Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics Using LC-MS/MS Technology and Expertise to Reduce Risk and Increase Your Speed to Market

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 1 Introduction

Oligonucleotides are a rapidly growing family of biopharmaceutical products whose therapeutic potential is now primed and waiting to be unlocked. First recognized for their ability to modulate gene expression more than three decades ago, advances in fundamental research and enabling technologies have doubled the number of oligonucleotide drugs on the market since the start of 2016, with many more expected in the years to come. This important class of therapeutics is coming of age, and for biopharmaceutical companies, the race is on to launch the next generation of innovative oligonucleotide products. Bringing safe and effective oligonucleotides to market relies on robust bioanalysis workflows. However, given their unique physicochemical properties, oligonucleotides require advanced and unique analytical strategies that go above and beyond those used to study conventional small molecule or even peptide and protein drugs. Implementing ineffective solutions that poorly meet these needs can cause substantial bioanalysis backlogs, lengthy study delays or even suboptimal development decisions based on incomplete or incorrect data. Even the largest and most experienced drug developers are not immune to these challenges. To accelerate the development of your oligonucleotide therapeutics and drive clinical success, you need robust bioanalysis solutions underpinned by industry- leading analytical expertise, cutting-edge technologies and proven workflows built on decades of experience.

Abstract Developing bioanalytical solutions to support oligonucleotide drug programs can be challenging due to the unique physicochemical properties of these molecules. This whitepaper outlines both the established and the unique liquid chromatography and extraction technologies Covance has developed to ensure you remain at the forefront of oligonucleotide innovation. Learn how you can benefit from the workflows to: ▶ Eliminate bottlenecks and accelerate timelines with state-of-the-art liquid chromatography, focused detection and extraction technologies that generate accurate and robust data ▶ Rapidly receive the results you need through world-class scientific expertise based on decades of experience in solving complex bioanalytical challenges ▶ Capitalize on the rapidly expanding oligonucleotide marketing with the support of technologies and expertise Now Is the Time to Get Ahead of the Oligonucleotide Therapeutics Field

Oligonucleotide therapeutics However, following extensive encompass a wide range of research efforts and a large number different modalities, with antisense of clinical trials, the last few years oligonucleotides (ASOs), small have seen an upsurge in the number interfering RNA (siRNA), micro of oligonucleotide drugs that RNA (miRNA) antagomirs/mimetics have been granted FDA approval, and oligonucleotide aptamers among signaling the start of a new era in the most widely utilized families.1 oligonucleotide therapeutics. Since Each class has been shown to operate 2016, the number of oligonucleotide through different mechanisms, products on the market has more which researchers are exploring to than doubled and a wealth of develop new treatments across a promising candidates are currently broad range of therapeutic fields, in late-stage clinical trials. The oligonucleotide including cancer,2 neurodegenerative This growth shows no sign of conditions3 and infectious diseases.4 therapeutics market stopping, with the oligonucleotide is expected to grow Despite the breadth of targets and therapeutics sector expected to mechanisms of action, translating increase at an annual rate of by 10% annually to all this potential into marketable 10% between 2018 and 2022 to reach $760 million drugs has had something of a an estimated $760 million.6 The by 2022. Now is the slow start (see market milestones oligonucleotide market is currently time to expedite timeline).5 Prior to 2016, just wide open and a new generation of three oligonucleotide therapeutics safe and effective therapeutics now your research in this (pegaptanib, mipomersen and within reach. This represents an exciting drug area, fomivirsen) had been approved enormous opportunity to capitalize before the market by the U.S. Food and Drug on this growing market and get fully matures.6 Administration (FDA), with the latter ahead of the competition. discontinued due to improvements in HIV treatments.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 3 Market Milestones in the Development of Oligonucleotide Therapeutics5

FDA Fomivirsen (Vitravene) Approval Developers: Isis (now Ionis) 1998 Pharmaceuticals & Novartis Ophthalmics

Pegaptanib (Macugen) FDA Approval Developer: NeXstar Pharmaceuticals 2004

FDA Mipomersen (Kynamro) Approval Developer: Isis (now Ionis) 2013 Pharmaceuticals

Defibrotide (Defitelio) FDA Approval Developer: Jazz Pharmaceuticals 2016

FDA Eteplirsen (Exondys 51) Approval Developer: Sarepta 2016 Therapeutics

Nusinersen (Spinraza) FDA Approval Developers: Ionis Pharmaceuticals & Biogen 2016

FDA Onpattro (Patisiran) Approval Developer: Alnylam 2018 Pharmaceuticals, Inc.

