Day 1 – June 25
Lars Holmberg, PeptiSystems AB (Plenary session)
Past and future challenges related to process development and manufacturing of oligonucleotides and peptides
Process development and manufacturing of peptide and oligonucleotide therapeutics have made great progress in the last decades thanks to improvements in different areas such as chemistry, instrumentation, analysis, and raw material quality, cost, and availability. This presentation will cover challenges in the areas that have been overcome in the past, the ones that still exist and how they can be addressed.
Thomas Müller-Späth, Chromacon (Joint Oligo & Peptide Session)
A systematic approach for MCSGP optimization
MCSGP process (“Multicolumn Countercurrent Solvent Gradient Purification”) represents a scalable manufacturing solution to match the increasing demand of oligonucleotide and peptide drugs. It displays several advantages over traditional singlecolumn chromatography such as increased yield, elimination of re-chromatography and reduced eluent consumption. The MCSGP process is usually designed based on a singlecolumn batch chromatogram. The initial design leads to the first set of operating parameters. To minimize process development time, a systematic approach is desirable. In this presentation new gradient design and systematic optimization procedures are demonstrated in an oligo purification case study. The procedures are verified using simulations based on a mechanistic model. The results show that the development and optimization procedures significantly streamline and standardize the development of MCSGP saving valuable time on the pathway to scale-up. Moreover, the presented case study shows the potential of mechanistic modelling in complementing experimental process design and accelerating the investigation of certain aspects of process characterization, such as robustness analysis.
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Oligonucleotides
Day 1 – June 25
Afaf El-Sagheer, Oxford University and Suez University
Biocompatible Artificial Backbones: A journey of mRNA adventures from modifying to destroying
Chemical ligation of DNA strands can be used to produce artificial DNA backbones which have potential for use in therapeutics and synthetic biology. Next-generation sequencing analysis of modified DNA templates that can be read through and copied accurately by DNA polymerases explained our backbone modifications and confirmed our hypothesis of the ability of these modifications in mimicking the native phospahate backbone. These results provide insights into the design of biocompatible backbone mimics that could be used in the assembly of large modified DNA and RNA constructs and used in various applications including Crispr gene editing. The transient nature of RNA makes it important in therapeutics and as biomarker for research and diagnostics. Its fragility on the other hand requires its conversion to DNA prior to detection. Reverse transcriptases are biased to specific RNA sequences, while ligases are inhibited by RNA secondary structures induced by the ligation conditions. We demonstrate that chemical ligation of DNA oligonucleotides hybridized to a complementary RNA template to form an artificial squaramide backbone is a high-yielding reaction that can be used to quantify very small amount of RNA. This reaction requires mildly buffered, monovalent salt solutions with no extra chemical reagents, and can be performed at a range of temperatures within minutes. We describe the careful design of a three-component ligation system and demonstrate its use in qPCR to detect long RNAs in complex systems and achieve 0.3 attomole detection limits, (on going work). A novel mRNA modification strategy can be used to investigate translation processes in cells. Our results demonstrate a significant enhancement in mRNA stability and protein output.
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Juliette Nourry, DPM laboratory
Studies of interactions between aptamers/antibodies and bacteria through direct and indirect analytical method
Aptamers are simple DNA or RNA sequences selected by SELEX procedures. These recognition elements exhibit efficacy across a wide range of analytes. In recent decades, there has been an increasing interest in the detection of bacteria by aptamers with the aim of developing new therapeutic and diagnostic tools. Numerous sequences targeting various bacteria have been reported in the literature. In this study, the interactions between aptamers/antibodies targeting ATCC 700728 and ATCC 8739 Escherichia coli strains were investigated in detail using both direct (flow cytometry) and indirect (fluorescence spectroscopy) methods. Many assays were carried out by varying several parameters such as the concentration of aptamers/antibodies, concentration of bacteria, nature of the buffer, temperature, and incubation time to find the optimal conditions to obtain the best interaction. Specificity studies were undertaken using two control strains (an untargeted Escherichia coli ATCC 700728 or ATCC 8739 and an ATCC 12228 Staphylococcus epidermidis) and the Minimum Inhibitory Concentrations (MICs) of the aptamers against the bacteria tested were assessed. Observing interactions through an indirect method has proven to be challenging due to the very low specific surface area of the cells and to the presence of intracellular content in the media. Conversely, employing flow cytometry demonstrated that this method is more suitable to study the interactions between bacteria and biomolecules. The main asset of flow cytometry is its rapid and direct detection, also the sample can be analysed immediately after incubation without treatment. However, in that case, the data processing method may impact the study’s conclusions. Finally, based on our results, the advantages, and the limitations of aptamers/antibodies as recognitions elements for bacteria was discussed.
