Main Conference Day 3 - 14 November 2024Thursday, 14 November 2024 - CET (Central European Time, GMT+01:00)

The pharmaceutical market is getting more diverse with therapeutic oligonucleotides including siRNA. The characterization and analytical control strategy rely mainly on orthogonal LC and MS methods. This presentation will focus on the method development of novel LC methods and their hyphenation to MS(/MS) for investigation and QC release.

With an increasing number of guide RNA programs emerging in the commercial market, including for in-vivo applications, proper impurity characterization and control of guide RNA impurities are essential to ensure the safety and reliability of a therapy. Enter Next Generation Sequencing (NGS), which not only allows scientists to determine sequence-specific impurities, but also capture the fingerprint of the guide RNA impurity profile.

We present in-line mid-IR PAT as a tool for the optimisation and control of manufacturing scale solid-phase oligonucleotide synthesis (SPOS). IR data collected at the lab-scale was used to optimise the detritylation reactions of a 16-mer ASO in a cycle-by-cycle manner ultimately providing significant reductions in the amount of detritylation reagent solution and acetonitrile washes used. In addition, mid-IR PAT highlighted potentially critical differences between development and manufacturing scale synthesisers demonstrating the potential for this technology for monitoring and control. Finally, we share our views how this technology can be used in the future to further optimise SPOS.

Changing from the well-established DMF-based SPPS platform to non-toxic binary solvent mixtures causes both chemical and practical challenges, but also provides new tools and opportunities for process optimisation.

The presentation will showcase the selection and synthesis of relevant siRNA product-related impurities. Development of a chromatographic method for separation of impurities through HILIC will be demonstrated. The ability of HILIC to complement other separation modes (AEX, IP-RP) will be discussed.

Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical method used in the pharmaceutical industry for detecting and characterizing impurities in drug substances and products. For Bepiroversin; an oligo-based drug by GSK, we built the BEPI LCMS Impurity application; a web application that captures raw LCMS data, calculates the results of full-length impurities content drug substance/product, and compiles detailed reports of impurity analysis, including validation results and compliance with regulatory guidelines.

Modified messenger RNA constitutes an interesting new approach for transient protein expression in different therapies, including the recently approved SARS-Cov-2 vaccines. However, the details of the intracellular delivery of such macromolecules using so-called lipid nanoparticles remains unknown. In this work we have prepared lipid nanoparticles (LNPs) of two different ionizable lipids (DLin-MC3-DMA and DLin-DMA), cholesterol, distearylphophatidyl choline (DSPC) and a PEG lipid. We then dosed these two LNPs intravenously in mice measuring LNP uptake, mRNA delivery and the concurrent protein expression in liver cells, i.e. hepatocytes, liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). The in vivo data clearly showed that although uptake of lipid and delivered mRNA is very similar for both types of LNPs, the protein expression in hepatocytes is order of magnitude different. In order to rationalize these in vivo observations, mRNA LNPs were characterized by several techniques e.g. 13C-NMR and small-angle x-ray scattering. Previously, we have shown that LNPs have a core-shell structure and here we focused our efforts into studying the core of LNPs, as bulk phases. By careful analysis of the inverse hexagonal phase structure of both ionizable lipids, we put forward a hypothesis on why DLin-MC3-DMA LNPs outperforms DLin-DMA LNPs in vivo.

Traditional peptide synthesis relies on an orthogonal protecting group strategy, employing excess coupling reagents and generating significant waste. To enhance sustainability, our collaboration with PeptiSystems explores the potential of flow-through column technology. Initial experiments show promising results, reducing amino acid and coupling agent consumption, while maintaining comparable crude purities. In addition, coupling times were decreased, and the PMI significantly improved using high loaded resins without compromising the overall process. This initiative aligns with Corden Pharma’s commitment to greener and more efficient manufacturing.

Cys is a key amino acid in many biological processes and hence, in therapeutic peptides. The sulfur present in Cys, and also in Met, are prone to suffer side reactions, which can ruin a large-scale production. The minimization or abolition of the most common side reactions on Cys and Met residues such as t-butylation, oxidation, and racemization of Cys-terminal carboxylic peptides will be presented.

The elimination of orthogonal protective groups for Arginine and Histidine in SPPS, allows to increase single amino acid incorporation atom economy and the overall ideality factor in green solvents binary mixture. The typical impurities generated during the cleavage from the resin coming from protective group residues are suppressed simplifying the final purification process.

Avecia has successfully validated 15 programs for a diverse range of oligonucleotides, including both modified and unmodified DNA/RNA, with phosphorothioate, phosphodiester, or mixed backbones, and various phosphate modifications. This presentation will detail Avecia’s approach to process validation, including stages and strategies employed. Supporting documents for validation will also be explained, and an overview of Avecia’s capabilities and production scales will be provided

Synthesis of long oligonucleotides have gained a lot of attention in the last several years due to the development of various genome editing technologies. At Tessera, we have developed a robust, efficient and high-yielding processes, both in high throughput format as well as gram-scale, to access quality oligonucleotides to support our Gene Writing platforms. Our methods are also compatible with a variety of chemical modifications which provides us a wider design space to improve editing efficiency.

Pioneering advancements in guide RNA manufacturing since 2016, BioSpring is a global supplier of commercial, clinical, and preclinical guide RNA. In this talk, our leading manufacturing expert will guide you through what it takes to scale GMP guide RNA manufacturing for clinical and commercial use, the extensive development involved, and the engineering and innovation that goes into evolving reliable high-resolution analytical methods and achieving high purity, even in long and complex guide RNA constructs.

EMA guideline on the development and manufacture of synthetic peptides addresses specific aspects regarding the manufacturing process, characterisation, specifications and analytical control for synthetic peptides which are not covered in other guidelines. The Pharma industry has just finished commenting on the 1st draft of the new EMA guidance directly or via different pharma associations. The feedback included industry reflections on important topics such as; expectations to have limits for impurities in individual purification fractions, expectation that peptide impurities limit from the European pharmacopoeia is applied to early clinical trial, potential for impurities chromatographic peaks grouping with overall limit and considerations related to bioassay, immunogenicity and API Starting materials. This presentation will provide an insight into the most important and critical feedback provided back to EMA by the industry as the presenter participated in putting and submitting those comments via an European industry association.

In 2022 FDA issued a product-specific guidance for Nusinersen advocating for the establishment of diastereomeric composition sameness between generic API and the Reference Listed Drug (RLD). This recommendation presents a formidable challenge for generic industry as Nusinersen drug substance, with its intricate structure, is a mixture of about 130,000 stereoisomers. TAPI approaches for development of generic Nusinersen drug substance manufacturing process, analytical characterization and sameness assessment will be presented.

Growing interest in the application of peptide drugs in major metabolic diseases space has opened up enormous opportunities for revisiting existing manufacturing strategies. To meet future needs of peptides estimated to be in multi ton quantities, it is important to overcome challenges in the most widely used SPPS as well as to develop alternative synthetic technologies and isolation processes.

We will present our first results on a new linker concept based on the safety-catch principle. This allows the cleavage of the peptide from the resin without the concourse of any acid. This safety-catch linker is stable to both bases, such as piperidine, and acids, such as trifluoroacetic acid (TFA). Still, at the end of the synthetic process, it is converted into a linker labile to bases. Thus, this linker is compatible with both Fmoc and Boc strategies before the cleavage is chemically manipulated to alter it to be labile in the presence of a base.