Givlaari (Givosiran) FDA Approval Developer: Alnylam 2019 Pharmaceuticals, Inc.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 4 Experience and Expertise Are Essential When Developing Oligonucleotide Bioanalysis Workflows

Developing and validating The analysis of oligonucleotides Given these challenges, many bioanalytical methods for using traditional LC-MS techniques biopharmaceutical companies choose oligonucleotide therapeutics by is also more challenging than to work with external partners to liquid chromatography-mass for other types of molecules. access the specialist techniques, spectrometry (LC-MS) is widely Oligonucleotides are comprised of technologies and expertise necessary regarded as being more challenging just four alternating nucleobases, for successful oligonucleotide than conventional small molecule meaning that chromatographic bioanalysis. Moreover, with analysis.7 Without capable separation is more mass- and bioanalysis workflows involving technologies and specialist know- size-based than most other extensive protocol development how, bioanalytical strategies may chromatography applications. and careful method optimization, deliver inaccurate or unreliable These molecules are also highly outsourcing to dedicated specialists results and leave oligonucleotide polar and multiply charged, can be a notably more cost-effective development programs vulnerable making conventional reversed- route to achieving successful results to unexpected issues and delays. phase chromatography unsuitable. in the shortest possible timeframe. These potential challenges arise The pronounced polarity and due to the unique physical multiple charging also produce characteristics associated with sensitivity challenges, especially in this biotherapeutic class. high-aqueous reversed-phase LC Outsourcing conditions, where the nebulized oligonucleotide Firstly, the isolation of evaporation process to produce oligonucleotides from biofluids bioanalysis discrete gas phase ions in the mass may often be complicated by ex vivo to dedicated spectrometer source component instability. Oligonucleotides also is that much more difficult due specialists can bind strongly to proteins and can to the high concentration of undergo nonspecific binding be a more water. Furthermore, as some (i.e., secondary binding) to cost-effective oligonucleotides can be thermally equipment and labware. Careful labile at low pH, analytical route to your consideration must therefore be paid parameters such as ion source to methods to minimize unreliable achieving temperature must be systematically quantification and ensure the success in the optimized. As such, it can be difficult collection of accurate data. to develop reliable quantitative shortest possible analysis methods using the LC-MS timeframe. techniques commonly employed for small molecule analysis.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 5 Benefit from Advanced Oligonucleotide Bioanalysis Techniques

To accelerate innovation and hit development deadlines, it’s essential your bioanalysis workflows incorporate the most capable oligonucleotide extraction, separation and detection techniques. Drawing on a global network of expertise with decades of experience in analytical problem-solving, we’ll work together to develop a robust bioanalysis strategy that meets your needs. This includes optimized methods are underpinned by state-of-the-art technologies, ensuring we deliver the results you need to make key decisions, on time and on budget.