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Claire McLeod, CPI
An overview of the oligonucleotide programme at CPI’s Medicines Manufacturing Innovation Centre
Oligonucleotide manufacturing, especially towards larger scales, is challenging and there are known limitations in the scalability and sustainability of the manufacturing process. The Grand Challenge 3 project is part of the Medicines Manufacturing Innovation Centre’s programme of pharmaceutical Grand Challenges – joint projects involving pharmaceutical and technology innovation companies. Grand Challenge 3 is developing a novel liquid phase approach to oligonucleotide synthesis. Another novel approach to manufacturing innovation comes from the GC3 Biocatalysis project which is developing a one-pot enzymatic approach to oligonucleotide synthesis. Alongside the Grand Challenge 3 projects CPI has several other manufacturing innovation projects, including a joint project with Intellegens to accelerate oligonucleotide process development using machine learning tools. The recent announcement of CPI’s new oligonucleotide centre to be opened in Glasgow, will provide a hub for these different projects. The centre aims to drive innovation, support clinical manufacture, and develop a highly trained workforce through tailored training programmes. An overview of the progress on the portfolio of projects will be discussed as well as an overview of the next challenges and opportunities to be addressed through the oligonucleotide manufacturing innovation centre of excellence.
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Patrick Endres, Tosoh Bioscience commercial speech
Therapeutic oligonucleotides & chromatography – what’s new
• Chromatographic approaches for purification and analysis
• Choosing appropriate strategies for different impurity classes
• Selected case studies
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Nadim Akhtar, AstraZeneca
European Pharma Oligonucleotide Consortium (EPOC)
European Pharma Oligonucleotide Consortium (EPOC) is a cross industry forum for developing CMC best practice to speed access to revolutionary medicines through collaboration, driving quality and efficiency in the development of oligonucleotides. Since its inception EPOC has published position papers on multiple topics and continues to be a thought leader on this exciting modality. This presentation will share EPOC success story and position on topics published to date
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Mark Hail, Novatia LLC
Automated LC-MS Strategies for High-Throughput and Detailed Analysis of Oligonucleotides
The developments of electrospray ionization (ESI) and modern liquid chromatography-mass spectrometry (LC-MS) methods and instrumentation have greatly expanded the capabilities for analyzing complex biomolecular samples. Traditionally, processing and interpretation of the mass spectral data from biomolecular samples has been laborious and time consuming. The development of new data processing strategies, involving robust algorithms for reliable and automated charge deconvolution with intuitive and informative results display, has enabled new application areas where ESI/MS can play a vital role. In this presentation, automated analytical strategies will be described that demonstrate both high-throughput (e.g., 3000 samples/day) and detailed LC-MS analysis applications of oligonucleotides. LC-MS with automated column switching allows for rapid desalting of oligonucleotides and analysis times as short as 25 sec/sample. ProMass Deconvolution software is used to automatically process the data after each sample is acquired. These systems enable high-throughput mass confirmation for quality control of expected synthesis products. The same instrument platform can be automatically switched from high-throughput mode to LC-UV-MS profiling mode for more detailed information such as purity assessment and impurity identification. These systems may be connected to either nominal mass instruments (e.g., ion trap) or high-resolution orbitrap analyzers for automated accurate mass analysis. The utilization of high-resolution orbitrap mass analyzers provide additional confidence in quality control with mass accuracy < 3 ppm mass error without any loss of sample throughput, while also unlocking several other important applications. Some of these applications include detailed accurate mass analysis of oligonucleotide therapeutic metabolites, MS/MS sequence confirmation, LC-MS characterization of 5’ capped mRNA, and bottom-up sequence confirmation of the open reading frame of mRNAs up to 4300 nts long.
