Main Conference - Day 2 (May 16)Thursday, 16 May 2024

The rapidly growing number of therapies approved and in advanced clinical trials is placing unprecedented demands on our capacity to manufacture oligonucleotides at scale. Existing methods of chemical synthesis rely on iterative coupling, capping, oxidation and deprotection to achieve stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. This talk will describe transformative biocatalytic approaches to efficiently produce oligonucleotides in a single operation, where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks, aqueous conditions and can be used to produce diverse oligonucleotide sequences containing a range of pharmaceutically relevant modifications.

Therapeutic RNA oligonucleotides have shown tremendous potential to manage and treat disease, yet current manufacturing methods may not be able to deliver on this promise. Here, we report the development and optimization of a novel, aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methodologies. Our platform is made possible by reversible terminator nucleoside triphosphates and an enzyme capable of their incorporation. We show that many common therapeutic RNA modifications are compatible with our process and demonstrate the enzymatic synthesis of natural and modified oligonucleotides in both liquid and solid phases. Our platform offers many unique advantages over chemical synthesis, including the realization of a more sustainable process to produce therapeutic RNA oligonucleotides.

Chemically modified synthetic RNA used for therapeutic applications such as antisense oligonucleotides and RNA based genome engineering are difficult and expensive to synthesize using current phosphoramidite chemical synthesis methods. Thus, new RNA synthesis technologies designed to significantly increase yields and efficiency while greatly reducing costs are needed. We demonstrate rapid and efficient one step enzymatic synthesis of chemically modified RNA oligonucleotides over 150 nt in length with >90-95% purity. A newly discovered RNA polymerase efficiently synthesizes RNA with 100% 2’-fluoro, 2’-O-methyl or alpha-phosphorothioate modified ribonucleotides. We anticipate that scaling up of this versatile enzymatic technology will allow for significant reductions in the cost of synthesizing chemically modified RNA oligonucleotides and mRNA based therapeutics while greatly increasing yields for large scale production.

Cyclic peptides are diverse molecules that are now a focus in drug discovery efforts. Their molecular size, between small molecules and biologics, provides attractive scaffolds to screen against some challenging targets, including protein-protein interactions and those considered to be “undruggable” proteins. With messenger ribonucleic acid (mRNA) display screening technology now able to produce trillions of peptide molecules for screening and quickly identify tight binders against targeting proteins, an exciting time of cyclic peptide drug discovery has come. We have been working on cyclic peptide drug discovery since 2010 and have successfully identified two compounds derived from mRNA display that have entered clinical trials. One of them is a complement C5 inhibitor, zilucoplan. Here we present the discovery of zilucoplan, starting from hits identification via mRNA display screening against C5, followed by medicinal chemistry modifications to improve the potency, plasma stability and PK properties, leading to the clinical candidate.

Bicycles are low molecular weight bicyclic peptides constrained via a chemical scaffold. The pharmacologic and pharmacodynamic properties of Bicycles are highly suited to the delivery of potent payloads such as toxins, radionuclides and immune agonists in oncology. This presentation will focus on the application of Bicycle tumor targeted immune cell agonists (Bicycle TICA^TM) that simultaneously bind to overexpressed cell-surface targets on tumor cells and activating receptors on immune cells to drive highly specific anti-tumor activity.

GSK has developed a templated oligonucleotide assembly platform that takes advantage of engineered DNA ligases to make single stranded oligonucleotides. The process eliminates the need for chromatography yet produces oligonucleotides with purity that exceeds that typically seen for solid supported synthesis. Data on application of this platform to different oligonucleotide types and progress on scale up will be presented.

Enzymatic de novo DNA synthesis promises higher oligo quality, length, and could simplify manufacturing processes due to the absence of hazardous reagents and waste. We discuss a novel approach for enzymatic DNA synthesis based on polymerase-nucleotide conjugates, describe early use cases of the technology and how the system enables direct synthesis of oligos longer than 1000 nucleotides.

