chromatography
`ultra-violet
`ultra-violet/resonance Raman spectroscopy
`
`variable temperature
`
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`

`

`The Editors
`
`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 pharma-
`ceutical 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 chem-
`istry (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
`
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`xvi
`
`Analysis of Oligonucleotides and their Related Substances
`
`(both genotoxic and standard impurities) and in vitro approaches to assess genotoxi-
`city. 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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`

`

`The Contributors
`
`Hagen Cramer, PhD
`
`Hagen Cramer, PhD, has worked in the field of oligonucleotides
`since 1989. He has published 22 peer-reviewed papers including
`a book chapter, holds four patents and has authored one book. Dr
`Cramer received his PhD from the University of Konstanz,
`Germany in the laboratory of Professor Dr Wolfgang Pfleiderer in
`1995. After completing his PhD, he joined Dr Paul Torrence’s
`group at the National Institutes of Health for post doctorate work on chemically
`modified 2-5A analogues and 2-5A antisense. Before joining Girindus in 2005, Dr
`Cramer served as Scientific Director for Gemini Technologies and as Director of
`Chemistry for Ridgeway Biosystems. At NITTO DENKO Avecia he currently is
`Director of Operations where he directs the company’s operations through a team of
`functional managers and group leaders, with a focus of timely delivery of Active
`Pharmaceutical Ingredients (APIs), adherence to cGMP procedures, safety, customer
`satisfaction, employee relations as well as setting and achieving plant performance
`metrics.
`
`Kevin Finn, PhD
`
`Kevin Finn completed his undergraduate education in chemistry at
`Ohio University where he carried out undergraduate research with
`Professor Mark McMills in the area of Rh-catalysed tandem
`reactions. Kevin received his PhD from Brock University (Ontario,
`Canada) in 2006 under the supervision of Professor Toma´sˇ
`Hudlicky´. His thesis work involved the development of chemo-
`enzymatic methods toward the synthesis of synthesis of morphine alkaloids. Kevin
`accepted a post-doctoral position in Freiburg, Germany with Professor Reinhard
`Bru¨ckner, where he was engaged in total synthesis of light-harvesting carotenoids.
`Since 2008, Kevin has been employed at NITTO DENKO Avecia in the Oligonucleo-
`tides Process Development Group. His title is Scientist, and his focus is scale-up,
`technology transfer and process validation of therapeutic oligonucleotides.
`
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`

`xviii
`
`Analysis of Oligonucleotides and their Related Substances
`
`Nanda Sinha, PhD
`
`Dr Nanda D. Sinha received his doctoral degree in organic
`chemistry from Patna University (Patna) India. His postdoctoral
`works at the university of Massachusetts and Yale University were
`to develop newer methods for synthesis of natural products and
`their analogues. Later, he joined the laboratory of Professor
`Hubert Koester at the University of Hamburg to pursue research work in the field of
`nucleic acids chemistry and oligonucleotides. There he co-invented ß-cyanoethyl
`phosphoramidite chemistry for the synthesis of oligonucleotides in 1983 and also
`developed a method for the solid phase synthesis of methylphosphono-oligonucleo-
`sides. In 1986, Dr Sinha joined Biosearch Inc. as a Research Scientist in California.
`Subsequently at Millipore MA he was in charge of nucleic acids chemistry. There he
`developed many newer reagents and chemistries for the refinement of oligonucleotides
`synthesis. Dr Sinha, in 1996, co-founded Boston Biosystems Inc. for large-scale
`synthesis of oligonucleotides under cGMP to support therapeutic and diagnostic
`applications. The company was acquired by Avecia Inc. in 1999. At Avecia, he served
`as a Vice-President of Research & Development. He was instrumental in the scale-up
`siRNA synthesis, purification and developed a very efficient activator for synthesis
`based on Saccharin. Dr Sinha has contributed four chapters in different books in the
`field of oligonucleotides and analogues. He has co-authored more 45 scientific
`publications and is co-inventor and inventor of more than 10 US Patents.
`
`Bernhard Noll, PhD
`
`Dr Bernhard Noll is a Consultant for Analytics & CMC (Chem-
`istry, Manufacturing & Controls) (www.NBChem.de) in the Bos-
`ton area (MA, USA), providing strategic and technical expertise
`on analytics, validation, process development, scale-up and tech-
`nology transfer. Prior to his current role, he served as CMC
`Project Manager for ThromboGenics NV (Leuven, Belgium),
`contributing to the company’s successful first BLA submission. His responsibilities
`included planning and coordinating of CMC activities at external contractor sites, and
`writing and review of technical and regulatory documentation.
`Prior to joining Thrombogenics, he served for several years as Associate Director
`Analytics CMC, at Roche Kulmbach GmbH (Kulmbach, Germany), playing a leading
`role in the IND preparation of Roche’s first siRNA therapeutic. Dr Noll started his
`professional work in 1998 as a Scientist at Coley Pharmaceutical GmbH, where he
`helped the company to evolve from a small start-up to a major player in the field of
`nucleic acid therapeutics. In his last position at Coley, he served as Associate Director
`in Chemistry and Analytics in Du¨sseldorf, Germany. Dr Noll obtained his diploma in
`Chemistry at the Technische Universita¨t Darmstadt in 1993 and received his PhD in
`Biochemistry at the University of Mainz in 1997.
`
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`

