The presentation will showcase Biogen’s efforts to scale LPOS to 2.5kg and then to 25 kg in readiness to GMP manufacturing. Highlight the synthetic and analytical challenges and then look ahead to new technologies developed in-house that could help to simplify the manufacturing process.

Isolating oligonucleotides as aqueous solutions can enhance safety, sustainability, and efficiency of drug manufacturing process. The design of the drug substance manufacturing facility should incorporate principles from both small molecule and biologics manufacturing. A careful selection of drug substance matrix can facilitate forward processing to drug product and lower exposure risk compared to powder. Recognizing solution oligonucleotides as drug substance can streamline manufacturing process and improve efficiency.

Disulfide-constrained peptides have gained interested due to their intrinsic stable properties and the plasticity of the loops protruding from the core of the peptide. Capitalizing on these intrinsic properties we are building a powerful display platform to produce peptide leads for clinically validated targets.

My laboratory is involved in the discovery and development of cyclic peptides for therapeutic applications. In recent years, we have begun to address the long-standing goal of developing target-specific peptides that are membrane-permeable and orally available. To this end, we are focusing on the generation of cyclic peptides that have a relatively small size (< 800 Da) and a limited polar surface area, so that they have a high chance of passively crossing membranes. To generate sub-kilodalton cyclic peptides that bind to disease targets of interest, we have established an approach based on nanomole-scale cyclic peptide synthesis and high-throughput screening of crude products (1, 2). In short, we generate thousands of peptides by solid-phase peptide synthesis and combinatorially diversify them by reacting them with a myriad of chemical building blocks. In this approach, all reagents are transferred in nanolitre volumes by acoustic dispensing and reactions are performed at the nanomole scale, allowing tens of thousands of cyclic peptides to be synthesised and screened in a short time. Recently, we have shown that cyclic peptides developed using this approach can achieve good oral availability (3). In my talk, I will explain the approach to the synthesis and screening of cyclic peptide libraries, show examples of libraries and their screening, present nanomolar ligands we have developed against different proteins, including a protein-protein interaction target, and show data on the membrane permeability and oral availability of the peptides. [1] S. Kale, et al., Science Advances. 2019, 5 (8). [2] S. Habeshian, et al., Nature Communications. 2022, 13 (3823) [3] M.L. Merz, et al., Nature Chemical Biology. 2023.

Membrane receptor proteins are essential in cellular signaling. In recent years, there has been a surge of interest in discovering peptide binders to membrane receptors due to their potential use for diagnostic, therapeutic, and theranostic purposes. Phage display technology is frequently employed to identify peptide binders to recombinant proteins. Improper folding of recombinant proteins, a well-known challenge, would hinder the identification of peptide binders. In our lab, we developed a cell-based model to overexpress membrane proteins for phage display biopanning to ensure reliable identification of cyclic peptide binders. In this presentation, we show different examples of novel CLIPS^TM peptides with excellent binding affinities and selectivities to the protein of interest that were identified using our novel cell-based model. We illustrate the techniques that enable cell-based biopannings using a benchmark membrane protein. Additionally, we will share preliminary data on a “real” difficult-to-target protein for which a recombinant alternative is currently unavailable. The work presented convincingly illustrates that cell-based biopanning is a reliable model for discovering peptide binders to membrane-bound proteins that are difficult to target using the recombinant form of that same protein.

The currently largest European manufacturing plant for Oligonucleotides has been built in Switzerland in 2020-2022. Since the first batch started in August 2022 several drug substance batches have been successfully produced. The main challenges encountered during the design phase, including low bioburden related activities, consumables selection, management of water and solvents are discussed. In addition selected cases encountered during manufacturing of Leqvio drug substance are presented.

Oligonucleotides are dream molecules for an NMR spectroscopist. So many aspects related to both structure and content can be studied by NMR. Total content determination is performed by the internal standard ³¹P platform qNMR method for both drug substance and drug product. Correct hydrogen bonding in the loop in one RNA strand, is furthermore confirmed by using a 2D ¹H-¹⁵N platform method at natural abundance.