High-Performance Ion-Pairing Methods State-of-the-Art LC-MS Systems Reversed-phase ion pair chromatography (RP-IPC) Issues around cross-reactivity and interference from is a well-established technology for oligonucleotide other metabolites found in biological samples can bioanalysis.8 It is used to separate polar organic present a serious challenge when using analytical molecules and employs the same types of eluents and techniques such as ELISA. LC-MS and LC-FD based on hydrophobic stationary phases as conventional reversed- tandem chromatographic separations with either mass phase liquid chromatography. However, unlike standard spectrometry or fluorescence detection offer much higher reversed-phase techniques, RP-IPC utilizes an ion- levels of analytical specificity, eliminating the ambiguity pairing reagent (typically an alkylsulfonate, alkylsulfate or associated with false positives. alkylammonium salt), which binds with the analyte(s) to You’ll gain access to high-end triple quadrupole enable chromatographic retention and resolution from UHPLC-MS platforms, including Sciex Triple Quad™ other components. 6500+ systems that facilitate sub ng/mL limits of By varying the type and concentration of the ion-pairing quantitation for the determination and quantification reagent in the mobile phase, among other parameters, of oligonucleotides, as well as Sciex Triple Quad™ 4500 the chromatography and charge envelope can be systems for exploratory and early development work. moderated according to the analytical requirements. These systems are recognized for their high sensitivity in negative ion mode, which is particularly well suited You’ll have access to developed assays utilizing very for detecting oligonucleotide analytes. Such technologies effective ion pairing systems consisting of triethylamine give you complete confidence in the bioanalytical data you (TEA) and hexafluoroisopropanol (HFIP), specifically need to make critical drug development decisions. for oligonucleotide analysis. TEA is a mild base that acts as the ion pair reagent, improving chromatographic In addition, Thermo Fisher QExactive UHPLC-HRAM performance. HFIP is a weak acid that neutralizes TEA, systems are available and can offer both quantitative data improving droplet volatility and helping to desolvate and for GLP analysis while also collecting full-scan data files remove TEA and mobile phase solvents from the ion that can be utilized for exploratory mining of metabolites source to prevent signal suppression while enabling the that may be present in the extracts without having to separation of common metabolites. This combination reanalyze the samples. of reagents delivers very accurate results and eliminates delays to your program by ensuring very reliable performance and throughput.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 6 Advanced Ion Pairing-Free Methods Through the continual optimization of analytical methods, we’ve developed Novel Column Chemistries unique, next-generation bioanalysis workflows thateliminate the need for and Extraction Kits 9 ion-pairing reagents completely. These workflows, which employ separation Extend your team with a bench technologies based on hydrophilic interaction liquid chromatography (HILIC) of scientists that stay on top as well as ion pairing reagent-free sample preparation and extraction strategies, of the latest technologies to are designed to deliver more reliable data and maximize instrument uptime. incorporate the best methods HILIC is a variant of normal-phase liquid chromatography that utilizes a into workflows and work with hydrophilic stationary phase and a hydrophobic and water-miscible mobile vendors to evaluate new column phase to achieve reliable analyte separation (Figure 1). The hydrophilic chemistries and extraction kits stationary phase ligands used in HILIC systems include bare silica or so that these can be applied polar cyano, amide, diol, zwitterionic or aminopropyl chemistries.10,11 This where suitable. For example, combination of stationary and mobile phases results in the partitioning of many of our oligonucleotide analytes between a water-deficient bulk mobile phase and an immobilized bioanalysis workflows employ water-rich layer adsorbed onto the surface of the column (Figure 2). Waters oligonucleotide separation technology (OST) columns, which are based on C18-functionalized second-generation hybrid-silica FIGURE 1: HILIC Uses Elements of the Three Main LC Methods: BEH Technology™ particles. Normal-Phase Liquid Chromatography (NP-LC), Reversed-Phase Phenomenex® Clarity® Oligo-XT Liquid Chromatography (RP-LC) and Ion Chromatography (IC) columns, specifically designed for robust oligonucleotide separation, can also be used as an alternative. Advanced Type of stationary phase chemistries Charged stationary NP-LCIanalyt C such as these deliver exceptional compounds phase oligonucleotide resolution e and optimal throughput and adsorbent HILIC reliability, ensuring you have the answers you need to make timely eluent development decisions. Type of organic Concentration In addition, your bioanalysis solvent RP-LC of bu er program also benefits from ® Mobile phase Type of Phenomenex Clarity OTX™ composition ion-strength extraction kits for sample preparation workflows. By Bu er employing a mixed-mode solid pH value phase extraction (SPE) sorbent in conjunction with carefully formulated buffers, these kits are capable of isolating oligonucleotides from various interferences such as genomic DNA, large proteins, salts and sugars to ensure more accurate, reliable and consistent oligonucleotide quantification.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 7 FIGURE 2: HILIC Retention Mechanism, Showing Separation of Cytosine

Hydrophilic Electrostatic partitioning interaction

Si - NH + NH + O 2 2 NH + ACN 2

H2O H H H H O H O H H H O H H2O NH2+ ACN H H Hydrogen O approximately bonding 90% CAN H H H O H H H O O Si H H H O O O H H H H O H HH NH2+ H H O O H H H H O O O H O O H H H O O O H H - H O O O H O O O H H O H H O O O H H H

Water Acetonitrile:water (90:10)

H2O ACN:H2O (90:10) H H N O N O

90%90% ACACNN N N Si H N O- Si O O NH + NH + Si H O 3 3 - N Si O N Si O O O O NH O 3+ N H H Si Si Si O Si NH O 3+ O O H O H Si O Si O H O O O H O Si O H O O H O Si Si Si H H O H O O O H H H O O Si H2O O H Si Si O O Si O O O- H H O O Si H Si Si O- Si O O O O O Si O Si Si H O O Si Si Si Si O O O H O O H O Si H Si O O H O O- Si O Si Si Si O O- O H O O Si Si OO Si Si Si O O O O Si O Si Si Si