`The Contributors
`
`xix
`
`Ingo Ro¨hl, PhD
`
`Dr Ro¨hl studied chemistry at the University of Oldenburg (Ger-
`many) from 1992 to 1997, after which he gained his Diplom
`thesis work in combinatorial organic chemistry. From 1998 to
`2000, he completed his PhD work, focussing on the isolation and
`identification of new sex pheromones from marine invertebrates.
`From 2001 to 2003, Dr Ro¨hl became head of the analytical chemistry group at the
`NOXXON Pharma AG, Berlin. The company is focused on the development of the so-
`called Spiegelmers – RNA aptamers based on the non-natural L-ribose configuration
`in the sugar backbone. From 2003 to 2007, he moved to Alnylam Europe AG, where
`he was head of analytical chemistry with a focus on siRNA analytics. From 2007 to
`2011, he joined Roche Kulmbach GmbH as associate director of analytical chemistry
`at the Roche RNA Center of Excellence. His main responsibilities include leading the
`analytical QC group to support the in-house synthesis department, CMC development
`for clinical RNA candidates and DMPK and tissue distribution of siRNA. Currently,
`Dr Ro¨hl is Director of Analytics and DMPK at the Axolabs GmbH in Kulmbach,
`Germany.
`
`Patrick Limbach, PhD
`
`Patrick A. Limbach was born in 1966 and obtained his BS degree
`in 1988 from Centre College (Kentucky, USA). In 1992 he
`obtained his PhD degree in Analytical Chemistry from The Ohio
`State University, under the supervision of Alan G. Marshall. He
`spent two years as a post-doctoral researcher in the laboratory of
`James A. McCloskey at the University of Utah. In 1995 he
`accepted his first faculty position in the Department of Chemistry at Louisiana State
`University in Baton Rouge, Louisiana. In 2001 he moved to his current location in the
`Department of Chemistry at the University of Cincinnati. He is a Professor of
`Chemistry and is an Ohio Eminent Scholar. His research interests include the develop-
`ment of new mass spectrometry methods for analysing RNAs, the identification of
`unknown modified nucleosides, the characterisation of RNA-protein complexes and
`investigating the role of modified nucleosides in biological systems.
`
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`xx
`
`Analysis of Oligonucleotides and their Related Substances
`
`Elena Bichenkova, PhD
`
`Dr Bichenkova graduated in Chemistry (BSc – 1983) with a PhD
`in NMR structural studies of nucleic acids (1993, Russia), Dr
`Elena Bichenkova continued her research in the USA at Purdue
`University (1992, 1994) and then at the University of Texas
`(1996) collaborating with the first-rank NMR laboratory of
`Professor David G. Gorenstein. After being awarded a Royal Society/NATO Postdoc-
`toral Fellowship in 1996, she joined the University of Manchester as a Research
`Fellow. In January 2004 Elena was appointed by School of Pharmacy & Pharma-
`ceutical Sciences as a Lecturer in Medicinal Chemistry followed by promotion to the
`Senior Lecturer level in 2009.
`
`Daren Levin, PhD
`
`Dr Levin received his MSc from Northeastern University in
`Chemistry and Chemical Biology and his Ph.D. from Northeast-
`ern University in Analytical Chemistry with a focus on the
`development of differential mobility spectrometry – mass spec-
`trometry instrumentation and its use for the analysis of proteins
`peptides and oligosaccharides. Prior to joining GlaxoSmithKline
`he worked at Alkermes Inc. on the analytical development and control strategy of their
`novel inhaled and injectable microsphere products encompassing small molecule,
`protein and peptide therapeutics. Since joining GlaxoSmithKline in 2006 he has taken
`a lead role in the CMCs of oligonucleotide therapeutics. He has co-authored an
`industry and regulatory agency led white paper on oligonucleotide therapeutic drug
`substance specifications and has also authored several peer-reviewed articles and
`presented at several conferences on the analytical method development/validation,
`control strategy and stability of oligonucleotide therapeutics.
`
`ChemGenes Exhibit 2024 - Page xx
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`