Herantis Pharma has developed a novel D-amino acid peptide HER-096 that mimicks the active site of the neuroprotective and neurorestorative CDNF protein, and efficiently penetrates the blood-brain barrier allowing peripheral administration. Subcutaneously administered HER-096 has shown great efficacy in a preclinical Parkinson’s disease animal model. A phase 1a clinical study demonstrated good safety & tolerability profile and efficient blood-brain barrier penetration in healthy volunteers.

Using the streaMLine platform, we have developed GUBamy a long-acting amylin analogue with attractive therapeutic applications. GUBamy is an amylin receptor selective analogue with improved physical and chemical properties allowing formulation at neutral pH. GUBamy shows 10 % weight loss in DIO rats, and its pre-clinical PK makes it suitable for once weekly dosing in humans. GUBamy is currently in phase I.

Improving understanding of processes offers dual benefits for oligonucleotide development. Firstly, it establishes a potential platform for creating similar molecules with different sequences, thereby accelerating process development time /efforts. Secondly, it ensures robust processes that yield high-quality APIs. While Quality by Design (QbD) is often equated with Design of Experiments (DoE), it actually encompasses overall process design, with DoE being just one aspect. We'll explore how embracing QbD principles can optimize process understanding and utilization of data, including examples of how mathematical modeling systematically gathers data, enhances process understanding , and contributes to process characterization in solid-supported synthesis.

While alternative methods are being explored, solid-supported synthesis remains the most established and practical approach for producing oligonucleotides, especially in smaller quantities. Before 2016, the main focus was on introducing oligonucleotides as therapeutics. However, with over 15 approvals in an 8-year period, and considering the potential for a platform approach, developing a strategy for process characterization would greatly benefit the overall oligonucleotide portfolio and streamline the filing process with a deeper understanding. This study emphasizes the importance of the Quality by Design (QbD) concept from development through to product lifecycle. Using case studies to model specific unit operations like TFF, we demonstrate how this approach can provide regulatory benefits.

Recent RNA approvals highlight the need for robust analytical methods to monitor critical quality attributes (CQAs), ensuring control of manufacturing processes and product quality. Oligonucleotide mapping via liquid chromatography / mass spectrometry is effective for CQA assessment, however, sample preparation approaches and data handling present a time-consuming challenge. A novel digestion procedure, data acquisition and data processing workflow was developed to produce fragment assignments for sgRNA and mRNA, including measurement of key CQAs

QurAlis is developing precision therapies, targeting genetic drivers in sub-forms of ALS. Two of QurAlis’ programs are in early phase clinical trials. One of these programs is a splice modulator ASO delivered by intrathecal injection. The talk will focus on how to successfully prepare for IND/IMPD regulatory submissions with regulatory expectations changing over time.

In recent years there has been increased interest in using peptides as therapeutics, especially since the introduction of peptide drug substances Semaglutide and Tirezepetide into the market. This talk will describe strategies we use at AstraZenca to develop close to commercial processes to deliver peptide drug substance, where the key performance indicators are cost of goods, throughput and sustainability.

Advancements in real-time process analytical technologies (PAT) have significantly improved insights into chromatographic processes. However, the complexity of model building, associated equipment and mathematical algorithms often requires extensive development and restricts applications to late-stage processes.

We present a streamlined PAT approach based on selected wavelengths in the UV/Vis range, tailored to early-stage development of purification processes for peptides, and for transfer to multi-purpose production facilities. The approach presented ensures fast development cycles and increases flexibility in large-scale production.

Novartis' first marketed siRNA oligonucleotide, inclisiran, is approved in 90+ countries around the globe, including EU and US, for the treatment of hypercholesterolemia. In this presentation, we will share our post-approval experiences with Health Authorities, discuss the key CMC aspects that require careful consideration and planning and highlight the learnings and best practices that can facilitate a successful and efficient regulatory CMC post-approval process for siRNA oligonucleotides.