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Xavier Gerard, Thermo Fisher Scientific commercial speech
Enhancing the Activity and Delivery of Oligonucleotide Therapeutics with Modified Phosphoramidites and Nucleotides
Oligonucleotide therapeutics are a promising new class of biopharmaceuticals, revolutionizing the landscape of molecular medicine. They offer versatile capabilities, including the modulation of gene expression, gene activation, and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs gaining approval over the past two decades. However, further therapeutic development is challenged by unfavourable pharmacokinetics, pharmacodynamics and biodistribution properties for many clinical applications. Chemical modification represents one of the most effective approaches to enhance oligonucleotide drug activity and delivery. In many cases, these modifications are brought by modified phosphoramidites, which are used as building blocks to chemically synthesize oligonucleotide therapeutics. Phosphoramidites can be modified in a variety of ways, including chemical linkers, to covalently attach the final therapeutic product to a ligand, and thus conferring improved activity, stability and delivery properties. Thermo Fisher has a decades long commitment to being at the forefront of innovations in modifying phosphoramidites and nucleotides. This presentation aims to highlight the breadth of modified nucleotides, phosphoramidites and chemical linkers available from Thermo Fisher, which have the potential to be used in innovative strategies to improve the performance and delivery of therapeutic oligonucleotides, paving the way for more effective treatments for a variety of diseases. Not only are these compounds manufactured to the highest quality standards, but they are backed by a comprehensive quality management and documentation system.
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Day 2 – June 26
Hagen Cramer, QurAlis
Strategies for Overcoming Challenges in the Development of Oligonucleotide Therapeutics
RNA therapeutics have catalyzed a revolutionary transformation in the medical field, offering unprecedented potential in disease prevention and treatment. However, despite their great promise, challenges remain which include the following: (1) delivery, due to their high molecular weight and polyionic nature, (2) off-target effects, based on their tertiary structure or sequence analogy with other genes (3) low stability caused by their susceptibility to degradation by nucleases, and (4) unwanted immunostimulatory effects. Chemical modifications have been developed to overcome some of these challenges. A selection of those will be discussed in this presentation.
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Aurélien Thomen, CAMECA’s NanoSIMS commercial speech
Advances in Subcellular measurements of oligonucleotides and peptides with the CAMECA NanoSIMS
There is increasing interest in moving away from animal testing and moving towards human organoids. However, oligonucleotides are difficult to measure in vitro with conventional mass spectrometry. This is partly due to that fact that these molecules tend to become trapped in subcellular structures. In situ techniques are thus necessary to measure the concentration in those structures. Here we present recent results from a new methodology measuring the subcellular concentration of Anti-Sense Oligonucleotide peptide conjugates. The methodology requires isotopic and/or element labelling the conjugate and measuring the labelled part with quantitative imaging. The quantitative imaging is performed with the NanoSIMS 50L ion microscope commercialized by CAMECA. The key to this methodology is the combination of spatial resolution of the NanoSIMS and the sensitivity provided by the right labeling strategy. Typical labeling strategies consist in attaching Iodine element or 34S isotope to the ASO peptide conjugate. The ultimate lateral resolution below 50 nm of the NanoSIMS 50L reveals the organelles content by their labelled signal converted in concentrations by simple calibration. The ability to measure organelles content provides a new view of peptide and oligonucleotide uptake at subcellular level which promises to be a critical metric for the design of these therapeutics. We will conclude by introducing our new NanoSIMS-HR product with a better lateral resolution down to 30 nm and an upcoming cryosystem allowing more accurate measurements on frozen biological sample.
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Sritama Bose, Nucleic Acid Therapy Accelerator (NATA)
Novel phosphoramidite linkers for the synthesis of oligonucleotide conjugates
One of the major challenges limiting oligonucleotide-based therapeutics to reach its full potential is the limited ability of these relatively large and often highly charged molecules to effectively cross cellular membranes, making it difficult for them to reach therapeutic concentrations at the site of action (in the cytosol or nucleus). Chemical conjugation of oligonucleotides with transporter molecules is one of the most convenient approaches employed to improve the intracellular delivery and therapeutic potential of these agents. We have developed a set of novel vinylpyrimidine linkers that have been successfully incorporated in oligonucleotides using standard phosphoramidite chemistry on an automated oligonucleotide synthesiser. Our linkers are linear or cyclic, including carbocyclic and heterocyclic scaffolds with varying ring sizes. We have conjugated these novel linker containing oligonucleotides to thiol bearing small molecules and protein to prepare oligonucleotide conjugates via thiol-ene click reactions. This novel approach could have numerous uses including a) to attach PK/PD improving moieties to therapeutic oligonucleotides; b) to assist in tissue specific delivery and c) act as an imaging tool or probe by attaching fluorescent moieties. In a broader context, the method could be used to conjugate two or more oligonucleotides; and to attach thiol modified biopolymers, biomolecules or small molecules to modified oligonucleotides. The modification could also find application in attaching oligonucleotides to thiol functionalised surfaces, including the surface of nanomaterials to build spherical nucleic acids.Our method can be used as a generic method for preparing thiol-reactive oligonucleotides that is compatible with conventional solid-phase oligonucleotide synthesis protocols. Factors influencing the reactivity of these linkers are also being explored such as position of the double bond, substitution on the double bond, type of ring (purine/pyrimidine) etc which may have effect on the stability and binding/therapeutic efficiency.