`

`Foreword
`
`Working in the field of synthetic oligonucleotides is extremely fascinating. Synthetic
`oligonucleotides are an exciting class of therapeutic products that are under develop-
`ment for a variety of indications. Many of them have been developed to address
`significant medical needs that are so far unmet.
`Oligonucleotides are a unique class of molecules with diverse mechanisms of
`action different from small molecules but also different from biopharmaceuticals. The
`specific chemical properties and the large sizes compared to small molecules make
`the analysis of synthetic oligonucleotides and the quantification of their related
`substances and degradation products one of the most challenging tasks for analytical
`chemists.
`Characterisation and quality control testing are required throughout the clinical
`development of oligonucleotides intended for therapeutic use. Detailed information on
`structural characterisation studies that supports the designation of these properties or
`characteristics should be provided in submissions to regulatory agencies worldwide.
`What to control is one of the key questions in connection with regulatory
`submissions during clinical development and marketing authorisation applications.
`Identifying critical quality attributes (CQAs) is vital during pharmaceutical
`development. A critical quality attribute is a physical, chemical, biological or micro-
`biological property or characteristic that should be within an appropriate limit, range
`or distribution to ensure the desired product quality. Impurities are an important class
`of potential drug substance CQAs because of their potential impact on drug product
`safety. This is described in detail in the International Conference on Harmonisation
`Quality Guidelines on pharmaceutical development (ICH Q8) and the development
`and manufacture of drug substances (ICH Q11). Consequently, a major focus is
`knowledge and control of impurities. How to apply these principles to the analysis of
`oligonucleotides is a core feature of the present book.
`Synthetic oligonucleotides are not included in the scope of the ICH specification
`guidelines – neither for synthetic drug substances (ICH Q6A) nor for products of
`biotechnology (ICH Q6B). They are also excluded from the ICH Guidelines on
`impurities (ICH Q3A and Q3B) and therefore consequently from the scope of ICH
`Q11. Nevertheless, in ICH Q11 it is clearly stated that this guideline might also be
`appropriate for other types of products, such as oligonucleotides, following consulta-
`tion with the appropriate regulatory authorities.
`Critical quality attributes are part of the overall target product profile that is based
`on the desired clinical performance. The extent of characterisation is linked to the
`
`ChemGenes Exhibit 2024 - Page xxi
`
`