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 8 HILIC is capable of separating Alongside this approach, we have highly polar, zwitterionic or developed high-recovery mixed-mode Access the deep, charged analytes without the need SPE workflows that are free from for ion-pairing reagents, and the ion-pairing reagents. This approach specialized technique can be coupled with MS is ideal to support subsequent HILIC expertise and analysis to avoid the accumulation separation methods and is based on technologies of problematic residues which can the use of a SPE plate consisting of occur using ion-pairing techniques.9 weak anion exchange moieties on necessary to help HILIC is a more resilient method a polymeric base (WAX). Following you advance your that cuts instrument maintenance extensive method optimization, we oligonucleotide and reduces downtime, while also derived at a protocol that utilizes offering improved data reliability, two potent wash steps and delivers programs and minimizing the need for repeat exceptionally clean extracts, stay ahead of analyses. As such, HILIC offers at least comparable to those the competition. a very effective platform to that can be achieved using accelerate the development of your conventional mixed-mode SPE oligonucleotide therapeutic. protocols. Combined with the HILIC separation protocol, this method offers a highly flexible and effective approach for quantitative oligonucleotide analysis.9

To Drive Success in Oligonucleotide Bioanalysis, Seamless Support Is Essential

The unique physicochemical properties of Get bioanalytical solutions that give you the answers you oligonucleotides can present bioanalysis challenges need in the shortest possible timeframe though active beyond those encountered with traditional small engagement and a deep understanding of your specific molecule or peptide and protein workflows. So, when service requirements. Decades of experience in working it comes to outsourcing oligonucleotide bioanalytical with many of the world’s leading biopharmaceutical workflows, you need an experienced partner that has the companies to overcome complex bioanalysis challenges proven in-house technical expertise and bioanalytical ensure you get the right results, the first time around. problem-solving experience to provide seamless support – becoming an extension of your own team.

You need a partner who’ll be an extension of your team – not an isolated service provider who’s only prepared to run conventional tests and little more. Leverage a world-class reputation for solving complex bioanalytical challenges and the specialist know-how and technologies to advance your oligonucleotide programs.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 9 Located Where You Need Us

Harrogate, UK Huntingdon, UK Salt Lake City, Utah Madison, Wisconsin Indianapolis, Indiana Chantilly, Virginia Somerset, New Jersey Shanghai, China

Global Expertise in Oligonucleotide Bioanalysis

To cut through bioanalysis bottlenecks and access the data you need to progress the next generation of oligonucleotide therapeutics to market, partner with the global experts in solving complex bioanalytical challenges. Through decades of planning and executing successful bioanalysis projects for many of the world’s leading biopharmaceutical companies, we know that to overcome critical development challenges, it’s essential to invest in the most capable workflows and the skills of the expert teams that use them. State-of-the-art technologies, industry-leading bioanalytical expertise and global capacity ensure we unlock the potential in your molecule.

Overcoming Bottlenecks in Quantifying Oligonucleotide Therapeutics 10 References 1. A Khvorova, JK Watts, The chemical evolution of oligonucleotide therapies of clinical utility, Nature Biotechnology, 2017, 35(3), 283–248.

2. Y Morita, M Leslie et al. Aptamer Therapeutics in Cancer: Current and Future, Cancers (Basel), 2018, 10(3), DOI: 10.3390/cancers10030080.

3. DR Scoles, SM Pulst, Oligonucleotide therapeutics in neurodegenerative diseases, RNA Biology, 2018, 15(6), 707–714.

4. RL Juliano, The delivery of therapeutic oligonucleotides, Nucleic Acids Research, 2018, 44(14), 6518–6548.

5. CA Stein, D Castanotto, FDA-Approved Oligonucleotide Therapies in 2017, Molecular Therapy, 2017, 25(5): 1069–1075 and Alnylam Pharmaceuticals, Inc.

6. Technavio, Global Oligonucleotide Therapeutics Market 2018–2022, June 2018, https://www.technavio.com/report/global- oligonucleotide-therapeutics-market-analysis- share-2018.

7. Panel discussion on oligonucleotides, Chimica Oggi-Chemistry Today, 2018, 36(2)

8. JV Bonilla, GS Srivatsa, Handbook of Analysis of Oligonucleotides and Related Products, 2011, CRC Press.

9. R MacNeill, T Hutchinson et al. An oligonucleotide bioanalytical LC–SRM methodology entirely liberated from ion-pairing, Bioanalysis, Vol. 11, No. 12, 2019, https://www.future-science.com/ doi/10.4155/bio-2019-0031.

10. The Bioanalysis Glossary 3rd Edition, Bioanalysis, 2018, 10(21) Suppl. 1.

11. P Jandera, P Janás, Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review, Analytica Chimica Acta, 2017, 967: 12–32.

12. B Buszewski, S Noga, Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique, Analytical and Bioanalytical Chemistry, 2012, 402(1): 231–247. Learn more about our bioanalytical solutions at www.covance.com/BIOA

Covance is a business segment of LabCorp, a leading global life sciences company, which provides contract research services to the drug, medical device and diagnostics, crop protection and chemical industries. COVANCE is a registered trademark and the marketing name for Covance Inc. and its subsidiaries around the world.

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