This talk will focus on a platform of novel P(V) reagents for the synthesis of nucleic acids and other phosphorus containing molecules. The historical development of these reagents in collaboration with BMS will be described along with their application in a variety of different contexts: Chimeric oligonucleotides with numerous internucleotide bonds, cyclic dinucleotides, bioconjugation, di-and tri-phosphates, radical chemistry, and even a unique way for the peptide universe to benefit from this chemistry.

ProteinQure has designed a series of de-novo peptides including the first known cyclic binders to SORT1 using our proprietary computational platform. Sortilin (SORT1) is a member of the vacuolar protein sorting 10 protein (Vps10p). As a cell surface receptor, SORT1 is able to mediate efficient endocytosis of extracellular ligands to the lysosomal compartment. Numerous reports have identified enriched SORT1 expression in the brain. We sought to exploit SORT1-dependent internalization of peptides as a platform for rapid and specific siRNA delivery into CNS cells. Using PQStudio (our proprietary computation-enabled design capabilities), we generated high affinity SORT1 targeting peptides that exhibit efficient receptor-dependent internalization. Alternative computational approaches such as AlphaFold2 and large language models failed to recapitulate the peptide design. Peptide-siRNA conjugates molecules exhibit potent and durable knockdown in all regions of the brain in mouse models, thereby highlighting the potential of SORT1-engaging peptides for nucleotide delivery.

Conventional delivery technologies for genetic medicine face challenges: off-target delivery, innate immune response, unable to repeat dose, or costly manufacturing. NanoGalaxy platform consists of a diverse library of hydrophilic polymers and, through systematic and iterative screening, has been used to identify NPs with selective delivery to the nervous system via intrathecal administration and to the innate immune system via intravenous administration. This presentation will introduce NanoGalaxy platform and share the delivery results of genetic medicine payloads.

To overcome limitations of intracellular delivery of biologics, we designed a family of cyclic cell-penetrating peptides that form the core of our Endosomal Escape Vehicle (EEV™) technology. EEV peptides efficiently delivered oligonucleotides to skeletal and cardiac muscle in preclinical models of Duchenne muscular dystrophy. Additionally, EEV-modified lipid nanoparticles enhanced the delivery of mRNA and gene editing in primary human immune cells. These findings demonstrate the potential of the EEV platform to efficiently deliver different types of biologic therapies to target cells and tissues.

The CRISPR/Casx system is widely recognized as a breakthrough technology for precisely editing DNA sequences, allowing for the removal, addition, or alteration of genetic material. Comprising two crucial elements, the system features the enzymatic scissor, Casx, and the guide RNA, known as single guide RNA (sgRNA), tasked with precision in genome targeting. sgRNA can be generated through cell transcription, in vitro transcription (IVT), or solid-supported synthesis. The growing demand for solid-supported synthesis, particularly for longmers (>100mer), to meet therapeutic needs presents unique challenges compared to ASO or siRNA synthesis. This presentation will delve into the outcomes of process optimization for longmer preparation, shedding light on the impact of key parameters.

The analytics needed to support the characterization and release of CRISPR-based therapeutics represent a broad landscape of techniques and methods and have unique scientific challenges. The analytical needs will change significantly across ex vivo vs. in vivo approaches, delivery modalities, and several of potency assays may be needed, and will typically be unique for each indication. Clinical phase associated validation requirements and current regulatory guidance documents must also be considered.

Not all oligonucleotides drug substance manufacturing processes are equal. Within the industry there are ambiguities regarding microbial control for Oligos, which have characteristics of upstream synthesis similar to Small Molecules and downstream purification similar to Large Molecules. Therefore, there is no one size fits all when it comes to the application of a microbial control concept. What happens when in-process bioburden samples from a processing step exceed the control limits or preliminary target while the final API release results are well within specification? This presentation will cover lessons learned where a risk based holistic microbial approach was utilized to determine impact on patient safety and material quality of a clinical phase GMP manufactured oligonucleotide DS.