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Matthew Simon, Denali Therapeutics Inc.
Targeting Transferrin Receptor to Transport Antisense Oligonucleotides Across the Blood-Brain Barrier
Antisense oligonucleotides (ASOs) are a promising therapeutic option for many neurological disorders, however, the blood-brain barrier (BBB) prevents central nervous system (CNS) delivery of ASOs with systemic administration. Consequently, current ASO therapies are typically delivered to the brain via direct injection into cerebrospinal fluid (CSF), raising safety and biodistribution concerns. Utilizing transferrin receptor (TfR), which is enriched at the BBB and undergoes transcytosis, we have developed a molecule to enable transport of ASOs into the brain called the “oligonucleotide transport vehicle” (OTV). Systemically delivered OTV drives significant, cumulative, and sustained knockdown of the ASO target across multiple CNS regions and all major cell types. Further, systemic OTV delivery enables more uniform ASO biodistribution and knockdown compared to other clinically relevant ASO delivery routes including a standard, high affinity TfR antibody, or direct ASO delivery to the CSF. Together, our data support systemically delivered OTV as a potential therapeutic platform for neurological disorders.
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Sophie Corbet, Dupont – commercial speech
Chromatography techniques for oligonucleotides purification
Oligonucleotides are short synthetic DNA sequences are used for diagnostic and therapeutic purposes. They are manufactured through multi-step chemical synthesis processes which are prone to generate impurities. In this presentation we compare oligonucleotide purification via both anion exchange and reverse phase chromatography strategies.
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Wen-Hsuan Chang, Acurastem
Accelerating ASO Optimization through Humanized Preclinical Models on a Patient-based Platform
In the dynamic landscape of central nervous system (CNS) disorders, the pace of scientific advancement often outstrips our ability to engineer corresponding preclinical models. AcuraStem addresses this challenge through its innovative iNeuroRx® technology platform, which harnesses patient-derived neuronal models (hiPSCs) to faithfully replicate disease pathology without the need for genetic manipulation. Complementing this approach, we employ humanized mouse models tailored for specific disease contexts, facilitating confirmation of in vivo target engagement and mechanism of action—a strategy we term patient-based therapeutics development. Neurodegenerative diseases present particular hurdles due to age-dependent pathology and the diverse genetic backgrounds of patients. Leveraging our platform, we’ve advanced three programs, with one nearing Investigational New Drug (IND) status. Our focus is on disorders like Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), where significant unmet medical needs persist.
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Holger Stalz and Claudia Scaccabarozzi, Agilent commercial speech
Accelerating your biotherapeutic workflows: from automated sample quality analysis to high-quality software solutions
During our workshop, Agilent Specialists will introduce you to new products to automate and accelerate your daily analytical lab routine. Quality assurance of proteins encompasses two main aspects: confirming the accuracy of the manufactured products, and identifying any impurities present in the samples. The New Agilent ProteoAnalyzer system is an automated quality control instrument designed to provide accurate and precise quality measurements of protein samples. The system analyzes proteins based on migration time and fluorescence intensity using parallel capillary electrophoresis with sodium dodecyl sulfate. Rapid and consistent sizing and quantification results can be achieved for both reduced and nonreduced protein samples. The ProteoAnalyzer system analyzes up to 12 samples simultaneously in one run in approximately 30 minutes. Accurate sizing can be achieved for both small and large samples, ranging in size from 10 to 240 kDa. Samples can be detected over a wide concentration range, from 2 to 2,000 ng/µL. The ProteoAnalyzer is a robust system, well-suited for protein applications in biotherapeutics and synthetic biology workflows. The newly introduced Agilent OpenLab CDS 2.8 SW version comes with a set of features to enable the automatization of biopharma workflows. If you want to know how Spectral Deconvolution of your biopharma samples can be automated, or if you are interested about the tools to facilitate and accelerate the analysis of Synthetic Oligos addressing synthesis related impurities detection, we will provide answers during our presentation on OpenLab 2.8.