`

`xxii
`
`Analysis of Oligonucleotides and their Related Substances
`
`level of risk associated with each phase of drug development. It is expected that the
`oligonucleotide molecule will have been well-characterised before a marketing
`authorisation application is submitted to the regulatory agencies.
`New and improved state-of-the-art analytical technologies and techniques are
`becoming available on an on-going basis and should be applied during development
`for both characterisation and routine control. Analytical methods for characterisation
`during development and control for release and/or stability testing are covered
`excellently and comprehensively in the present book. The authors of each chapter are
`practitioners of the art and leaders in the field of oligonucleotide analysis.
`Clinical qualification is considered the most
`important aspect when setting
`acceptance criteria in specifications. For critical attributes the acceptance criteria
`should not be wider than what has been qualified to yield a safe and efficacious
`product. Therefore,
`identifying differences in impurity profiles using orthogonal
`analytical methods for the detection and quantification of impurities in early stages of
`drug development and the application of good science will be extremely helpful in the
`course of development.
`For successful research and development, it is necessary to acquire new skills
`and knowledge in the field of oligonucleotide analysis. Therefore, there was a dire
`need for such a book as this which provides various analytical technologies that are in
`frequent use in modern research.
`The book is subdivided into seven chapters. An introduction to oligonucleotides
`is followed by a comprehensive chapter on oligonucleotide impurities and their origin.
`Chapter 3 provides an overview on the separation of oligonucleotides and related
`substances. Chapter 4 deals with the analytical characterisation of oligonucleotides by
`mass spectrometry while Chapter 5 is concerned with the analytical characterisation
`of oligonucleotides by NMR spectroscopy. Chapter 6 focuses on the analytical
`characterisation of oligonucleotides using thermal melting curves. The last chapter of
`the book is devoted to very important topics in connection with oligonucleotide
`stability and degradation. Each chapter is composed as an independent unit enabling
`the reader to pick the topic of immediate interest.
`The technological achievements over the last decades are tremendous; this book
`provides the reader with broad perspectives and a wealth of current knowledge related
`to oligonucleotide research.
`The reader of this book is also familiarised with the actual status of instrumenta-
`tion, i.e. the current state of the art and the different latest techniques. All techniques
`are described in a clear manner and by means of examples and case studies including
`explanations of the theoretical background.
`The authors must be congratulated for producing what is considered a truly
`outstanding and unique work, thereby rendering a most valuable service to scientists
`working in the field of oligonucleotides in support of therapeutic development.
`
`Bonn, December 2012
`Dr Rene´ Thu¨rmer, Pharmaceutical Assessor
`BfArM – Federal Institute for Drugs and Medical Devices
`Kurt-Georg-Kiesinger-Allee 3, 53177 Bonn, Germany
`
`ChemGenes Exhibit 2024 - Page xxii
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`

`

`Preface
`
`Over the last two decades, research into the development of synthetic oligonucleotides
`as therapeutic medicines has grown significantly. These biopolymers have the potential
`to be used as highly specific, targeted medicines to treat a wide range of diseases,
`particularly where there is a genetic origin or basis, such as Duchene muscular
`dystrophy, thrombotic thrombocytopenic purpura and cystic fibrosis.
`This growth in oligonucleotide research has been catalysed by a number of key
`historical events. First was the 2006 Nobel Prize for Fire and Mello’s ground-
`breaking discovery of RNA interference, which signalled the possibility of silencing
`genes. Second was the sequencing of the human genome, which has helped science
`to create a map of key gene groups that is implicated in many genetic disorders.
`Third was the regulatory approval of oligonucleotide-based drugs such as fomivirisen
`sodium (Vitravene) in 1997, an antisense oligonucleotide used to treat cytomegalo-
`virus retinitis in acquired immune deficiency syndrome patients, and,
`in 2004,
`pegaptanib octasodium (Macugen), an angiogenic pegylated aptamer approved for
`the treatment of neovascular age-related macular degenerative disease. A wide range
`of oligonucleotide therapeutic classes now exist; these include short interference
`RNA (siRNA), antisense oligonucleotides (ASOs), oligonucleotide ligands (aptamers
`and Speigelmers), immunomodulatory oligonucleotides (IMOs), micro interference
`RNA-blocking oligonucleotides, RNA decoys, ribozymes and DNA enzymes. By far
`the most cited molecules are the siRNAs, ASOs, aptamers and IMOs.
`Synthetic oligonucleotides have a relatively simple linear structure consisting of
`repeating nucleotides of between 10 and 21 units (or mers; i.e. 10mer) in length. Their
`molecular weights range from 3.5 to 7 KDa. Uniquely, the biological properties of
`oligonucleotides are principally a function of their primary and secondary structure.
`Each nucleotide unit is linked together by a phosphate backbone and consists of a
`sugar residue (ribose for RNA or deoxyribose for DNA) and an organic purine
`(guanine and adenine) or pyrimidine (uracil, thymine and