There is currently a lack of guidelines and harmonization from Health Agencies on the control strategies that should be implemented for synthetic peptide active pharmaceutical ingredients. In the US, further ambiguity is perceived since synthetic peptides > 40 amino acids are registered as BLA and synthetic peptides ≤ 40 amino acids are registered as NDA. This can result in an increased regulatory risk at the time of filing and globally divergent, non-efficient approaches. The Peptides Working Group (WG), as a part of the International Consortium for Innovation & Quality (IQ) in Pharmaceutical Development, has conducted a survey of participating IQ Consortium companies on the control strategies applied for synthetic peptides based on their phase of development and number of amino acids for both DS and DP. In this presentation, a comprehensive analysis of the survey results will be presented along with recommendations on phase- and size-appropriate specification setting strategies. The compiled survey results from ten pharmaceutical companies revealed that while most respondents follow similar control strategies for ID testing, purity measurements, and assay testing, none of the survey questions received a unanimous response. Interestingly, the number of, and type of, analytical techniques utilized for each test differed when comparing the phase of development, the number of amino acids in the peptide, and whether it was for the DS or DP. The survey questions that surprisingly had the greatest variance were what limits companies set for their reporting, identification, and qualification thresholds throughout development as well as the rationale used to justify these limits. It is evident from the results of this survey that there is a lack of alignment amongst the pharmaceutical industry on what specifications and controls should be implemented for synthetic peptides. Ultimately, the knowledge acquired from the survey results in combination with previously published literature and unique company experiences has enabled the Peptide WG to put forth appropriate recommendations to achieve harmonization on control strategies for peptides.

Process analytical control strategies are built from the incoming starting material supply, process and purge capabilities, analytical control strategies, risk assessment tools and state of the art analytical methods. Different customers and regulatory agencies are requiring different levels of risk management and control. This presentation will showcase several peptide drug substance examples of managing customer and regulatory expectations, while assuring supply chain capabilities.

European medicines agency (EMA) published a draft guidance on the development and manufacture of synthetic peptides which will address multiple quality aspects including control for peptide purity and related impurities. The EMA guidance discusses the use of orthogonal methods to minimize the risk of undetected impurities. This presentation examines the analytical toolbox available to support peptide synthesis process development and provides a case study - building an impurities control strategy for an AstraZeneca phase 3 chemically synthesized peptide drug candidate. The formation, identification, quantitation, fate and purge of certain impurity classes are discussed in the context of developing the manufacturing process and control strategy for the synthetic peptide drug substance.

Entos Pharmaceuticals’ proprietary Fusogenix Proteo-Lipid Vehicle™ (PLV™) platform, enables precision non-viral and re-dosable delivery of all nucleic acids payloads (DNA, RNA, or combinations), ushering in the new era of genetic medicine. The Fusogenix platform combines the best attributes of current viral and nonviral delivery technology that allows for delivery of all nucleic acid modalities including gene editing tools.

The FORCE™ Platform was developed to enable TfR1-mediated delivery of oligonucleotides to muscle for the treatment of serious genetic muscle diseases. Preclinical data showed robust muscle delivery and target engagement in DM1 and DMD disease models. Initial data from the ACHIEVE trial in DM1 and DELIVER trial in DMD demonstrated clinical proof of concept.

The production of oligo drug substances faces two critical challenges: improving chemical purity and establishing sequence identity. In this presentation, we delve into Agilent Technologies’ large-scale proof-of-concept orthogonal chromatography approach. Additionally, we explore a novel MS-guided PAT (process analytical technology) designed for verifying synthesis reagents during synthetic nucleotide chain extension. This innovative approach has significant implications for achieving full sequence verification of a synthetic oligo drug substance, regardless of its length.

Acylated peptide are required in multi-tonne amounts for treating type-2 diabetes and obesity. This contribution will focus on cost and capacity models for fragment-based approaches and compare this with linear SPPS.

In the past, acetonitrile has primarily been recovered by multistage distillations. While these can be effective, they also come with some processing limitations and challenges. An alternative pilot scale system has developed which demonstrates a new approach to recovering this critical solvent for peptide manufacturing and greening processes.

Using the transferrin receptor as a mechanism to target and deliver siRNAs to muscle has now been demonstrated in multiple species and in clinical trials. Exciting new data from recent preclinical studies and clinical trials demonstrate the power of receptor-mediated uptake of therapeutics to broaden the scope of cell and tissue types that can be targeted with oligonucleotide therapeutics.