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David Laffan, AlphaLyncis
Towards a Sustainable Future for Oligonucleotide Manufacture
As theraupeutic oligonucleotides grow in significance in the pharmaceutical industry and the expected commercial volumes continue to increase, there is growing attention on the sustainability challenges of this modality. The current technology is energy intensive and highly wasteful in comparison to standard small molecule Drug Substances as well as using large volumes of hazardous reagents. This places a high burden on manufacturers in terms of waste management and costs, which could affect the overall sustainability of supply chains and the access to these medicines around the world. This talk will summarise the current technology with respect to sustainability of oligonucleotide synthesis, purification and isolation, and discuss them with reference to long term viability and resilience of supply chains. Standard industry measures, in particular PMI (Process Mass Intensity), will be used to provide comparison between current and potential approaches for oligonucleotide manufacture with standard small molecule manufacture. An understanding of potential future developments in oligonucleotide manufacture is essential for those designing commercial supply chains for these important modalities.
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Stefania Corti, University of Milan
Transforming Neurological Care: The Rise of Antisense Oligonucleotide Therapy
Antisense oligonucleotides (ASOs) represent a revolutionary therapeutic approach in neurology, offering new hope for patients with both genetic and acquired neurological disorders. These synthetic molecules precisely target RNA sequences to modulate protein production, critical for disease progression. By potentially decreasing toxic proteins, enhancing beneficial protein production, or correcting dysfunctional protein structures, ASOs can restore normal cellular functions, making them a versatile tool in neurotherapeutics. The transition of ASOs from laboratory research to clinical application involves overcoming significant delivery challenges to ensure these molecules effectively reach their targets within the nervous system. Advances in chemical modification have resulted in ASOs with enhanced structural stability and resistance to degradation, thereby improving their therapeutic potential. Additionally, cutting-edge delivery methods, including viral vectors, peptide and antibody conjugations, and nanocarriers, are being developed to enable ASO penetration through the blood-brain barrier. To date, the U.S. Food and Drug Administration (FDA) has approved six ASO therapies for three neurological conditions: spinal muscular atrophy, Duchenne muscular dystrophy, and hereditary transthyretin-mediated polyneuropathy. These approvals mark significant milestones in the clinical application of ASO technology, particularly in pediatric neurology, where there is a pressing need for effective treatments. Current research efforts are focused on expanding the range of neurological disorders that can be treated with ASOs. Through comprehensive preclinical and clinical studies, researchers are assessing the safety and efficacy of ASOs for a broad spectrum of neurological conditions, including pediatric disorders. This presentation will explore the latest developments in ASO technology, including our insights on mechanisms of action, design, chemical modifications, delivery strategies, and clinical applications, highlighting the transformative potential of ASOs in neurology.
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Clement Paris, AstraZeneca
Challenges and last advances to the development of therapeutics Oligonucleotide for the treatment Pulmonary diseases
The lung as an organ for therapeutics approaches encompassing synthetic oligonucleotides is largely unexplored. Oligonucleotide is an emerging new therapeutics modality for the treatment of chronic respiratory disease as idiopathic pulmonary Fibrosis (IPF), asthma or chronic obstructive pulmonary disease (COPD). Pulmonary delivery of oligonucleotide as drug represents a promising approach to respond to outstanding requirement for lung chronic disease due to the complex lung morphology. Oligonucleotides are design to inhibit specifically mRNA transcript and could potentially hit any disease related target consider as “undruggable” for other modalities like small molecules or biologics. Inhaled administration of oligonucleotide to the lung and specific lung cell type delivery remains challenging due to many physiological barriers. This presentation will focus on the biologicals barrier that affect pulmonary delivery and how chemical modification on the oligonucleotide can influence their properties as drug. The recent strategies implemented for the oligonucleotide delivery to specific cell type will be presented.
